Lakhtin V., Lakhtin M., Aleshkin V. INTERACTION OF

Lakhtin V., Lakhtin M., Aleshkin V. G.N. Gabrichevsky Research Institute for Epidemiology and Microbiology, Russia INTERACTION OF GLYCOSIDASES WITH CARBOHYDRATE-SENSITIVE REAGENTS Summary. The data on glycobiological aspects of 68 glycosidasases (interacting with carbohydrate-sensitive proteins and other reagents) from 430 sources are systemized and presented as a table. Key words: glycosidases, lectins, glycoconjugate recognition, biotechnology, microbiology, medicine. The data on at least 68 exo- and endoglycosidases from more than 430 sources are presented in the table (see below). Isolation of glycosidases using lectins The use of ConA-sorbents was especially effective for isolation of: shrimp chitinase (40 fold [f]) [905], bovine testicular lysosomal sialidase (25 f) [923], human placental lysosomal alpha- glucosidase (127 f) [960], human liver acid alpha-glucosidase (35-54 f) [707], flax linustatinase and linamarase as cyanogenic beta-glucosidases (32-44 f) [1276], human placental lysosomal alpha- glucosidase (146 f) [960], leguminous seed alpha-galactosidases (22-80 f) [972, 973, 977], human liver or placental acid beta-galactosidases (107-166 f) [960, 984, 985], feline or human liver lysosomal beta-galactosidase (45-78 f) [999, 1000, 1002, 1003], human liver acid beta-galactosidase of the patient with GM1-gangliosidosis (344 f) [1006], human placental acid beta-galactosidases (127-167 f) [1011, 924], wheat grain aleuronic alpha-mannosidase (49 f) [1034], mussel hepatopancreatic Zn2+-dependent alpha-mannosidase (71 f) [1037], human placental acid alpha- mannosidase (109 f) [960], goat kidney lysosomal alpha-mannosidase (27 f) [1056], mung bean beta- fructofuranosidase (40-90 f) [1107, 1109], pea beta-N-acetyl-D-glucosaminidases (70 f) [1031], human placental acid beta-N-acetyl-D-glucosaminidase (119 f) [960], ficus latex beta-N-acetyl-D- glucosaminidase (110 f) [1166], pea seed beta-N-acetyl-D-glucosaminidases (137-250 f) [1170], human placental acid beta-N-acetyl-D-hexoseaminidases (45-63 f) [1193, 1201], human spleen acid beta-N-acetyl-D-hexoseaminidases (40 f) [1208], human liver acid alpha-L-arabinofuranosidase (35- 54 f) [707], human lung alpha-L-iduronidase (540 f) [1242], and white mustard seed myrosinase (33 f) [1302]. The use of WGA-sorbents was especially effective in islolation of: wheat (endogenic) grain aleuronic beta-galactosidases (48 f) [991], human liver acid alpha-mannosidase (93 f) [707], fungal beta-N-acetyl-D-glucosaminidase (33 f) [968], wheat (endogenic) grain beta-N-acetyl-D- glucosaminidase (34 f) [1174], and human liver lactosylceramidase-II (75 f) [707]. LCA-sorbents were useful for isolation and multiple forms separation of: lentil alpha- galactosidase forms [979], bacteriophage hyaluronidase [1132], parasitical nematode beta-N-acetyl- D-hexososaminidase (10 f) [1182], and calf thyroid gland plasma membrane bound NAD- glycohydrolase [1295]. Other immobilized lectins were effective for the isolation of rat liver lysosomal alpha- glucosidase (RCA-sorbent, 36 fold) [617], influenza virion sialidase (CJA-I-sorbent) [918]. In many cases glycosidases were activated during lectin chromatography. Thus, slight activation (up to 10%) was observed in the cases of sorghum cyanogenic beta-glucosidase (dhurrinase) or black cherry prunasine hydrolases filtrated through ConA-sorbent [1275, 1278], human placental lysosomal alpha-glucosidase [960], chicken liver or bovine brain beta-galactosidases [995, 996], human cytosolic alpha-mannosidase filtrated through lectin sorbent [1042], monkey testicular

1 hyaluronidase [1139], or potato tuber beta-fructofuranosidase [1104]. Significant levels of glycosidase activation were registrated in the following cases: rabbit kidney neutral beta-glucosidase filtrated through lectin sorbent (18% activation) [962], human placental beta-galactosidase (50%) [985], human liver acid beta-galactosidase of the patient with GM1- gangliosidosis (few fold activation) [1006], rabbit kidney neutral beta-galactosidase filtrated through lectin sorbent (18% activation) [962], guinea pig liver acid alpha-mannosidase (42% activation) [1059], potato tuber beta-fructofuranosidase (84% activation) [1105]. Interestingly, activator(s) of glycosidases can be also activated during lectin chromatography, as it was shown for glucosylceramidase activator proteins from human spleen (up to 30%) [1148]. Thus, glycosidase activation can be influenced by elimination of glycosidase inhibitor(s) [1105], or the presence of endogenic activating factors. In a number cases, glycosidase activation was accompanied with significant levels of enzyme purification at the step of lectin chromatography [960, 985, 1005, 1106]. A lot of examples on separation of enzyme multiple forms can be found in the table. For example, mammalian lysosomal (acid) glycosidase forms with affinity to lectins can be simply separated from cytosolic (neutral) glycosidase forms without affinity to the same lectins [707, 958, 962, 1192, 1216]. Fungal purified glycosidase forms having similar sugar composition can be further separated on lectin sorbent using elution with sugar gradient [876]. A set of lectins allows to reveal or separate glycosidase forms, as it was demonstrated for human liver acid beta-galactosidase [1008], radish or castor bean beta-fructofuranosidases [1098, 1103], sweet wormwood or rat liver beta-N- acetyl-D-glucosaminidases [952, 1128], human intestinal sucrase-isomaltase [892]. Interestingly, mutual conversion of isoenzyme forms can take place [828, 1008]. Sometimes, glycosidases reveal strong binding to lectin sorbents. For example, immobilization of fungal beta-glucosidase on ConA-Macrosorb has taken place even in the presence of 0,5 M lectin- specific sugar and 10% ethanol [949]. In another cases the presence of ethylene glycol or borate together with sugar was needed for successful elution of glycosidases bound to lectin sorbents [1190, 1118]. Retaining of glycosidase activity of enzyme bound to lectin allows using lectin matrixes as affinity carriers for preparing biocatalysts (see Chapter 1). Sometimes, low affinity interaction between glycosidases and lectin sorbents result in enzyme or enzyme form retardation in eluates without use of sugar for elution [952, 968, 1098]. Interestingly, a number of glycopeptides and glycans from glycoproteins can be retarded during lectin chromatography, depending on glycan structure and lectin type (see Chapter 1). Coupled/ sequential column technique was applied for purification of the following glycosidases: bacterial cell wall zymogenic form of lysozyme (combination of immobilized hemoglobin and ConA, 62 f) [914], trichinella larval beta-N-acetyl-D-hexoseaminidases (filtration through DEAEC followed by affinity chromatography on LCA-sorbent) [1182], human liver lysosomal alpha-L-iduronidase (ConA and Blue agarose, 380 f) [1241]. Few cases include the use of lectin sorbents for immobilization of ligands interacting with glycosidases (for "sandwich" chromatography of glycosidases) [1069, 1070, 1238, 1253]. It shoud be noted that the use of lectins (RCA-I and others) in study of pathological forms of glycosidases may be of diagnostical significanca. Examples of glycosidases from human tissues and cell lines from patients with liver disorders, I-cell disease, mucolipidosis-II, GM1 gangliosidosis, leukaemic diseases can be found in the general table. Glycosidases can interact with endogenic lectins [1019, 1174, 1196, 1219]. As a result of glycosidase-lectin complexes, modulation of glycosidase activity can be revealed (see Chapter 1). Carbohydrate moiety of glycosidases As for other enzyme classes and esterases described above, carbohydrate moiety of glycosidases are characterized as wide varying one: from very low contents (<1% w/w) in cases of bacterial beta-

2 amylase [812], fungal beta-glucosidase [946], or rat liver mannosidase-I from Golgi apparatus [1271] up to 45-80% in the cases of Aspergillus cellulaes and beta-glucosidase [861, 938], yeast glucoamylase [847], beta-fructofuranosidase [1065] and exo-beta-1,3-D-glucanase [1231], or castor bean beta-fructofuranosidase [1102]. Interestingly, too low sugar content does not allow to interact with lectin properly. In such cases special conditions are needed. Thus, Man9GlcNAc-splitting alpha-mannosidase must be incubated with ConA-sorbent for 16 hours to increase its binding ability from 0 up to 70% [1271]. Specific interaction in this case was supported by experiment with EndoH-treated enzyme which lost ability for further interaction with lectin. On the other hand, polysaccharide-associated enzyme with relatively high content of sugars can not interact with the same lectin. Thus, Candida exo-beta-D-glucanase treated with beta-glucosidase reveals ability to be precipitated with ConA [1231]. However, native enzyme (68% sugars, mainly glucose and mannose) does not bind to ConA-sorbent nor be precipitated with non-immobilized lectin [1231]. In some cases carbohydrate composition of glycosidase sanples was characterized by the presence of L-rhamnose [813, 910], L-arabinose [971], or both sugars [976]. However, many microbial glycosidases include sugar residues which are typical for glycans linked to proteins [280, 692, 866, 898, 987, 1161]. That is why the use of carbohydrate-binding proteins in study of glycosidases would be of special interest for further glycan structure analysis or separation of glycosidase multiple forms. A set of lectins with specificities needed can be selected from the table. Carbohydrate moiety of glycosidases can be investigated with a set of standard glycosidases alone or in combination with lectin analysis as it was described and discussed above for other enzume classes and groups. In addition to EndoH, EndoF and sialidase, other following glycosidases were used for study of isolated enzymes: endo-beta-N-acetyl-D-glucosaminidase and glycopeptidase in the cases of human salivary alpha-amylases [828], beta-glucosidase in case of yeast exo-beta-glucanase [1231], alpha- mannosidases in the cases of Aspergillus oryzae glucoamylase [838], and Saccharomyces cerevisiae beta-fructofuranosidase [1078]. The size of carbohydrate moiety can be calculated using endoglycosidase-treatments (see general table). The use of tunicamycin-treated cells [1101, 1277], or TFMA-treatment for elimination of carbohydrate moiety [900, 1086] can be also useful for registration of enzyme multiple forms and evaluation of the sugar contents. For example, TFMSA-treatment can help in study of O-linked glycans resistant to EndoH, EndoF, or N-glycanases [900, 902]. Rhizopus oligosporus chitinases 50 and 52 kDa include 11,0 or 10,6% sugars resistant to EndoH but sensitive to TFMSA [900]. O- Glycans (mucin type, others) are found in many glycosidases [449, 814, 815, 835, 838, 898]. Tunicamycin- or EndoH-treatment resulted in abolishing of the investigated glycosidases in interaction with ConA [1101, 815, 821, 822, 1284, 1271]. Human liver acid alpha-mannosidase treated with exogenic sialidase revealed distinct displacement of isoelectric point (pI) from 6,0 up to 6,45 [1039]. Carbohydrate moiety can include phosphorylated and sulphated glycans recognized by endogenic receptor lectins [1196, 1219]. Interestingly, human urine precursor of alpha-glucosidase contains mannose-6-phosphate compared to the absence of this saccharide in the mature form [854]. Examples of recombinant glycosidases can demonstrate both the use of such enzymes as a good models in study of carbohydrate moiety and perspectives of the host cell systems in which investigated enzyme is expressed: murine alpha-amylase expressed in yeasts, with affinity to ConA and sensitivity to EndoH (ConA+EndoH+) [818], clostridial endoglucanase from yeasts (ConA+EndoF+) [904], glucoamylase STA2-encoded form-II from yeasts (EndoH+) [847], trichodermal endo-beta-4-glucanases from yeasts (ConA+) [877].

Other cases of modified glycosidase carbohydrate moiety include chemically galactosylated fungal glucoamylase and human lysosomal exo-alpha-1,4-glucosidase with affinity to galactose- specific lectins [841, 842], non-enzymatically glucosylated E. coli beta-galactosidase having decreased activity compared to the native enzyme [7]. It should be noted that highly purified endo- and exoglycosidases standardizied in their carbohydrate and/or glycan specificity can serve as new additional tools for analysis of carbohydrate moiety of carbohydrate-containing enzymes. Examples of such potential new effective glycosidases can be found in the table (EndoM and others). General comments for the following table on glycosidases Left column Enzymes are listed according to Enzyme Commission (EC) numbers [1791, completed]. Sources of enzyme are given in the following order: viruses, bacteria, lower plants (yeasts, fungi), higher plants, invertebrates, vertebrates (mammals). Within each group pointed above sources are given according to alphabetical order of latin genus and species (with exclusion of some vertebrates). Molecular or SU masses are pointed according to methods of their determination (in patenthesis). Sequential procedures or conversion processes are pointed. The most effective step of enzyme purification is marked as **, and steps of HPLC, FPLC, HPLAC are marked as *. Calculated carbohydrate moiety is also given if possible. Right column Lectin+ means interaction between lectin and enzyme or influence toward enzyme activity (complex, precipitate, sorption of enzyme to lectin sorbent, retardation of enzyme during lectin chromatography or LIE, inhibition or activation of enzyme). Relative affinity of enzyme to lectin can be also marked (2+,3+) in the case of a panel of lectin tested. Glycosidase+ or PNGase+ means splitting of GP. Sensitivity of carbohydrate moiety for stain is also shown (PAS+, perjodate, TFMSA, etc.).

Table Interaction of glycosidases and their protein modulators with carbohydrate-sensitive reagents

Enzyme, sources, procedures, Carbohydrate-sensitive Ref. carbohydr. moiety, comments reagents, comments

3.2.1.1. α-Amylase

Bacteria Bacillus stearothermophilus (CF) + Enzyme 48kDa, 13,2% hexoses (Phenol-H2S04) DEAEC [812]

Clostridium thermohydrosulfuricum E 101-69 (CF) alpha-Amylase with pullanase activity, forms I (370kDa, pI 4,28), II (330 kDa, pI 4,28) Starch**—DEAEC—HA—Superose12* —Superose6* Carbohydr.: 12 or 11%, for I or II, respectively; Rha, Glc, Gal, Man [813]

Lower plants Aspergillus oryzae (fungal CF) Taka-amylaseA 51kDa, 3 forms DEAE-Sephacel—ConA-Sepharose ConA+ (desorption with

0,1 MM) SDS-PAGE—Blot—Lectin ConA+, WGA+, ConA- (EndoH-treated enzyme) + 1 Asn-glycan: Man5GlcNAc2; EndoH 1 Ser/Thr-glycan [814,815]

Candida antarctica CBS 6678 (yeast CF) alpha-Amylase 50kDa, pI 10,3 Carbohydr. : 2,4% hexoses [816]

Pichia pastoris MPA 3625 (methylo- trophic yeast) Bacillus licheniformis unglycosylated alpha-amylase expressed in yeast Enzyme 30 kDa (highly glycosylated) Trypsinized Gps + + C8-RP*—Dot-Blot—Lectin-peroxidase ConA , WGA Asn-glycans of oligoMan type EndoH+ [817]

Saccharomyces cerevisiae (yeasts) Murine alpha-amylase expressed in yeasts, 2 forms around 55-60kDa (SDS-PAGE) ConA-Sepharose ConA+ (glycosylated form), ConA- (non-glycosylated form) Asn-glycans of oligoMan type EndoH+ (glycosylated form) [818] Schwanniomyces alluvius ATCC 26074 (yeast CF) alpha-Amylase 39kDa (SephadexG100), or 52kDa (SDS-PAGE), acidic pI PAS+ ConA-Sepharose ConA+ Carbohydr.: 17% hexoses [819]

Higher plants Avena sativa L. (oats), Hordeum vulgare L. (barley), Sorghum vulgare L. (sorghum), germinating seeds Purified alpha-amylase SDS-PAGE—Blot—ConA(or Xyl-specific ConA- Ab) Carbohydr.: absence of N- and EndoH- O-glycans [820]

Oryza sativa L. (rice germinating seeds) alpha-Amylase forms S and R from scutellum ConA-Sepharose ConA+ (for S; desorption with 0,5M MM), ConA- (for R) Asn-glycans of oligoMan type in S EndoH+ (for S), EndoH- (for R) [821,822] SDS-PAGE—Blot—ConA ConA+ (for S) [820]

Oryza sativa L. cv.Kuban-3 (rice germinating seeds) alpha-Amylase forms A and B (44kDa, SDS-PAGE, pI 4,0-4,4), and C (41kDa,

5 pI 4,6-4,8) beta€-Cyclodextrin-Sepharose4B, ConA-Sepharose4B ConA+ (for A and B; desorption with gradient 0-0,3M MM), ConA- (for C) [823] Oryza sativa L. cv.Nipponkai (cell suspension culture, CF) alpha-AmylaseH 44kDa (SDS-PAGE), pI 5,7 PAS+ Prepar.column-IEF—IEF-PAG, SDS-PAGE—Lectin-peroxidase ConA+, PNA-,WGA-,UEA-I- Carbohydr.: 2% neutral hexoses [824]

Triticum vulgare L. cv.Lutescence 70 (wheat seeds) alpha-Amylase around 40kDa Q-Sepharose*—ConA-Sepharose ConA+ (desorption with 0,5 M Man; separation from unbound protein inhibitor 18kDa) [825] Zea mays L. (maize germinating seeds) Purified alpha-amylase SDS-PAGE—Blot—ConA ConA- Carbohydr.: Ser/Thr-glycans [820]

Protozoans Entamoeba histolytica HM:1:IMSS (amoebial trophozoites) Cell alpha-amylase 31kDa, pI 5,7 SephadexG100—IE—ConA-Sepharose- ConA+ (washing with 4B—CM-TrisacrylM gradient 0-1M MM; desorption with 1M MM; 73%, 7.5-f) [826] Mammals Human pancreas alpha-Amylase 60kDa, pI 7,0 Carbohydr. : 1,61% hexoses [827]

Human saliva alpha-Amylase forms 64kDa, pI 5,9, and 61kDa, pI 6,4 ConA-Sepharose ConA+ (for 64kDa; separation from unbound 61kDa) Carbohydr.: 8,77% (64kDa), 2,18% (61kDa) [827] alpha-Amylase forms A (62kDa), and B (58kDa) SDS-PAGE—Blot—ConA-peroxidase ConA+ (62kDa), ConA-(58kDa) Carbohydr.(for A): 6.5%; or 4kDa in Endo-beta-N-acetyl-D-glucos- 62 kDa aminidase+, Glycopeptidase+ (62—58kDa) [828] alpha-Amylase forms 63 and 59kDa SephadexG75alphaConA-agarose ConA+ (for 63kDa; binding of 70% total

activity; desorption with 0,5M MG) [829] alpha-Amylase sialoform ConA-sorbent* ConA- [830]

Human serum alpha-Amylase from healthy adults Precipitation with WGA WGA+ (71%) [221] alpha-Amylase major forms pI 6,4 and 7,0, and minor form pI 5,9 ConA-Sepharose ConA+ (for pI 5,9) [827]

Human sera of patients with IgA-type myeloma alpha-Amylase two sialoforms with SU 60kDa Superose12*—ConA-sorbent* ConA- (for both forms) [830] 3.2.1.2. beta-Amylase

Bacteria Bacillus subtilis (CF) Enzyme 57kDa DEAEC Carbohydr. : 0,8% hexoses [812]

Higher plants Pisum sativum L. cv.Laxton's Progress No 9 (pea ethyolated seedling epicotyles) Monomeric beta-Amylase 55kDa (SephacrylS200), or 57kDa (SDS-PAGE), pI 3,5 PAS- DEAEC**—SephadexG75—PreparPAGE Affinity to ConA ConA- [831]

3.2.1.3. Glucan 1,4-alpha-glucosidase

Bacteria Bacillus stearothermophilus ATCC 12016 (cells) p-NP-alpha-D-glucopyranosidase 47kDa, pI 5,2 DEAE-Sephadex—SephadexG100—DEAE- Sephadex—HA**—SephadexG100— ConA-Sepharose ConA- (70%, 1,2-f; separation from GP) Carbohydr.: absence of neutral sugars [832]

Lower plants Aspergillus candidus (mutant fungal CF) Glucoamylase 73kDa(SDS-PAGE) similar to enzyme of the parent strain ConA-Sepharose, Norleucin-Sepharose, ConA+ (desorption with Phenyl-Sepharose, SepharoseCL6B, 0,3M MM) DEAEC, DEAE-Sephadex [833]

Aspergillus niger GlucoamylaseG1 52-110kDa, from commercial sample (Novo Industries, Bagsverd, Denmark) Trypsinized Gp BiogelP100—ConA-Sepharose ConA+ (desorption with 0,2M MM) [834] Carbohydr.(M/M): 73 hexoses (including 35 hexoses in Ser/Thr- glycans); glycan Tca3-1 (from Gp) similar to Taka-amylase-A glycan; Tca2-1 (from Gp) with oligomannoside Ser/Thr-glycan [834,835]

GlucoamylaseG2 treated with Staphylococcus aureus V8 protease Mixture of Gp ConA-Sepharose—BiogelP4 ConA+ (separation of Gp) Carbohydr.(M/M): 76 hexoses; Ser/Thr-glycans [836]

Glucoamylase 95-98kDa Carbohydr.: Man, Glc, Gal (Ser/Thr- glycans) [837]

Aspergillus oryzae Purified glucoamylase Carbohydr.: 91 alpha-Manp residues alpha-Mannosidase+ including O-2(3)-glycosylated (41 residues) and O-4-glycosylated (20); 2.6% hexosamines (of total carbohydr.) [838]

Glucoamylase sample (Tokyo Tanabe) modified with p-aminophenyl-Galp, neoGP Affinity to bovine-brain-synapto- somal-Mb-bound GM1-sensitive lectin BBL+ (specific binding) [839] Aspergillus terreus GTC 826 Glucoamylase with pI 3,4 PAS+ DEAEC—ConA-Sepharose ConA+ Carbohydr.: 7,5% [840]

Aspergillus sp. Glucoamylase sample (Koch-Light, England) native and chemically galactosylated (neoGP) forms Lectin-Sepharose4B RCA-I+, ConA- [841] Precipitation of native form with lectins ConA+, ConA- (in the presence of Cys) [842] Chalara paradoxa (black mold CF) Glucoamylase GP-forms A1 (76kDa, pI 3,80), A2 (77kDa, pI 3,75), A3 (78kDa,

8 pI 3,70), A4 (pI 3,65), A5 (pI 3,60), and A6 (pI 3,55) Carbohydr.: 6,8, 7,2, or 9.2% hexoses, for A1, A2, or A3, respectively [692]

Humicola grisea var.thermoidea (thermo- philic fungal CF) Glucoamylase form 2 65kDa (Biogel- P100), or 63kDa (SDS-PAGE) DEAEC—CMC—Aceton-precipitation Carbohydr.: 1,5% neutral hexoses [843]

Mucor javanicus (fungal CF) Glucoamylase 61kDa (SDS-PAGE), forms I and II ConA-Sepharose—SephacrylS200 ConA- (for I), ConA+ (for II; desorption with 0,1M MM) [844] Rhizopus sp. (yeasts) Purified glucoamylase three forms Carbohydr.: 7-11%(w/w) [845]

Saccharomyces diastaticus 5106-9A (yeast CF) Glucoamylase 66-80kDa, SU 68+59+53+14 kDa DEAE-Sephadex—CM-Sephadex—HA— SephacrylS200, ConA-Sepharose ConA+ (desorption with 0,25 M MM) Carbohydr.: 40% (68kDa), 30% (59kDa), and 23% (53kDa); Asn-glycans of oligoMan type: 27kDa in EndoH+ ([68+59+53]—41kDa) 68kDa, 18kDa in 59kDa, 12kDa in 53kDa [846]

Saccharomyces cerevisiae 5206-1B (MATa arg4 STA2) STA2-encoded glucoamylaseII 670kDa (HPLC), or 300kDa (SDS-PAGE), or 56+170kDa (SDS-PAGE for reduced II) Carbohydr.: 80% hexoses (mainly Man); Asn-glycans of oligoMan type EndoH+ [847]

Saccharomyces cerevisiae diploid strains SK1 and AP1 (yeast sporulated cells) Glucoamylase with SU 69+72+75+79kDa DEAEC—HA**—LCA-Sepharose4B LCA+ (30%, 4,0-f) [848]

Yeasts (others) Amyloglucosidase sample (Sigma, USA) ConA-alginate-gel (for biocatalysis) ConA+ [18] Crude-ConA-cross-linked with glutaraldehyde (for biocatalysis) ConA+ [12] ConA-SephadexG50 (for biocatalysis) ConA+ (>30% as immobilized activity) [19] Mammals

Human fibroblasts in culture alpha-Glucosidase ConA-agarose ConA+ (binding of 97% in the presence of 3mM MM) [849] Human kidney brush-border proximal tubule cell lysosomes Acid alpha-glucosidase mature forms 70 and 76kDa (SDS-PAGE), and precursor form 110kDa (SDS-PAGE) ConA-Sepharose4B—SephadexG100 ConA+ (for all forms; desorption with 1M MG) [850] Human liver Acid alpha-glucosidase Lectin-Sepharose4B ConA+ (complete binding; desorption of 90% with 1M MM; 35-54-f), WGA+ (binding of 90%; desorption of 10% with 1M GlcNAc), GSA-I+ (8%), RCA-I+ (5%), SBA+ (5%), UEA-I+(5%) [707] Human lung lamellar bodies without lysosomes Lysosomal-type acid alpha-glucosidase with SU 110+76+70kDa, 2 forms ConA-Sepharose4B—Blot—Ab ConA+ (70%) [851,852]

Human lung lysosomes Acid alpha-glucosidase ConA-Sepharose ConA+ (binding of >95%) [851] Human placental lysosomes Acid alpha-glucosidase 105kDa solubilized with trypsin ConA-Sepharose**—UltrogelAcA34— ConA+ (desorption with Sephadex-G100 0,1M MM; 99%, 12-f) [853]

Human urine Lysosomal type alpha-glucosidase precursor form 109kDa (SDS-PAGE), and mature forms 76 and 70kDa ConA-Sepharose4B**—SephadexG100 ConA+ (for all forms; desorption with 1M MG; 40%, 14-f) Carbohydr. (M/M): 3,5 or 0 Man-6-P, for 109 or 70-76kDa, respectively [854]

3.2.1.4. Cellulase

Bacteria Bacillus sp. KSM-635 (alkalophilic) Enzyme form 500kDa, SU 130kDa, and monomeric form 100kDa Carbohydr.: 2-3% GlcNAc, for both forms [855]

Bacillus sp. (mesophilic strain, CF) Cellulase 30kDa (SDS-PAGE), pI 4,8 PAS+ ConA-Sepharose ConA+ (desorption with 0,2M MM;14%, 3,4-f) Carbohydr.: 11.5% neutral hexoses [856]

Bacteroides cellulosolvens (CF) Cellulase 230kDa Carbohydr.: 6 glycans; terminal beta-Gal + (furanose form) with affinity to GSA-I-B4 GSA-I-B4 [857]

Cellulomonas fimi (CF) Monomeric cellulase forms CB1 (58kDa) and CB2 (56kDa) ConA-Sepharose—MonoQ* ConA+ (for both forms; separation from 48kDa) [858,859]

Cellulomonas sp. ATCC 21399 (CF) Cellulase EG-B 67kDa BiogelP60—CM-Sepharose6B—ConA- ConA+ (desorption of Sepharose6B 54% with 1M MM) [860]

Cellulase forms EG-A 78kDa, EG-CA 49kDa, EGCB 53,5kDa, and EG-I 118kDa Affinity to ConA ConA- (118kDa), ConA+ (other forms) [860] Lower plants Aspergillus aculeatus No F-50 (fungus) Avicellase forms FI 109kDa (SDS-PAGE), and FIII 112kDa Carbohydr.: 32% hexoses and 0,4% GlcN (for FI); 22% hexoses and 1,1% GlcN (for FIII) [280]

Aspergillus niger Cellulase (Type I,Sigma,USA), forms A, B, and C ConA-Sepharose ConA+ (B and partial C; desorption with 0,5-0,7M Man) Carbohydr.: 57% (for B), 48% (for C), highly glycosylated A [861]

Chaetomium cellulolyticum (fungal CF) CM-Cellulase ConA-Sepharose—ConA-Sepharose ConA+ (binding of >90%; desorption with 10mM MM; separation from strongly immobilized form) [862] Chaetomium thermophile var.coprophile IIS-110 Monomeric cellulase 38kDa + Carbohydr. (H2S04-ethanol-heating) [863]

Macrophomina phaseolina JARI 25 (fungal jute pathogen, CF) CM-cellulaseI 35kDa (SDS-PAGE)

DEAEC—SephadexG75—ConA-Sepharose- ConA+ (desorption with 4B—MonoQ*—Superose12* 0,1M MG; 20%, 1,7-f) [864] Monilia sitophila (fungal CF) Cellulase ConA-Sepharose ConA+ (desorption with gradient 0-0,5M MM) [865] Neurospora crassa mutant cell-1 (fungal CF) Cellulase forms I (46,5kDa), II (21,0 kDa), III (37,3kDa, pI 6,5), and IV (30,0kDa, pI 5,5) + PAGE (H2SO4-thymol) (for all forms) Carbohydr.: 29,4% (I), 5,3% (II), 6,4% (III), and 6,4% (IV) [866]

Penicillium funiculosum (fungal CF) CM-cellulase monomeric formI 56kDa BiogelP200—ConA-Sepharose ConA+ (desorption with 50mM MM or MG) [867] Penicillium purpurogenum (CF) CM-cellulase formI 72kDa, pI 4,3, and formII 50kDa, pI 3,9 BiogelP6—DEAE-Sepharose**—CM- Sepharose—ConA-Sepharose(or ConA+ (washing with 2mM MM; Chromatofoc if I)—SP-Sephadex desorption with 30mM MM; 50%, 1,1-f) [868] Phanerochaete pulverulentum (Chrysospotium lignorum, Sporothrichum pulverulentum) (fungus) Cellulase forms T1 32,3kDa (major), T2a 36,7kDa, T2b 28,3kDa, T3a 37,5kDa, and T3b 37,0kDa DEAE-Sephadex—BiogelP60—ConA- ConA+ (desorption of total Sepharose—SP-Sephadex activity with 25mM MM; 73%, 3.8-f; strong binding of T1) Carbohydr.: 10.5% (T1), 0% (T2a), 7.8% (T2b), 4.7% (T3a), 2.2% (T3b); Man and Glc (T1) [869]

Saccharomyces cerevisiae (baker yeasts, CF) Clostridium thermocellum endoglucanase A (97kDa) encoded by the "cel-A" gene in yeasts ConA-Sepharose (purification) ConA+ Carbohydr.: 45%; Asn-glycans of EndoH+ (97—53kDa) oligoMan type (44kDa in 97kDa) [870]

Schizophillum commune (fungus) Cellulase forms 40,6 and 39,4 kDa ConA-Sepharose4B ConA+ (desorption of 50% with 10mM MG, and next 50% with 0,5M MM and 0,1

M MG) [872] Heterodimeric cellulase-I of crude endoglucanase sample (Pulp and Paper Res. Inst., Canada) DEAE-Biogel—ConA-Sepharose ConA+ (desorption with 20mM MM) [873] Trichoderma reesei (fungal CF) Avicellase of mutant strain C-30 ConA-Sepharose4B (for biocatalyst) ConA+ (47%; slight activation of immobilized enzyme form) [874] Cellulase 43kDa, pI 4,0 ConA-Sepharose ConA+ (desorption with 10mM MM) [875] Cellulase forms 56-67kDa, pI 5,0-6,5, 2-7% hexoses [876]

Hydrophobic CM-cellulase 65kDa with high affinity to cellulose, forms pI 4,6+4,5+4,25+4,0 ToyopearlHW65 (hydrophobic chromatography) —ToyopearlHW55(gel- filtration) —MonoP*—ConA-Sepharose ConA+ (almost complete binding of each form; complete desorption with step- wise gradient 0-50mM MG; partial separation of forms) Carbohydr.: 9,2% Man, 1,8% Glc (pI 4,6); 8,0% Man, 2,5% Glc (pI 4,5); 8,1% Man, 4.0% Glc (pI 4,25); 15% Man, 3,8% Glc (pI 4,0 [876]

Monomeric cellulaseP4 64kDa, pI 4,75, of strain CDU-11 DEAE-SepharoseCL6B—DEAE-Sepharose- CL6B—Electrophoresis—ConA- ConA- (form P4-1), ConA+ Sepharose4B—SephadexG100 (form P4-2; desorption with 25mM MM; separation from beta-glucosidase) [877] Endoglucanase 25kDa (SDS-PAGE), PAS+ pI 7,5, of 7-day-old fungal strain QM 9414 (ATCC 29921) ConA-Sepharose ConA- (separation from 2 bound isozymes eluted with gradient 0-1M MG) [878] Trichoderma viride (fungus) Cellulase (Enzyme Developm.Corp., N.-Y.) ConA-Sepharose ConA+ (desorption with MG; separation from unbound cellobiase) [879] Crude CM-cellulase ConA-Sepharose—ConA-Sepharose ConA+ (desorption with 0,1M borate pH 6.5 instead of 0,1M MM; separation from cellobiase) [880]

Cellulase 223OA (Rome, Italy) for biocatalyst preparation ConA-Sepharose instead of CNBr- ConA+ (significant Sepharose or CNBr-glass-beads (for activation of immobilized immoblization) enzyme form) [881]

Trichoderma sp. endoglucanase 2 forms (60-66kDa) expressed in yeasts, resulted in hyperglycosylated forms 70-80kDa [871]

Higher plants Persea gratissima (avocado ripening fruits) Cellulase 54,2 kDa ConA-Sepharose4B—SephacrylS300, ConA+ (complete binding Cellulose CF11 and desorption with 0,1M MG) [882] 3.2.1.6. Endo-1,3(4)-beta-glucanase

Bacteria Bacillus subtilis 1.65 ICA (CF) Crude enzyme Precipitation with RSA RSA+ Activation with RSA RSA+ RSA-crosslinked with glutaraldehyde RSA+ (as sorbent) [883]

Flavobacterium dormitator var. glucoanalyticae FA-5 Monomeric enzyme 33kDa (SDS-PAGE or BiogelP100), pI 6,5 Carbohydr.: 10,3% hexoses (approx.17 residues/monomer) [884]

Yeasts Saccharomyces cerevisiae strains X14, TD28 Purified enzyme as GP [885]

3.2.1.8. Endo-1,4-beta-xylanase Lower plants Cryptococcus albidus CCY 17-4-1 (yeast cell walls) Crude enzyme ConA-Sepharose ConA+ (desorption with 0,25M MM; separation from unbound beta-xylosidase) [886] Humicola grisea var.thermoidea (fungal CF) Enzyme 23kDa (BiogelP60), or 25,5kDa (SDS-PAGE) DEAEC**—SephadexG200 Carbohydr.: 45% hexoses [887]

Irpex lacteus (Polyporus tulipiferac) (fungal cell walls)

Enzyme 38kDa, pI 7,6-8,0, from Driselase (Kyowa Hakko Kogyo Co., Tokyo) SephadexG25—DEAE-Sepharose—CL6B**— CM-SepharoseCL6B—Phenyl-Sepharose— UltrogelAcA54—ConA-Ultrogel ConA+ (desorption with 25mM MM using gradient 0-0,1 M MM) Carbohydr.: 23% hexoses (13 Glc : 1 Man, M/M) [888]

Monilia sitophila (fungal CF) Endoxylanase ConA-Sepharose ConA+ (desorption with gradient 0-0,5 M MM) [865] Penicillium capsulatum Raper and Fennell, CMI 291669 Cell-wall-bound enzyme forms A (28.5kDa, pI 5,0-5,2) and B (29,5kDa, pI 5,0-5,2) solubilized with Tween80 Sepharose6B—DEAEC; BiogelP60 Carbohydr.: 58.5% or 97 residues(for A); 33.4% or 63 residues(for B) [889]

Trichoderma koningii IMI 73022 Monomeric endoxylanase forms I (29kDa, pI 7,24), and II (17,7 kDa, pI 7,3) ConA-Sepharose ConA- (for both forms) Carbohydr. PAS- (for both forms) [890] Higher plants Cucumis sativus L. cv.Heinz 3534 Endoxylanase 96kDa SP-Sephadex—SephadexG100—ConA- ConA+ (low recovery,or Sepharose(or other sorbent) enzyme inactivation) [891] 3.2.1.10. Oligo-1,6-alpha-glucosidase

Human small intestinal cells in culture Sucrase-isomaltase mature form 245kDa (145+130kDa, trypsinized Gp), intermediate forms 212kDa with completely glycosylated core, and 210kDa with oligoMan type Asn-glycan Lectin-Sepharose LCA+ (desorption with 50mM MM), HPA+ (130, 145, 212, 245kDa; desorption with 10mM GalNAc), HPA- (210kDa) [892] Rat intestinal brush-border Mb Sucrase-isomaltase complex solubilized with papain

SephadexG200 Affinity to HTL-125I HTL+ [893]

Rat small intestine of suckling and postsuckling animals Soluble neutral glucoamylase-maltase forms I and II (postsucling rats), III and IV (suckling rats), Mb-bound I and III solubilized with proteinase, Mb-bound II and IV solubilized with Na-DOC ConA-Sepharose ConA+ (all forms; desorption of I, II, or III with 5-20mM MM, and IV with 5-55mM MM, using step-wise gradient 0-0,25 M MM) [894] Preweaning rat (13-18-day) and postweaning rat (25-day) small intestine

Soluble neutral glucoamylase-maltase forms 400 and 150kDa (13-day rats), 150kDa (18-day rats), and 400kDa (25- day rats) ConA-Sepharose ConA+ (all forms; different elution of forms with gradient of MM) [895] Suckling (11-day) rat small intestine Soluble neutral glucoamylase-maltase forms I (200kDa) and II (400kDa, SU 200+210kDa) Sepharose4B—Sepharose6B-Tris— DEAEC—LCA-Sepharose4B LCA+ (desorption with 50mM MM followed by 0,5M MM, for I and II, respectively) Asn-glycans of oligoMan type (I), EndoH+ (I), EndoH- (II), of complex type (II) EndoF+ (II) [896]

3.2.1.11. Dextranase Fungi Lipomyces starkeyi ATCC 20825 Enzyme forms 74, 71, 68, and 65kDa (SDS-PAGE), pI 5,6-6,1 CM-Sepharose—BiogelA0.5m**, Lectin-Sepharose ConA+ (strong binding, immibilization), LCA+ (non-strong binding), WGA- Carbohydr.: 8% hexoses [897]

Penicillium melinii QM 1931 Dextranase-F 40kDa Ser/Thr-glycans (>80% of total carbohydr. as Man and Glc) [898]

3.2.1.14. Chitinase

Bacteria Aeromonas hydrophila subsp. anaerogenes A52 (CF) Enzyme 110kDa, pI 4.60 PAS+ Chitin**—CM-Sephadex [899]

Lower plants Rhizopus oligosporus Saito IFO 8631 (filamentous fungus, CF) Chitinase forms I (50kDa), and II (52 kDa, SDS-PAGE) Chitin**—SephadexG75—DEAE-Toyopearl Thr/Ser-glycans released by TFMSA- TFMSA+ (50—44,5 treatment kDa, 52—46,5kDa) EndoH- Carbohydr.: Thr/Ser-glycans (5,5kDa in 50 kDa, 5,5kDa in 52kDa) [900]

Saccharomyces cerevisiae (baker's yeasts, CF) Mannan-associated chitinase 14kDa (SDS- PAGE, for reduced protein), 18% hexoses ConA-Sepharose ConA+ (desorption with 1M MM) [901] Native chitinase 130kDa (60kDa for nonglycosylated form) Chitin—SephacrylS200 Thr/Ser-glycans EndoH-, N-Glycanase- [902] Higher plants Coix lachryma-jobi (seeds) Chitinase 10,0 kDa (SDS-PAGE) as GP [903] Nicotiana tabacum cv. SR1, cv.Wisconsin 38 (tobacco) Serratia marcescens bacterial chitinase modified and secreted by transgenic tobacco Asn-glycans of complex type EndoH-, EndoF+ [904]

Invertebrate Artemia nauplii (brine shrimp) Chitinase 32kDa, pI 3,9 DEAE-SepharoseCL6B—UltrogelAcA44— ConA-Sepharose ConA+ (desorption with 0,5M MM; 4%, 40-f) [905] Mammals Human plasma 4-MUF-tetra-N-acetyl- -D-chitotetraoside hydrolase SephadexG200—DEAEC—ConA-Sepharose ConA- [906]

3.2.1.15. Polygalacturonase

Lower plants

Aspergillus niger (fungus) Endopolygalacturonase Blot—Lectin Lectin+ [907]

Botrytis cinerea (fungal CF) Monomeric endopolygalacturonase 36kDa, forms pI 8,8 and 4,9 Column-IEF—ConA-Sepharose ConA- (separation from the bound exopolygalacturonase) [908] Fusarium moniliforme FC-10 (phytopatho- genic fungus, CF) Monomeric polygalacturonase forms 41,5 and 45,0 kDa, PAGE Carbohydr.-sensitive stain+ [909] Galactomyces reesei (yeast-like fungus,CF) PolygalacturonaseL 33-40kDa, pI 8,4-8,5 Carbohydr.: 5,1% neutral sugars [910]

Kluyveromyces fragilis IFO 0288 (yeasts, CF) Polygalacturonase-F approx.40kDa, pI 5,0, Carbohydr.: 5,9% hexoses [910]

Trichosporon penicillatum SNO-3 PolygalacturonaseS 33-40kDa, pI 7,6-7,8, Carbohydr.: 1,7% hexoses [910]

Higher plants Lycopersicon esculentum (tomato ripening fruits) Polygalacturonase forms I (102kDa, SU 45+ 40kDa, SDS-PAGE), and II (81kDa, SU 45 kDa) ConA-Sepharose4B—MonoS*—Superose6* ConA+ (for I;desorption with 0,2M MM) [911] L. esculentum cv.VF 145B-7878 (tomato fruit cell walls) Polygalacturonase ConA-Sepharose4B ConA+ (desorption with 10mM MM; separation from pectinmethylesterase) [432] 3.2.1.17. Lysozyme

Bacteria Streptococcus faecium ATCC 9790 (mutants defective in autolysis cell walls) Latent zymogenic lysozyme 130kDa (SDS- PAGE), and active form 87kDa (SDS-PAGE) as trypsinized 130kDa ConA-Sepharose4B (for crude enzyme) ConA+ (binding of 80-100% of 130kDa; desorption with 50mM MM) [912-914] (Hemoglobin-Sepharose4B—ConA- Hemoglobin-ConA+ Sepharose4B)**—SephacrylS300 (82%, 62-f) [914] Carbohydr. (for 130kDa): 5,3% Glc, <1% Man [914]

Lower plants Saccharomyces cerevisiae (yeast CF) Hen egg-white lysozyme expressed in yeast, oligomannosylated form 18kDa, and poly- mannosylated form 71kDa CM-Toyopearl—CM-Toyopearl—SephadexG50 —ConA-Sepharose ConA+ (for both forms; desorption with 0,1 M MM) Asn-glycans of oligoMan-type EndoH+ (for both forms) [915,916] Birds Hen egg-white Lysozyme Precipitation with ConA ConA+ (partial inhibition of precipitation in the presence of 50mM Man or Glc) [917] Mammals Human plasma Lysozyme SephadexG200—DEAEC—ConA-Sepharose ConA- [906]

3.2.1.18. Exo-alpha-sialidase

Viruses Influenza virions from allantois fluid of hen embrios Sialidase associated with hemagglutinin activity CJA-I-Sepharose CJA-I+ (desorption with 0,2M lactose) [918] Bacteria Bacteroides fragilis SBT 3182 (cells) Sialyl-alpha-2,8-specific sialidase forms PAS2+ (50kDa), 50 and 55kDa (SDS-PAGE) PAS+ (55kDa) DEAE-Toyopearl—HA**—MonoS*— Superose6* PVDF-Mb—Lectin-biotin (binding) ConA2+ (55kDa), ConA+(50kDa) PVDF-Mb—Lectin-biotin UEA-I+ (inhibition of 18% of enzyme, for 50kDa, but not 50kDa; among 7 lectins) [919] Protozoans Trypanosoma brucei brucei EATRO 427 (procyclic trypomastigotes) Mb-bound sialidase 67KdA (HPLC or SephadexG100) solubilized with TritonCF54 SephadexG150—Q-Sepharose*—Superose- 12*—MonoQ*, ConA-Sepharose ConA+ (strong binding without desorption; immobilization) [920] T. brucei brucei TREU-667-stock, Trypanosoma cruzi (trypomastigote lysate) alpha-2,3-Sialidase associated with sialo- transferase, solubilized with NP40, 63kDa (SDS-PAGE) ConA-Sepharose—MonoQ*—Superose12*— ConA+ (partial desorption

Superose6* with MM) [308,921]

Trypanosoma cruzi strain Y (promastigotes) Cell surface trans-sialidase solubilized with OG ConA-Sepharose—MonoQ* ConA+ (desorption with 0,5M MM) [922] Mammals Bovine testicular lysosomes Acid neuraminidase associated with beta-galactosidase ConA-Sepharose—beta-Galp-Sepharose ConA+ (desorption with 1M MG; 40%, 25-f) [923] Human placenta Lysosomal acid neuraminidase associated with acid beta-galactosidase activity, SU 76+66kDa ConA-Sepharose—p-Aminophenyl-thio- ConA+ (desorption with beta-Galp-CH-Sepharose 1M MG) [924,925]

Acid neuraminidase with SU 78+46kDa ConA-Sepharose4B — p-Aminophenyl-thio- beta-Galp-CH- Sepharose — Sorbent* ConA+ (desorption with 1M MG) [926,927] Porcine testis Sialidase >1,000 kDa (Sepharose6B) ConA-Sepharose —p-Aminophenyl-thio- ConA+(desorption with —Galp-Sepharose 0,5M MM; 69%, 11-f) [928] Rabbit erythrocyte Mb Ganglioside-sialidase 54kDa(SDS-PAGE), or 48kDa (SephadexG100), solubilized with TX100 DEAEC—EAH-Sepharose**—Octyl- Sepharose—SephadexG100—ConA- ConA+(retardation upon Sepharose—N-(p-aminophenyl)-oxamate- filtration without sugar; agarose—Heparin-Sepharose 74%, 1.4-f) [929]

Rat liver cells Cytosolic Sia-2-alpha-3-hydrolizing sialidase 43kDa (SDS-PAGE) ConA-Sepharose ConA- [930]

3.2.1.20. alpha-Glucosidase

Lower plants

Aspergillus niger (fungus)

Purified crystallized alpha-glucosidase (Amano Pharm.Co., Ltd., Japan) SDS-PAGE—Blot—Lectin-biotin ConA2+, DSA+(slightly) Asn-glycans of oligoMan type with EndoF+ terminal1-alpha -2-Gal(furanose form) [931]

Higher plants Musa sapientum L. cv.Giant Cavendish (preclimacteric green banana pulp) Acid alpha-glucosidase 70kDa (Sephacryl- S300) CMC**—ConA-Sepharose—SephacrylS300 ConA+ (complete binding; desorption with 15mM MG; 44%, 6,1-f) [932] Neutral alpha-glucosidase 42kDa, forms I and II (SephacrylS300) DEAEC—ConA-Sepharose—Sephacryl- ConA- (97%; separation S300—HA** from the bound acid alpha-glucosidase) [932] Glycine max L. cv.Tamanishiki (soybean cotyledon callus cells in culture, cell walls) alpha-Glucosidase forms I (70kDa) and II (115kDa) solubilized with 2M NaCl CMC—ConA-Sepharose ConA- (for I), ConA+ (for II; desorption with 0,1M MM) [63] Invertebrates Aedes aegypti Rockfeller strain (vector mosquito larval salivary glands) Extracellular alpha-glucosidase 68kDa ConA-Sepharose ConA+ (binding of 94%; complete desorption with 0,2M MM) [933] Apis mellifera (honeybee venom) alpha-Glucosidase 160kDa (SDS-PAGE), pI 4,9 SephadexG75—DEAEC—SephadexG200 Carbohydr.: 2.6% hexoses [588]

Birds Japanese quail embrionic and adult muscle Neutral alpha-glucosidase forms: adult (155 kDa, HPLC), embrionic 450 and >1,000 kDa DEAEC—HA—ConA-Sepharose ConA+ (embryonic forms; desorption with 1M MG), ConA- (adult form) [934] Mammals Rat small proximal and distal intestine Maltase-sucrase proximal (I) and distal (II) forms Lectin-Sepharose RCA-I+ (binding of 38 or 64%, for I or II, respectively), WGA+ (55 or 28%) [530] Rat small intestine of suckling and nonsuckling animals Soluble neutral maltase-glucoamylase forms I and II (nonsuckling rats), III and IV (suckling rats); Mb-bound I and III, solubilized with proteinase; Mb-bound II and IV, solubilized with

Na-DOC ConA-Sepharose ConA+ (all forms; desorption of I-III with 5-20mM MM, and IV with 5-55mM MM using gradient 0-0,25M MM) [894] Rat preweaning (13-18-days) and postweaning (25-day) small intestine Soluble neutral maltase-glucoamylase forms 400+150kDa (13-day), mainly 150kDa (18-day),and 400kDa (25-day) ConA-Sepharose ConA+ (different elution profiles of forms using gradient of MM) [895] Rat small intestine of 11-day suckling animals Soluble neutral maltase-glucoamylase forms 200 and 400kDa, SU 200+210kDa Sepharose4B— (Sepharose6B-Tris— DEAEC)**—LCA-Sepharose4B LCA+ (desorption of 200kDa with 50mM MM followed by 0,5M MM for 400kDa) Asn-glycans of oligoMan type (200kDa) EndoH+ (200kDa), EndoH- and complex type (400kDa) (400kDa), EndoF+ (400kDa) [896] 3.2.1.21. beta-Glucosidase

Bacteria Bacteroides succinogenes S-85 (rumen bacterial CF) Cl--stimulated cellobiosidase 75kDa, pI 6.7 DEAE-Sepharose—HA—Chromatofoc** Carbohydr.: 8-16% hexoses [935]

Lower plants Alternaria alternata (fungal autolyzed culture, CF) beta-Glucosidase 160kDa, SU 80+70kDa ConA-Sepharose ConA+ [936]

Aspergillus niger (fungus)

Cellobiase250L (NOVO Lab.,Inc.,Wilton,CT) ConA-Sepharose4B (purification and ConA+ (immobilized active preparing biocatalyst) form; without desorption by 1M Man; desorption at pH 3,5) [861,937] Cellobiase 210kDa, pI 3,4-3,7, from cellulase complex Novozym188 (NOVO, Copenhagen) DEAE-SepharoseCL6B—ConA-agarose ConA+ (washing with 50mM —Chromatofoc—SephacrylS200—IEF MM; desorption with 0,5 M MM) [436] Aspergillus terreus (fungal cellulase producent) Cellular beta-glucosidase 275kDa, solubilized

22 by sonication DEAE—CM—ConA-agaros—DEAEC ConA+ (desorption with 0,1M MM; 33%, 7.0-f) Carbohydr.: 65% hexoses [938]

Candida wickerhamii NRRL Y-2563 (yeasts) beta-Glucosidase 198kDa, SU 94kDa ConA-agarose ConA+ Carbohydr.: 30.5% hexoses (mainly Man) [939]

Dictyostelium discoideum (mold lysosomes) Acid beta-glucosidase glycans Immobilized mammalian liver lectin Liver-lectin+ (desorption with (receptor for Man-6-P) 0,5mM Man-6-P [940]

Humicola grisea var.thermoidea (fungal conidial mycelium) beta-Glucosidase 56kDa (SephadexG100), or 54kDa (SDS-PAGE) SephadexG100**—DEAEC Carbohydr.: 23% hexoses [941]

Monilia sitophila (Mont.) Sacc., Monila sp. (fungal CF) beta-Glucosidase 46,6kDa, pI 8,87 ConA-Sepharose ConA+ (binding of >90%; desorption with gradient 0-0,5M MM) [865] Neurospora crassa mutant cell-1 (fungal CF) beta-Glucosidase 75,5kDa, pI 8,7 + Carbohydr. (H2SO4-thymol) [866]

Penicillium herquei Banier and Sartory (fungal CF) beta-Glucosidase forms G1 (125kDa, pI 5,0), and G2 (122kDa, pI 5,2) DEAE-Biogel—CM-Biogel—DEAE- Biogel—Chromatofoc—BiogelA0.5m Carbohydr.: 12.7% (G1), 16.1% (G2) [942]

Penicillium oxalicum ATCC 64198 (autolized culture) beta-Glucosidase 133kDa (SDS-PAGE), pI 4 SephacrylS200**—DEAE-Biogel— CM-Biogel—MonoQ*, ConA-Sepharose ConA+ (strong binding, immobilization) [943] Saccharomyces cerevisiae (parent strain DBY640—derivative strains PRY98, PRY107) (yeast CF) Exoglucanase Biogel-TSK-DEAE-5PW, ConA-Sepharose4B ConA+ (desorption with gradient 0-50mM MM)

Asn-glycans of oligoMan type EndoH+ [944]

Schizophyllum commune (white-rot fungus, CF) beta-Glucosidase forms 95,7 and 93,8kDa (SDS-PAGE) ConA-Sepharose4B ConA+(washing with 10mM MG; desorption with 0,5M MM and 0,1M MG; partial separation from cellulase) [872] Trichoderma koningii (fungal CF) beta-Glucosidase 39,8kDa: glycosylated form pI 5,53 and nonglycosylated form pI 5,85 UltrogelAcA44—DEAE-Sepharose—ConA- ConA+ (recovery >90%; 3% of admixtural cellulase) [945] Carbohydr.: 2% hexoses (form pI 5,53)

Trichoderma reesei QM 9414 (fungal CF, plasma Mb) beta-Glucosidase 75kDa (ultracentrifugion), or 82kDa (SDS-PAGE) DEAE-Sephadex**—DEAE-Sephadex—SP- Sephadex—ConA-agarose(Glycosylex-A) ConA+ (desorption with 50mM MM; 84%, 1,2-f) Carbohydr.: < 1% hexoses [946]

ConA-precipitation of enzyme from CF ConA+ (approx.70%) [947] Mycelial protoplast plasma Mb-bound beta-glucosidase 70kDa (gel-filtration), or 72kDa (SDS-PAGE) ConA-Sepharose ConA+ (desorption with sugar) [948] Trichoderma reesei C-30 (fungal CF) beta-Glucosidase (NOVO Lab.,Connecticut, USA) ConA-Sepharose4B (for immobilization) ConA+ (44%) [874] ConA-Macrosorb(Kieselguhr-based support) (for preparing biocatalyst) ConA+ (immobilization in the presence of 0,5M Glc and 10% ethanol) [949] Trichoderma viride (fungal CF) Cellobiase from commercial cellulase (Enzyme Developm.Corp.,N.-Y.) ConA-Sepharose ConA- (separation from the bound CM-cellulase) [879] Cellobiase forms pI 5,2-5,7, from crude cellulase ConA-Sepharose—ConA-Sepharose ConA-ConA- (recovery 51%; separation from cellulase) [880] beta-Glucosidase forms A (98kDa, pI 6,1), PAS+ (A), PAS- (B) and B (70kDa, pI 7,2) Lectin-agarose ConA+ or UEA-I+ (complete binding), PNA+ (29%), RCA-I+ (30%), SBA+ (22%),

WGA+ (22%) [950] Cellobiase from commercial cellulase complex (Rome, Italy) ConA-Sepharose (for immobilization) ConA+ (up to 2-f-activation of immobilized form) [881] Higher plants Amigdalus communis var.dulcis (sweet amigdalus seeds) beta-Glucosidase (I) and its polyethylene- glycol(PEG)-conjugate (II) (38% amino- groups of Lys linked to PEG) ConA-Sepharose ConA+ (for I; desorption with 0,5M MG), ConA- (for II) [951] Artemisia annua L. (sweet wormwood) beta-Glucosidase 100-120kDa, pI 4,2-4,4 CMC—ToyopearlHW55—Lectin-Sepharose ConA+ (strong binding (for purification, immobilization, and in active form without preparing biocatalyst) desorption by MG, immobilization; separation from beta-galactosidase), LCA+ (immobilization in active form; separation from other beta-glycosidases) [952,953] Glycine max L.(soybean cells in culture, cell walls) Homogenic beta-glucosidase (SDS-PAGE) Dot-blot—Lectin(or alpha-1,3-Fuc-, or ConA3+, GNA3+, SBA+, beta-1,2-Xyl-specific Ab) TPA+ Asn-glycans of complex type with Xyl and/or without Fuc [431]

Hordeum vulgare L. (barley seed meal) Monomeric beta-glucosidase 53-55kDa, forms pI 8,5+8,8+9,0+9,2 DEAEC**—Blue-Sepharose—ConA- ConA+ (binding of >50%; Sepharose desorption with 0,1M MG) [954] Vigna radiata L.(Phaseolus aureus) (mung bean mature seeds) beta-Glucosidase 170kDa SephacrylS200—ConA-Sepharose ConA+ (desorption with 0,1M MG using gradient 0-0,3M MG; separation from beta- galactosidase, invertase, beta-xylosidase, alpha-L- fucosidase, alpha-L- arabinosidase) [955] Invertebrates Euphausia superba (antarctical krill) beta-Glucosidase forms I and II (150kDa, PAS+ for both forms) DEAEC**—ConA-Sepharose—Ultrogel- ConA- (for II; 18%), AcA34—PreparPAGE ConA+ (for I; desorption with

gradient 0-0,4M MM; 8,6%, 4,7-f) [340] Littorina littorea L.(periwinkle digestive gland) beta-Glucosidase associated with beta-galactosidase and alpha-fucosidase SephadexG200—DEAEC**—GalN-beta-amino- caproyl-agarose—ConA-Sepharose ConA+ (desorption with gradient 0-0,5M Man; 38%, 3,0-f) [956] Macrotermes muelleri (termite gut digestive tract infected with symbiotic fungus, Termitomyces sp.) beta-Glucosidase forms A (126kDa, PAGE; PAS+(for A) SU 62kDa, SDS-PAGE) and B (115kDa, PAGE; or 120kDa,SDS-PAGE) [957]

Mammals Human brain, kidney, liver, spleen Acid and neutral beta-glucosidases ConA-Sepharose ConA+(for acid enzyme; desorption with 1M MG; separation from unbound neutral enzyme) [958] Decreased level of acid beta-glucosidase from patients with Gaucher's disease ConA-Sepharose ConA+( desorption with 1M MG) [958] Human liver beta-Glucosidase BiogelA0.5m—ConA-Sepharose4B ConA+ (desorption with 0,75M MM) [959] Human placenta Lysosomal acid beta-glucosidase solubilized with Triton WR1339 ConA-Sepharose ConA+ (desorption with 1M MM; 107%, 146-f) [960] Human skin fibroblasts Acid beta-glucosidase without neutral enzyme ConA-Sepharose ConA+ (desorption with 1M MG) [958] Human spleen beta-Glucosidase, SU 63+61,5+56kDa, pI 4,9 and 5,5-6,5, solubilized with TX100 ConA-Sepharose4B ConA+ (complete desorption with 1M MG) [961] Rabbit kidney Neutral beta-glucosidase 47kDa associated with beta-galactosidase and alpha-fucosidase ConA-Sepharose4B—DEAEC—Sephadex- ConA- (separation from G200**—HA the bound acid beta-glycosidases; 118%, 1.3-f) [962] Rat liver Lysosomal acid beta-glucosidase RCA-Sepharose4B; RCA-I-Sepharose4B RCA+ (desorption with

(Medac, FRG) 0.25M Lac; 11%, 36-f) [617] Sheep liver Neutral beta-glucosidase 95kDa, pI 4,6 , associated with beta-galactosidase and alpha-fucosidase ConA-Sepharose4B—DEAEC—Sephadex- ConA- (70%; separation G200—HA—Butyl-agarose** from the bound lysosomal glycosidases) [963]

SephadexG200—DEAEC—ConA- ConA- Sepharose—PreparIEF [964]

3.2.1.22. alpha-Galactosidase

Lower plants Aspergillus niger 2.3 (fungal CF) alpha-Galactosidase ConA-Sepharose ConA+ [965]

Aspergillus oryzae (fungus) alpha-Galactosidase DEAEC—ConA-Sepharose4B ConA+ (desorption with 0,2M MM; separation from unbound endo-beta-N-acetylglucosaminidase) [966] Aspergillus tamarii IP 1017-10 (fungal CF) Monomeric alpha-galactosidase 56kDa HA**—DEAEC—DEAEC, ConA-Ultrogel ConA+ (desorption with 0,2M MG) Carbohydr.(M/M): 6 Man, 1,5 Gal, 1 GlcNAc [967]

Cephalosporium acremonium 237 (mold CF) alpha-Galactosidase 290kDa, pI 4,96 DEAEC—Lectin-agarose ConA+ (strong binding, immobilization; partial desorption with 0.2M MG; partial separation from beta-N-acetyl-D- glucosaminidase), LCA+ (strong binding, desorption with 0,2M MG), WGA+ (retardation upon elution without sugar), PHA-

Stabilization with ConA, in solution ConA+ Carbohydr.: 23% hexoses (Man, Gal), GlcN, Sia [968,969]

Dictyostelium discoideum (slime mold, aggregating and vegetative cells) alpha-Galactosidase ConA-Sepharose ConA+ (desorption with 1M MG; 5,5- or 4-f, for

aggregating or vegetative cells,respectively) [970] Higher plants Artocarpus integrifolia (A. heterophyllus) (jack fruit, seeds) Galactomannan-hydrolizing alpha-galactosidase 35kDa, SU 9.5kDa ConA-Sepharose ConA- Carbohydr.: 5.5% neutral sugars (Gal, Ara), without aminosugars [971]

Astragalus glycophylus (milk vetch) alpha-Galactosidase ConA-Sepharose ConA+ [972]

Canavalia ensiformis L.(jack bean, cell protein bodies) alpha-Galactosidase ConA-Sepharose4B ConA+ (binding of <50%; desorption with 0,1M Glc at pH 5,0; approx. 80-f; separation from alpha-mannosidase) [973] Cicer arietinum (chick pea) alpha-Galactosidase ConA-Sepharose ConA+ [972]

Citrullus battich (water melon, juce) monomeric alpha-galactosidase 45kDa DEAE-Sephadex-**—CM-Sephadex— SephadexG100, ConA-Sepharose ConA+ [974]

Cyamopsis tetragonolobus (germinating guar) Cationic alpha-galactosidaseC2 160kDa, SU 42kDa CM-Sephadex** —SephadexG100—ConA- ConA+ (desorption with Sepharose 20mM MM; 33%, 4,5-f) carbohydr.: 28% neutral sugars (Rha, Ara, Gal, Glc, Xyl) [975,976]

Anionic alpha-galactosidaseA 97kDa, SU 42kDa Carbohydr.: 32% neutral sugars (Rha, Ara, traces of Gal) [588]

Glycine max L.(soybean seeds) alpha-Galactosidase 160kDa, SU 40kDa ConA-agarose**—Mel-agarose ConA+ (desorption with 0,1M MM; 57%, 22-f) [977] QAE-Toyopearl—CM-Sepharose—ConA- ConA+ (desorption with Sepharose** 1,3mM MM using gradient 0-7,5mM MM) [978] Lens culinaris L.(Lens esculentum) (lentil seeds)

28 alpha-Galactosidase forms I (160kDa, SU 40 kDa) and II (40kDa, capable to aggregate into I) SephadexG100—ConA-Sepharose**— ConA+ (desorption with CMC(or SephadexG100) 0,1M MM; 88%, 32-f) [972] alpha-Galactosidase of protein bodies LCA-agarose LCA+ [979]

Lodoicea maldivica (mature coconut kernel endosperm) alpha-Galactosidase 21kDa (gel-filtration), pI 3,8 Capranilide-Sepharose4B Carbohydr.: 12% hexoses, 1.2 Glc (M/M) [980]

Medicago sativa (Medicago trunculata) (alfalfa) alpha-Galactosidase ConA-Sepharose ConA+ [972]

Trigonella foenum-graecum (Trigonella procumbens) (fenugreek) alpha-Galactosidase ConA-Sepharose ConA+ [972]

Vicia faba (fava bean, seeds) alpha-Galactosidase forms I (160kDa), IIa (42,6kDa), and IIb (41kDa) SephadexG100**—SephadexG100— ConA-Sepharose—CMC—CMC ConA+ (desorption with 0,5M MM; 56%, 2,3-f, for I; 32%, 6.0-f, for IIa+IIb) Carbohydr.(M/M): 6,7 Man, 13 Glc, 2,7 Xyl, 2,1 GlcN (160kDa); 2,3 Man, 1,5 Glc, 0,9 Xyl, 0,7 GlcN (42,6kDa); 3,2 Man, 5,1 Glc, 1,9 Xyl, 0,94 GlcN (41 kDa) [981,982]

Vigna radiata (Phaseolus aureus) (mung bean, mature seeds) alpha-Galactosidase forms I (162kDa) and II (42kDa) SephacrylS200—ConA-Sepharose**— ConA+ (desorption with Mel-agarose 0.1M MM; 74%, 23-f) [983]

SephacrylS200—ConA-Sepharose ConA- (25%), ConA+ (desorption of 70% with 40mM MG using gradient 0-0,3M MG; separation from glycosidases) [955] Mammals Hamster CHO cells Human lysosomal alpha-galactosidase expressed in CHO cells (Lysosomal Man-6-P-specific receptor lectin 215kDa)-Affigel-10 (0.4mg Lectin+ (desorption with

29 lectin/ml gel) 5mM Man-6-P; decreased binding of more aggregated form) [231] Human liver Acid alpha-galactosidase Lectin-SepharoseB ConA+ (binding of 96%; desorption 0f 90% with 1M MM; 35-54-f), WGA+ (92%;desorption of 50% with 1M GlcNAc), SBA+ (87%;without desorption), GSA-I+ (19%), RCA-I-, UEA-I- [707] alpha-Galactosidase forms A (major), and B (minor) ConA-Sepharose4B—CMC ConA+ (desorption with 1M MG; 75%, 107-f) [984] Human liver from patients with Fabry disease alpha-Galactosidase forms A (traces) and B (major) ConA-Sepharose4B—CMC ConA+ (binding of 70%, low specific activity) [984] Human placenta alpha-Galactosidase forms A (103kDa, SU 57.7 kDa, pI 4,7),and B (117kDa, SU 47,7kDa, pI 4,4) ConA-Sepharose**—DEAEC—Sephadex- ConA+ (desorption with G200—SP-Sephadex—(HA,for A)—Butyl- 0,2M MM; 150%, 166-f; agarose separation from inhibitor) Carbohydr.: 6,15% hexoses, 0,65% Sia (for A); 5,11% hexoses, 0,20% Sia (for B) [985]

Lysosomal acid alpha-galactosidase solubilized with Triton WR1339 ConA-Sepharose ConA+ (desorption with 1M MM; 92%, 111-f) [960] 3.2.1.23. beta-Galactosidase

Bacteria Escherichia coli Gel-entrapped beta-galactosidase in the presence of free RCA-I Activation with RCA-I RCA-I+ (up to 3-f) [986]

Non-enzymatic glucosylated beta- galactosidase (7,2%-decreased activity compared to non-glycosylated enzyme) [7]

Lower plants Alternaria tenuis (fungal CF) beta-Galactosidase 142kDa (SDS-PAGE), or 150kDa (BiogelP300), or 176kDa (ultracentrifugion), pI 4,6 DEAEC**—DEAEC—HA—beta-Galp- thio-carbamoyl-Sepharose4B

Carbohydr.: up to 30% (Gal, Glc, Man, Rha, GlcNAc) [987]

Aspergillus niger (fungus) beta-Galactosidase Metallized-glass-slide—Enzyme—Lectin ConA+, RCA-I+, WGA+, Carbohydr.: 0,5% Gal, 0,3% Glc, 10,2% Man) SBA- [194]

Aspergillus oryzae RT-102 beta-Galactosidase Asn-glycans including Man6GlcNAc [988]

Penicillium canescens 20171 (fungus) beta-Galactosidase 100kDa (ToyopearlHW55), CMC—CMC** Carbohydr.: 7,5% hexoses [989]

Higher plants Artemisia annua L.(sweet wormwood) beta-Galactosidase 100-120kDa, pI 4,2-4,4 CMC—ToyopearlHW55—Lectin-agarose ConA+ (desorption of 50% with 10mM MM), LCA+ (retardation of elution), HPA-, PHA-, RCA-I-

Precipitation with lectins ConA+ or PSA+(separation of enzyme forms) [952] Hordeum vulgare (barley seed meal) beta-Galactosidase 70kDa, SU 42+33kDa, pI 4,95+5,05+5,40+5,70 DEAEC**—CMC—SephadexG100—ConA- ConA+ (desorption with Sepharose 0,1M MG) Sialidase- [954] Lens culinaris L.(Lens esculentum) (lentil seeds) beta-Galactosidase LCA-agarose LCA- [979]

Lilium auratum (goldband lily, pollen) beta-Galactosidase forms I (450kDa), and II (>4,500 kDa) DEAEC—SephadexG100—Sepharose6B— ConA-Sepharose ConA+ (for both forms; desorption with gradient 0-0,5M MM) [990] Triticum vulgare L.(wheat resting seeds, aleurone layers) beta-Galactosidase forms I, II, and III WGA-Sepharose**—DEAEC WGA+ (desorption of I, II, or III with 50, then 100 followed by 200mM GlcNAc, respectively; total recovery 29%, 48-f) [991]

Lectin-Sepharose ConA+ or WGA+ (partial binding) [992]

Vigna radiata (Phaseolus aureus) (mung bean, mature seeds) beta-Galactosidase 162kDa SephacrylS200—ConA-Sepharose ConA+ (desorption of 95% with with 0,15M MG using gradient 0-0,3M MG; separation from a number of glycosidases) [955]

Vigna sinensis (L.) Savi (black-eyed pea, germinating seeds) beta-Galactosidase 58,8kDa DEAEC—SephadexG100**—ConA-Sepharose ConA+ (desorption with 0,1M MG; 88%, 1,3-f) [993] Invertebrates Drosophila melanogaster (fruit fly, adult insects) Dimeric beta-galactosidase-I 160kDa HA—DEAE-Trisacryl**—SephacrylS300— ConA-Sepharose ConA+ (desorption with 130mM MM using gradient 0-0,2M MM) [994] Euphausia superba (antarctic krill) beta-Galactosidase 155kDa, forms I and II DEAEC**—ConA-Sepharose—Ultrogel- ConA+ (for I; desorption AcA34(for II)—PreparPAGE with gradient 0-0,4M MM), ConA- (18%,for II) [340] Littorina littorea L.(periwinkle digestive gland) beta-Galactosidase associated with beta- glucosidase and alpha-fucosidase activities SephadexG200—DEAEC**—GalN-beta- amino-caproyl-agarose—ConA-Sepharose ConA+ (desorption with 0,5M Man; 40%, 3,2-f) [956] Birds Hen liver beta-Galactosidase >200kDa (SDS-PAGE) ConA-Sepharose**—DEAEC—Sepharose- ConA+ (desorption with 6B—n-Aminophenyl-thio-beta-Galp-agarose 0,1M MM; 109%, 12-f) [995]

Mammals Bovine brain GM1-ganglioside beta-galactosidase forms I (600-700kDa, SU 32+20kDa), and II (SU 68+ 62kDa, SDS-PAGE) ConA-Sepharose**—p-Aminophenyl-thio- ConA+ (for both forms; beta-Galp-Sepharose—SephadexG200 desorption with 0,75M MM; 103%, 25-f) [996] Bovine liver Acid GM1-ganglioside beta-galactosidase forms I (600-700kDa), II (200kDa), and III (110kDa) ConA-Sepharose—SephadexG200 ConA+ (all forms; separation from the bound aryl beta-hexosaminidase) [997] Bovine testis

Lysosomal acid beta-galactosidase associated with acid sialidase activity, SU 64kDa ConA-Sepharose—p-Aminophenyl-thio-beta- ConA+ (desorption with Galp-agarose, WGA-Sepharose 0,75M MM or MG; 72%, 12-f; or 47%, 30-f), WGA- Carbohydr.(mkM/mg protein): 0,16 Man, jack-bean- or fungal 0,16 GlcN alpha-mannosidase+ [923,998]

Feline brain GM1-ganglioside beta-galactosidase 250kDa (SephadexG200) p-Aminophenyl-thio-beta-Galp-agarose**— p-Aminophenyl-thio-beta-Galp-agarose— ConA-Sepharose ConA+ (desorption with 0,75M MM; 82%, 2.0-f) [999] Feline liver Lysosomal acid beta-galactosidase 700+130 kDa (Sepharose6B),or 115kDa (ultracentrifugion), SU 62kDa (SDS-PAGE, for reduced protein) ConA-Sepharose**—DEAE-sorbent—Gal- Sepharose—ConA-Sepharose ConA+ (desorption with 0,75M MM; 93%,78-f) [1000] GM1-ganglioside beta-galactosidase 250kDa ConA-Sepharose**—DEAEC—Cellex-E— ConA+ (desorption with p-Aminophenyl-thio-beta-Galp-agarose 0,75M MM; 65%,7-f) [1001] Human liver Acid beta-galactosidase forms A and B Lectin-Sepharose4B ConA+ (binding of >95%; desorption with 0,75M MM at 22oC; 83%, 78-f; separation from unbound neutral galactosidase), WGA+ (60%; desorption with GlcNAc), LCA- [1002] Acid beta-galactosidase Lectin-Sepharose4B ConA+ (binding of 91%; desorption of 86% with 1M MM; 35-54-f), WGA+ (90%; desorption of 68% with 1M GlcNAc), GSA-I+ (90%; desorption of 58% with 1M Gal), SBA+(55%; low desorption),RCA-I+ (19%),UEA-I+(8%) [707] Neutral beta-galactosidase Lectin-Sepharose4B ConA+(55%; desorption of 33% with 1M MM), SBA+(35%; low desorption), GSA-I+ (28%), WGA+(27%), RCA-I-, UEA-I- [707] GM1-ganglioside beta-galactosidaseA ConA-Sepharose4B**—DEAEC—Sephadex- ConA+ (for both forms of

G150—Ecteola-Cellulose A; desorption with MM; 58%, 45-f) [1003] Acid beta-galactosidase 83kDa and GM1- ganglioside beta-galactosidase 18,5kDa ConA-Sepharose4B—p-Aminophenyl-thio- ConA+ (desorption with beta-Galp-Sepharose4B** 0,75M MM; 56%, 42-f) [1004] Acid beta-galactosidase forms A2 (150kDa, SU 65kDa), and A3 (660kDa) ConA-Sepharose**—Sepharose6B—p- ConA+ (desorption with MM; Aminophenyl-thio-beta-Galp-Sepharose4B 56%, 22-f) [1005] Carbohydr.(for A2): 7,5% including Sia, Gal, GlcN, and Man (1,1:1,0:1,7: 2,7, respectively, M/M) [1005]

Acid beta-galactosidase two forms (SDS- PAGE) BiogelA0.5m—ConA-Sepharose4B ConA+ (for both forms; desorption with 0,75M MM) [959] Acid beta-galactosidase 65kDa(SDS-PAGE) DEAE-Sepharose*—ConA-Sepharose— ConA+ (desorption with p-Aminophenyl-thio-beta-Galp-Sepharose — 0,5M MM; 67%, 5,2-f) MonoQ(*)** [1006]

Acid beta-galactosidase 63+800kDa (Sepharose6B), SU 63+31+20kDa ConA-Sepharose —p-Aminophenyl-thio- ConA+ (50%, 20-f) beta-Galp-Sepharose** Carbohydr.: 9% (Man,Gal,NeuAc,GlcNAc); Asn-glycans: 60% of lactosamine-type biantennary sialylated; Man5-6GlcNAc2 [1007]

Acid beta-galactosidase forms A1(monomeric), A2(dimeric), and A3(multimeric) Lectin-Sepharose ConA+(all forms; binding of >95%; desorption of 60% with 1M MG)

WGA-Sepharose —WGA-Sepharose WGA —WGA (partial conversion A3—A1, or A1—A3) [1008] Neutral beta-galactosidase (lactosylceramidase-II) forms 37,5-39,5kDa, pI 4,25 and 4,85 ConA-Sepharose—DEAEC—Sephadex- ConA- (53% of both forms; G100**—IEF—beta-Galp-Sepharose4B separation from the bound acid galactosidase) [1009] Human liver from patient with adult form of GM1-gangliosidosis Acid beta-galactosidase 60kDa(SDS-PAGE) DEAE-Sepharose*—ConA-Sepharose4B** ConA+ (desorption of the activated enzyme with 0,5M MM; 486%, 344-f) [1006] Human liver from a patient with I-cell disease Acid beta-galactosidase ConA-Sepharose ConA+ (up to 37%-decreased

binding compared to normal liver enzyme) [1010] Human placenta Lysosomal acid beta-galactosidase associated with sialidase, SU 76 and 64-66kDa ConA-Sepharose—p-Aminophenyl-thio- ConA+(desorption with 1M beta-Galp-Sepharose(or Ab-titration) MG; 43%, 127-f; or 67%, 64-f) [924,925] Acid beta-galactosidase forms A (500kDa), and B (100kDa) ConA-Sepharose**—SephadexG200— ConA+ (75%, 167-f) Mercurial-Sepharose(for A)—DEAE- Sephadex—Mercurial-Sepharose [1011] beta-Galactosidase 170kDa, SU 70+35+23kDa ConA-Sepharose—Hexosaminyl-Sepharose- ConA+ (desorption with 4B—p-Aminophenyl-thio-beta-Galp- 0,75M MM; 67%, 36-f) Sepharose4B** [1012]

Monkey brain (Macaca radiata) Acid beta-galactosidase forms A (120kDa, SDS-PAGE), and B (1,200 kDa as aggregated A) ConA-Sepharose**—DEAE-Sephadex— ConA+ (for A and B; Sepharose6B—Ecteola-Cellulose(for A) desorption with 0,5M MG; 86%, 53-f) [1013,1014] Murine peritoneal macrophages in culture Lysosomal acid beta-galactosidase 82kDa (SDS-PAGE) ConA-Sepharose—Ab-precipitation ConA+ (desorption of 90- 95% with 0,5M MM) [1015] Porcine testis Acid beta-galactosidase associated with sialidase activity, approx. 600kDa, SU 63+31+20kDa ConA-Sepharose—p-Aminophenyl-thio- ConA+ (desorption with beta-Galp-agarose** 0.5M MM;74%,12-f) [928]

Porcine tissue Lysosomal acid beta-galactosidase multimeric form-I (acid pH), or monomeric form-II (neutral or slightly alkaline pH) ConA-Sepharose ConA+ (binding of up to 33%, for Sialidase-treated I), ConA-(for II) [1016] Rabbit kidney Lysosomal acid beta-galactosidase ConA-Sepharose**—p-Aminophenyl-thio- ConA+(desorption with beta-Galp-agarose 1M MM;102%, 42-f) [1017]

Neutral beta-galactosidase associated with beta-glucosidase and fucosidase, 47kDa ConA-Sepharose4B—DEAEC—Sephadex- ConA- (separation from

G200**—HA the bound acid beta- galactosidase; 118%,1.3-f) [962] Rabbit spleen Lysosomal acid beta-galactosidase forms I (75kDa), IIa (600kDa), and IIb (120 kDa) DEAEC—ConA-Sepharose—Sephadex- ConA+ (desorption of I or G200**—GlcNAc-Sepharose II with 1M MM; 44%,6.8-f, for I; 30%,2.9-f, for II) [1018] Rat epididymal fluid Acid beta-galactosidase immature form Affinity to Mb-bound epididymal lectin REL different from from Man-6-P- REL+ specific receptor [1019]

Rat epididymal tissue Neutral beta-galactosidase 50kDa HA—DEAEC—SephadexG100—ConA- ConA+ (desorption with agarose** 0.25M MM; 205%, 5,7-f) [1020]

Rat liver Lysosomal acid beta-galactosidase RCA-Sepharose4B, RCA-I-Sepharose RCA+ (desorption with (Medac, FRG) 0,25M Lac; 18%, 60-f) [617] Acid beta-galactosidase 122kDa ConA-Sepharose**—HA—SephadexG200 ConA+ (washing with 2,5% sucrose; desorption with 1M MG; 72%, 6.1-f) [1021] Sheep brain, kidney, spleen Lysosomal acid beta-galactosidase ConA-Sepharose ConA+ (desorption with 1M MM;separation from unbound gluco-, xylo-, fuco sidase, and L-arabinosidase) [1022] 3.2.1.24. alpha-Mannosidase

Lower plants Dictyostelium discoideum (slime mold) Lysosomal acid alpha-mannosidase glycans Immobilized mammalian liver Man-6-P- specific lectin receptor Lectin+(desorption with 0,5mM Man-6-P) [940] Tetrameric alpha-mannosidase mature form 240kDa, SU 58+54kDa, and precursor form, SU 150kDa), from strain AX2 Asn-glycans phosphorylated and sulfated (58 and 54kDa); non-sulfated EndoH- (partial resistance gycans (150kDa) of 240kDa) Asn-glycans of oligo-Man type (30kDa in EndoH+ (150—120kDa) 150kDa) [1023]

Puccinia graminis-fungal-rust-infected wheat leaves alpha-Mannosidase 10 forms 2-Dimential-PAGE—Blot—ConA ConA+ (all forms) [102]

Higher plants Acer pseudoplatanus L.(sycamore, protoplasts of cell culture) alpha-Mannosidase ConA-Ultrogel ConA+ (low desorption with MG) [1024] Canavalia ensiformis L.(jack bean, seed cotyledons and protein bodies) Tetrameric alpha-mannosidase with heavy SU68kDa possessing Asn-glycan of oligo- Man type ConA-Sepharose—ConA-Sepharose ConA- (73-77%), ConA+ (27%; desorption with 0,2M MM or pNP-alpha-Manp) [1025] ConA-Sepharose (for biocatalyst) ConA+ [1025]

SDS-PAGE—ConA-125I ConA+(68kDa) [1026] alpha-Mannosidase 220kDa, SU 44+66kDa ConA-Sepharose**, DEAEC, CMC, Phenyl- ConA+ (50-f; desorption Sepharose with gradient of pH or NaCl; irreversible inactivation of sorbent with crude extract) [1027]

DEAEC**—Phenyl-Sepharose—ConA- ConA+ (desorption with Sepharose gradient 0-1M NaCl at pH 5,0; 110%, 2,5-f) [1027]

Lectin-Sepharose4B ConA- (at pH 8.0; separation from other glycosidases), ConA+ (complete binding at pH 5,0; desorption with 1M NaCl; separation from the bound galactosidase), LCA+ or PSA+ (desorption with at least 5mM Glc) [973] alpha-Mannosidase ConA-Sepharose4B—ConA-Sepharose4B ConA+ (depending on pH, ionic power and hydrophobic contribution) [1028,1029] Carbohydr.: 2,66% Man, 0,84% GlcN; Asn-glycans (heavy SU) including EndoH+ + GlcMan9GlcNAc2 in ConA -form [1025,1030]

Lens culinaris L. (Lens esculentum) (lentil seed protein bodies) alpha-Mannosidase of albumin-type LCA-Sepharose LCA+ [979]

Malus domestica Borhk, cv.Golden delicious (apple, climacteric stage of ripeness) alpha-Mannosidase

MonoQ*—ConA-Sepharose4B ConA+ (binding without desorption by 0,3M MM; separation from beta-N-acetylhexosaminidase) [1030] Pisum sativum L. (pea seed cotyledons) alpha-Mannosidase ConA-Sepharose ConA+ (complete strong binding, immobilization; desorption of traces with 0,3M MG; separation from beta-N- acetylglucosaminidase) [1031] alpha-Mannosidase 300-320kDa, pI 4,3 BiogelP200**—ConA-Sepharose— ConA+ (desorption with 1M DEAE-Sephacel—Chromatofoc— MG) SephadexG75 [1032]

Prunus serotina Ehrh. (black cherry mature seeds) alpha-Mannosidase 150kDa, pI 4,8-5,2 DEAEC—SephacrylS200—ConA- ConA+ (complete strong Sepharose4B binding; desorption of <25% at pH 4,5-8,5 using 0,5M NaCl and/or 1M MM or MG; active immobilized form) [1033] Triticum aestivum L.(wheat leaves inoculated with the rust fungus, Puccinia graminis) alpha-Mannosidase forms 2-Dimential-PAGE—Blot — ConA ConA+ (10 protein spots with active enzyme) [102] Triticum aestivum (wheat resting seed aleurone layers) alpha-Mannosidase 197kDa (SephadexG200), PAS+ or 188kDa (ultracentrifugion) ConA-Sepharose**—DEAEC—Sephadex- ConA+ (desorption with G200—DEAEC 0,2M MM; 74%,49-f) Carbohydr.(%): 20,1 Glc, 3,09 Man, 1,7 Gal, <1 GlcNAc; Asn-glycans of oligoMan type EndoH+ (elimination of 33% carbohydr.) [1034] Vigna radiata (Phaseolus aureus) (mung bean, seedlings) Microsomal arylmannosidase forms A (237kDa, major SU 60+55kDa, minor SU 79+39+35kDa), and B (243kDa, major SU 72+55+45kDa, minor SU 42+39kDa), solubilized with TX100 DEAEC—Phosphocellulose**(A+B)—HA —ConA-Sepharose—SephacrylS300—PAGE ConA+ (desorption with 0,3 M MM; 54%, 1,8-f, for A; 71%, 2,4-f, for B) Carbohydr. in all SU (not for 45 and 42kDa) Asn-glycans of oligoMan type EndoH+ (elimination of up to 50% carbohydr.)

ConA-Sepharose ConA+ (washing with 18mM MG; desorption with 0,5M MM; separation of glycans) [1035] Vigna radiata (mature seeds) alpha-Mannosidase 79kDa SephacrylS200—ConA-Sepharose ConA- (32%), ConA+ (65%; desorption with 0,1M MG using gradient 0-0,3M MG; separation from a number of alpha- and beta-glycosidases) [955] Protozoans Trypanosoma cruzi strain YP3 (trypanosomal epimastigotes) alpha-Mannosidase 240kDa(Sepharose-4B), SU 58kDa(SDS-PAGE) DEAEC—Sepharose4B—PreparIEF— SephacrylS200—ConA-Sepharose** ConA+ (desorption with 0,2M MM; 82%, 25-f) [1036] Invertebrates Mytilus edulis L.(mussel hepato- pancreas lysosomes) Zn2+-dependent alpha-mannosidase 240kDa (SephadexG200) ConA-Sepharose**—ManN-Sepharose— ConA+ (desorption with CM-Sephadex—PAGE 0,5M MM; 27%, 71-f) [1037]

Mammals Bovine liver Microsomal arylmannosidase (pNP-alpha)- mannosidase) solubilized with Lubrol- PX followed by TX100 N-5-Carboxypentyl-1-deoxymannojirimycin- sorbent—ConA-Sepharose ConA+ (binding of >90%; partial separation from processing mannosidase-I) [1038] Human liver Acid alpha-mannosidase Lectin-Sepharose4B ConA+ (complete binding and desorption with 1M MM;up to 54-f), WGA+ (95%; complete desorption with 1M GlcNAc; 93-f), GSA-I+ (54%;desorption of 90% with 1M Gal), SBA+ (28%), RCA-I+ (26%), UEA-I+ (25%) [707] Acid alpha-mannosidase forms A (220kDa, pI 6,0), and B (300+460kDa, pI 5,45) ConA-Sepharose—ManN-Sepharose4B** ConA+ (complete binding of —FucN-agarose, DEAEC(A+B) both forms; desorption of 80-87% with 0,5M MM; 7-f; separation from unbound neutral cytosolic enzyme) Carbohydr. (for A): Sia Sialidase+ (pI 6,0—6,45) [1039,1040] Acid alpha-mannosidase forms A (SU 62+26

39 kDa), and B (SU 26+58+62kDa), A and B as immunologically identical ConA-Sepharose**—DEAEC ConA+ (desorption with 0,5 M MM; 53%, 4,3-f, for 62 and 58kDa), ConA- (26kDa) [1041] Neutral cytosolic alpha-mannosidase ConA-Sepharose ConA- (100%) [1039]

Neutral alpha-mannosidase Lectin-Sepharose4B ConA+ (27%), WGA+(42%), UEA-I+ (69%; low desorption with 1M Fuc), GSA-I+(32%), RCA-I+ (19%), SBA+(18%) [707] Human lymphoid B-cells including B-cell nonHodgkin lymphoma; lymphoid T-cells from patients with Cezari syndrom Neutral cytosolic alpha-mannosidase ConA-sepharose**—UltrogelAcA34 ConA- (103%, 13-f) [1042]

Human lymphoblastoid cells transformed with Epstein-Barr virus, from peripheral lymphocytes of normal individuals and patients with I-cell disease alpha-Mannosidase RCA-I-agarose RCA-I+ (up to 100%-binding) [1043] Human placenta Lysosomal acid alpha-mannosidase 205kDa with a number of pI-forms ConA-Sepharose4B**—DEAEC—Prepar PAGE, ConA-Sepharose—IEF ConA+ (desorption with 0,5 M MM; 87%, 39-f) Carbohydr.: 13,5% (64 Man, 20 Gal, C.perfringens sialidase+ 8 Fuc, 33 GlcNAc, 19 NeuAc, M/M) [1044]

Acid alpha-mannosidase solubilized with Triton WR1339 ConA-Sepharose ConA+ (desorption with 1M MM; 85%, 109-f) [960] Human skin fibroblasts from patients with cystic fibrosis alpha-Mannosidase sialoforms WGA-agarose WGA+ [1045]

Monkey brain (Macaca radiata L.) Acid alpha-mannosidase ConA-Sepharose—Phosphomannan- Sepharose, ConA-Sepharose—RCA-I- ConA+ (desorption with MG), Sepharose RCA-I+ (binding of 48%; desorption of 35% with 0,5 M Lac; separation from unbound form) [1046] Carbohydr. (for ConA+RCA-I+-form): Sialidase- (ConA+RCA-I+-form)

Asn-glycans of oligoMan- and complex type [742]

Neutral alpha-mannosidase ConA-Sepharose—Co2+-chelating- ConA- (separation from sorbent acid alpha-mannosidase) [1047]

Murine liver and spleen Tetrameric acid alpha-mannosidase (SU 65kDa) as lysosomal particulate form ConA-Sepharose—Blot—Ab ConA+ (desorption with sugar) [1048] Porcine kidney Lysosomal acid alpha-mannosidase Lectin-agarose RCA-I+ (binding of 85%), Rabbit-serum-MBP+ (complete binding of native but not perjodate-treated enzyme) Asn-glycans with terminal Gal, GlcNAc and/or Fuc [1049,1050]

Rat liver Acid alpha-mannosidase Lectin-agarose RCA-I+ or Rabbit-serum-MBP+ (complete binding) [1050]

Lysosomal acid alpha-mannosidase 335kDa (gel-filtration), or 200kDa (ultracentrifugion), and cytosolic neutral enzyme Phosphocellulose—HA—Dowex-50W— 2-Acetamide-2-deoxy-D-glucono-1,4- lactone-Sepharose**—ConA-agarose ConA+ (desorption with 0,1 (Miles-Yeda) M Man; separation from unbound cytosolic enzyme) PAGE PAS+(for acid and neutral enzyme) [1051] Cytosolic neutral alpha-mannosidase 113 kDa (Sephacryl-S200),or 108kDa (SDS- PAGE) ConA-Sepharose—Co2+-chelating- ConA- (97%; separation from Sepharose—DEAE-Trisacryl-M**— lysosomal acid enzyme) SephacrylS200 [1052]

3.2.1.25. beta-Mannosidase

Lower plant Thielavia terrestris NRRL 8126 (mold CF)

Five mannan-degrading enzymes including beta-mannosidase 72kDa (SDS- PAGE) and enzyme forms M1 (52kDa), M2 30kDa), M3 (55kDa), and M4 (89kDa) Sp-Sephadex—PreparPAGE Carbohydr.: 6-36% hexoses [1053]

Higher plants Triticum aestivum L.(wheat resting seed aleurone layers) beta-Mannosidase Affinity to WGA-125I WGA+ [1054]

Mammals Bovine kidney Lysosomal beta-mannosidase 100+110kDa (SDS-PAGE) ConA-Sepharose**—Ab-sorbent—TSK- ConA+ (desorption with butyl-column—MonoS* glycoside; 53%, 129-f) [1055] Goat kidney Lysosomal acid beta-mannosidase ConA-Sepharose**—SephadexG100— ConA+(desorption with MonoS*—MonoQ* sugar; 80%, 27-f) [1056]

Goat liver Lysosomal acid beta-mannosidase forms A and B, and neutral beta-mannosidase ConA-Sepharose ConA+ (for acid enzyme,but not neutral enzyme) [1057] Lysosomal acid beta-mannosidase forms pI 5,0+5,5+5,9, from adult liver, or forms pI 5,4+5,8, from 60-day fetal liver ConA-Sepharose ConA+ (48%,for adult enzyme; desorption with 0,5M MG; separation from non-lysosomal enzyme), ConA+ (binding of >50%, for fetal enzyme) [1058] Guinea pig liver Acid beta-mannosidase 110 or 130kDa(gel- filtration at pH 5,0 or 8,0, respectively) ConA-Sepharose**—SephadexG200— ConA+ (desorption with 0.5 DEAEC—GlcNAc-Sephadex—CM-Sephadex M MM; 142%, 8,8-f) [1059]

Human placenta beta-Mannosidase ConA-Sepharose4B**—Ultrogel- ConA+ (desorption with AcA34 —HA gradient 0-0,5M MM; 34%, 6,9-f; partial separation from alpha-mannosidase) [1060] Human urine Acid and neutral beta-mannosidases ConA-Sepharose—DEAE-Trisacryl ConA+ (binding of >70% of acid enzyme; desorption with 0,5M MM; separation from unbound neutral enzyme) [1061] 3.2.1.26. beta-Fructofuranosidase

Lower plants Aspergillus niger (fungal CF) beta-Fructofuranosidase ConA-Sepharose ConA+ (separation from amylolytic and proteolytic

activities) [1062] Tetrameric beta-fructofuranosidase forms I (302kDa,pI 4,5), II (310kDa, pI 4,9), and III (324kDa, pI 5,2), SU 85kDa, from strain M79 DEAE-Trisacryl**—SephadexG150—IEF Carbohydr.: 33% (I), 40% (II), 20% (III) [1063]

Aureobasidium sp. ATCC 20524 (fungal CF) EnzymeP1 318kDa (SephadexG200) with SU 94kDa (SDS-PAGE, for deglycosylated protein) DEAE-CellulofineA800—SephadexG200— SephadexG200 Asn-glycans: 33% in 104kDa EndoF+ (318—214kDa) [1064] Kluyveromyces fragilis ATCC 12424 (yeast CF) beta-Fructofuranosidase four forms ConA-Sepharose(better than DEAEC)— ConA+(all forms; desorption IEF) with MM) [1065]

ConA-Sepharose (for biocatalyst) ConA+ (highly active immobilized form) [1066] Carbohydr.: 66% hexoses [1620]

Phytophthora megasperma Drechs. var. sojae A.A.Hildebrand (fungus) beta-Fructofuranosidase DEAEC—DEAEC—L-Norleucine— Sepharose**—Agarose, ConA- ConA+ (complete binding; Sepharose desorption of 93% with 0,2M MM) Carbohydr.: 25%, Man and GlcN as >97% of total sugars [1067]

Saccharomyces cerevisiae (baker's yeasts, cells) beta-Fructofuranosidase ConA-Sepharose ConA- (20%), ConA+(desorption of 5% with 0,2M MM, next 33% with 0,5M MM, next 22% with 1M MM) [1068] beta-Fructofuranosidase Type VI (Sigma, USA) sConA-Sepharose6B (for immobilizat- sConA+ (covalent immobiliz- ion) ation with glutaraldehyde) [1069] beta-Fructofuranosidase (Boehringer- Mannheim, FRG) sConA-Sepharose (for immobilization) sConA+ [1070]

(Crude-ConA)-SephadexG50 ConA+ (stabilization of 14% with glutaraldehyde) [19] Purified beta-fructofuranosidase Precipitation with ConA ConA+ (100%) [1071]

beta-Fructofuranosidase without admixtural mannan ConA-agarose(Sigma,USA), Micro- ConA+ (73%; highly thermo- crystalline-Cellulose, DEAE-Sephadex, stable immobilized active CM-Sephadex, for immobilization form) [1072,1073]

Crystalline beta-fructofuranosidase (Sigma,USA) ConA-Sepharose4B (5 or 20mg ConA/ml ConA+ (increased enzyme gel) thermostability of sorbent complex treated with glutaraldehyde) [16,1074] ConA-Sepharose (for biocatalyst) ConA+ (exchange of old enzyme for new portion by preliminary washing with 0,1M Gly-HCl pH 2) [21] beta-Fructofuranosidase-ConA complex ConA+ entrapped into Ca-alginate gels (for biocatalyst) [18] beta-Fructofuranosidase-ConA-Yeast-cell ConA+ (binding through complex entrapped into PAG (for components of multienzyme biocatalyst) complex) [22]

Invertase ConA-Cellulose(for enzyme ConA+ (immobilization and thermistor) catalysis) [1075,1076] beta-Fructofuranosidase-agarose —Rat- liver-MBP MBP+ [1077]

Saccharomyces cerevisiae (yeast CF) beta-Fructofuranosidase 270kDa Carbohydr.: Man- alpha- alpha-Mannosidase+ [1078]

Dimeric beta-fructofuranosidase 135kDa, SU 60kDa DEAEC—BiogelP200 Carbohydr.: approx.50% (570 Man, 36 GlcN, M/M); Asn-glycans of oligoMan type EndoH+ [1079]

Other enzyme multiple forms (di-, tetra-, and octameric) Glycans of SU: 10 or 2, for native or partially deglycosylated enzyme, respectively [1080] Influence of glycans on enzyme oligo- merization and stability [1080-1082]

Schizophyllum commune (fungal CF) Invertase 126kDa (SU 60kDa) SephadexG150 Activity influencing by lectin ConA-, LCA-, PLA-, RCA-I-, SWA-, WGA-

Carbohydr.: 31% neutral sugars [1083]

Schizosaccharomyces pombe 972h- (yeast fission cells) Soluble beta-fructofuranosidase ConA-Sepharose ConA+ (complete binding) Precipitation with ConA ConA+ Carbohydr.: Man and Gal [1084]

Higher plants Daucus carota L. cv.Nantaise (carrot dry seeds) Monomeric beta-fructofuranosidase-I, 65kDa, pI 3,8 ConA-Sepharose4B —DEAE- ConA+ (washing with 10mM Sepharose**—SephacrylS200—HIC- MM; desorption with 0,5M sorbent*—DEAE-Sepharose MM; 43%, 6-f) [1085]

Daucus carota L.(seedling cells in culture) Cell wall beta-fructofuranosidase 63kDa (SDS-PAGE), and soluble extracellular form with SU 52+54kDa (SDS-PAGE) Mixture of pronase and trypsinized Gp ConA-Sepharose—Reverse-phase-Vydac- ConA+ (desorption with 0,1 column-C18 M MM; separation of Gp) [1086,1087] Asn-glycans: 1 of oligoMan-type, and EndoH+, TFMSA+ 2 of complex type (complete elimination for TFMSA+-form) [1086]

Dianthus caryophyllus cv.white sim (carnation petals) Soluble beta-fructofuranosidase-I 215kDa DEAEC**—ConA-Sepharose—BiogelP200 ConA+ (desorption with 75 mM MM using gradient 0-0,25 M MM; 51%, 1,2-f) [1088] Hordeum vulgare L. cv.Larker (barley elongation stem tissue) Cytosolic soluble beta-fructofuranosidase 120kDa, SU 60kDa DEAEC—ConA-Sepharose**—Organo- ConA+ (desorption with 10mM mercurial-Sepharose—SephacrylS300 MM using gradient 0-0,25M MM) [1089] Cell-wall-bound beta-fructofuranosidase 120kDa, SU 60kDa DEAEC—ConA-Sepharose—Sephacryl- ConA+ (desorption with 4mM S300 MM using gradient 0-0,25M MM; 42%, 1,8-f) [1089] Hordeum vulgare (L.) Morex (barley 30-day seedling stem tissue) beta-Fructofuranosidase DEAE-Sephacel—ConA-Sepharose ConA+ (desorption with gradient 0-100mM Man; separation from fructan- exohydrolase) [1090]

Lilium auratum L. (goldband lily, pollen) Cytoplasmic beta-fructofuranosidase-I 450kDa (Sepharose6B) DEAEC**—SephadexG200—Sepharose6B ConA+ (complete binding; desorption with 0,30-0,45M MM using gradient 0-0,5M MM; 38%, 6,2-f) Carbohydr.: 11% hexoses [1091]

Lycopersicon esculentum L. (tomato fruits) Buffer-soluble invertase 50kDa (Superose12*), or 56kDa (SDS-PAGE), PAS+ 3 forms pI 5,65-5,80 CM-Sephadex—ConA-Sepharose**— ConA+ (washing with 2mM MM; SephacrylS200 desorption with 0,3M MM; 96%, 3,4-f [1092] Nicotiana tabacum L. SR1-C58 (tobacco gall cells) Cell wall beta-fructofuranosidase 63kDa (SDS-PAGE) solubilized with TX100 and NaCl SP-Sephadex(pH-gradient)**—SP- Sephadex(NaCl-gradient)—ConA- ConA+ (desorption with 50mM Sepharose MG using gradient 0-1M MG; 24%, 1,4-f) [1093] Oryza sativa L. (rice cell walls) Invertase 32kDa (SepharoseCL6B), or 30kDa (SDS-PAGE), pI 6,45 ConA-Sepharose**—SepharoseCL6B ConA+ (desorption with —MonoQ* gradient 0-1M MM; 75%, 18-f) [1094] Oryza sativa L. (rice seedlings) Soluble acid invertase 98kDa (Sephadex- G100), SU 46kDa Activation with lectins ConA-, LCA+ (54%), RCA+ (63%) [1095] Prunus persica (L.) Batsch var. vulgaris Maxim."Hakuto" (peach immature fruits) beta-Fructofuranosidase 120kDa, SU 64kDa DEAEC**—ConA-Sepharose—Sepharose- ConA+ (desorption with CL6B gradient 0-0,5M MM; 33%, 2,1-f) [516] Raphanus sativus L. (radish seedlings) Cytoplasmic soluble beta-fructofuranosidase 48,5kDa, 5 forms pI 6,0-8,3 SephadexG25—ConA-Sepharose4B— ConA+ (desorption with 5mM UltrogelAcA34—Ab-sorbent MG; 12-f [1096]

ConA-Sepharose4B (crosslinked with ConA+ (desorption of 90% glutaraldehyde) with gradient 0-2.5mM MM and 25mM MM) [1097,1098]

ConA-Sepharose4B (ConA-S), ConA- ConA-S3+, ConA-U2+,

Ultrogel (ConA-U), LCA-Sepharose4B LCA+ [1099]

LCA-Ultrogel, LCA-Sepharose LCA+ (retardation of elution without sugar) [1098] Other lectin sorbents APA- , PHA-, PNA-, RCA-I-, WGA- [1100,1101] LIE ConA+, LCA+, and/or WGA+ (multiple forms) [1098]

SDS-PAGE—Blot—ConA—Peroxidase ConA+ [1101] Carbohydr.: around 7,7% (depending on enzyme solubility) [1096,1098]

Cell wall beta-fructofuranosidase similar to cytoplasmic enzyme according to lectin chromatography Lectin-sorbent (6 types) Lectin+ [1101]

Raphanus sativus L. (radish cells in culture) Deglycosylated beta-fructofuranosidase from tunicamycin-treated cells Affinity to ConA ConA- [1101]

Ricinus communis L. (castor bean, young leaves) beta-Fructofuranosidase Activation with lectins in solution CJA+ (53%), ConA+ (46%), LCA+ (34%), PSA+ (44%), RCA-I+ (90%), RCA-II+ (63%), UEA-I+ (65%), VEA+ (33%), WGA+ (55%) Carbohydr.: 45% hexoses [1102] Saturation of enzyme with lectins in CJA+, ConA+, UEA-I+, WGA+ solution [1103]

Solanum tuberosum L. cv.Kennebec, cv.Katahdin (potato tubers) beta-Fructofuranosidase 60kDa, SU 30kDa ConA-Sepharose—DEAE-Sephadex**— ConA+ (desorption with SephadexG150—DEAE-Sephadex 30mM MM; 110%, 11-f) [1104] ConA-Sepharose—ConA-Sepharose ConA-ConA-(for unbound form) [1104]

SephadexG100—DEAEC—ConA- ConA+ (desorption with Sepharose** 0,1M MM; 184%, 35-f; separation from inhibitor) [1105] Inhibition with STA STA+(>50%; non-competedly) [1106] Carbohydr.: 10.9% hexoses including Jack-bean alpha-mannosidase+ Man-alpha- [1104,1106]

Triticum aestivum L. cv.Yecovo Rojo (wheat 4-day seedlings, colyoptyles and roots) Cytosolic soluble beta -fructofuranosidase 158kDa, SU 53kDa

DEAEC—ConA-Sepharose**—Sephacryl- ConA+ (desorption with 0,1M S300 MM using gradient 0-0,25M MM; 70%, 52-f) [1107] Urtica dioica L. (stinging nettle, leaves)

Cytosolic soluble acid beta-fructofuranosidase 58kDa, pI 4,6 ConA-Sepharose**—SephadexG75— ConA+ (desorption with 0,25 Superose12*—MonoQ* M MM; 64%, 19-f) [1108]

Cell-wall-bound acid beta-fructofuranosidase pI 9,3 ConA-Sepharose**—CM-Biogel— ConA+ (desorption with 0,25 Superose12* M MM; 76%, 18-f) [1108] SDS-PAGE—Blot—ConA—Peroxidase ConA+ [1108] Carbohydr.: Man-alpha- alpha-Mannosidase+ [1108]

Vigna radiata L. (Phaseolus aureus) (mung bean, mature seeds)

Alkaline beta-fructofuranosidase >250kDa ConA-Sepharose ConA+ (complete binding; desorption with 0,2M MM) [1109] SephacrylS200—ConA-Sepharose ConA+ (binding of 96%; desorption with 0,2M MG using gradient 0-0,3M MG; separation from alpha- and other beta-glycosidases) [955] Vigna radiata L.(mung bean germinating seeds) Acid and alkaline beta-fructofuranosidases ConA-Sepharose ConA+ (complete binding of both enzymes; desorption with 0,2M MM; 40-90-f) [1109] Zea mays L.(maize germinating seeds) beta-Fructofuranosidase soluble form 750- 9,000 kDa, and particulate-bound form 40kDa (solubilized with 1M NaCl) ConA-Sepharose ConA+ (for both forms; desorption with 0,2M MM) [1110] Protozoan Isotricha prostoma L. (rumen holotrich ciliate, pathogenic for sheeps) Tetrameric beta-fructofuranosidase 350kDa (SepharoseCL4B—Octyl-Sepharose)** — DEAEC—ConA-Sepharose4B ConA+ (desorption of major form with 0,1M MM, and two minor forms with 0,12 and 0,2M MM, using gradient 0-0,25M MM) Carbohydr. (major form): 2,9% Man, 1,4% Glc, 0,9% Gal, 3,1% GlcNAc [1111]

Mammals Rat intestinal brush-border beta-Fructofuranosidase of extract Inhibition with PHA (1, 10, 50 mg/ml) PHA+ (26, 45, or 60%, depending on increasing concentration of PHA) [1112] 3.2.1.28. alpha-alpha-Trehalase

Lower plants Dictyostelium discoideum NC-4 (ATCC- 4697) (slime mold cells) Trehalase 100kDa (gel-filtration), or 97kDa (SDS-PAGE) PAS+ SephadexG150—DEAEC—Chromatofoc —PreparPAGE—ConA-Sepharose ConA+ (desorption with 35- 40mM MM using gradient 0-0,2M MM) [1113] Saccharomyces cerevisiae (baker's yeasts) Cellular trehalase HA**—ConA-Sepharose ConA+ (desorption with 1mM MG; 79%, 2,1-f; separation from the bound invertase) [1114] Soluble acid trehalase 215kDa (gel-filtration), and neutral trehalase 170kDa ConA-Sepharose ConA- (95%, for 170kDa), ConA+ (83%, for 215kDa; desorption of 22% with 0,2-1,0M MM) [1068] Higher plant Glycine max L. cv.Maple Arrow (soybean callus culture of nodule cells infected with symbiotic bacterium, Bradyrhizobium japonicum 61-A-101) Trehalase 54kDa, pI 5,2 ConA-agarose**—MonoQ*—Superose12* ConA+ (almost complete binding; desorption with 0,2M MM; 60%, 7,7-f) [1115] Invertebrates Aldrichina grahami (blowfly pupae hemolymph) Trehalase 80kDa CMC—DEAEC**—SephadexG150—ConA- ConA- (82%; 1.1-f) Sepharose [1116]

Periplaneta americana (american cockroach thoracic muscle) Trehalase forms A (80kDa, cytosolic), and B (110kDa, particulate-bound solubilized with snake venom) ConA-Sepharose4B**—SephadexG150 ConA+ (desorption of A or B with 0,2M MM; 61%, 20-f, for A; 48%, 32-f, for B) Carbohydr.: 17-18% hexoses [1117]

Mammals Porcine kidney cortex brush-border Mb Monomeric trehalase 80kDa, pI 4,4-4,7, solubilized with TX-100 SephacrylS300—DEAEC—ConA- ConA+ (binding of 90%; Sepharose—Phenyl-Sepharose**—Tris- desorption with 0,5M MG in Sepharose—Tris-Sepharose—HA the presence of 0,1M borate and 0,1% TX100; 38%, 5,0-f) Carbohydr.(M/M): 2 GlcNAc [1118]

3.2.1.31. beta-Glucuronidase

Bacteria Mycobacterioum leprae beta-Glucuronidase ConA-agarose ConA- [1119]

Invertebrates Euphausia superba (antarctic krill) beta-Glucuronidase Q-Sepharose*—SephadexG25—ConA- ConA+ (complete binding; Sepharose—Superose6*—MonoQ*, desorption of 54% with LCA-Sepharose 0,25M MG), LCA [1120]

Reptiles Dasypus sp. (armadillo liver) beta-Glucuronidase ConA-agarose ConA+ (80%; desorption with MM) [1119] Mammals Monkey brain Lysosomal beta-glucuronidase with SU 80+72+62kDa ConA-Sepharose—Phosphomannan- ConA+ (complete binding; Sepharose, ConA-Sepharose**—RCA-I- desorption with MG or MM; Sepharose 80%, 22-f; separation from microsomal enzyme), RCA-I- [742,1046,1121] Carbohydr.: 12,2% hexoses (Man, Glc, and Fuc) [1121]

Microsomal beta-glucuronidase ConA-Sepharose ConA+ (elution with sugar, before lysosomal enzyme; 58%, 4.3-f) Carbohydr.: 30% hexoses (Man, Glc, and Fuc) [1121]

Murine liver of normal and inbread strains C57BL/6, LTS/A (which have not protein egastin usually associated to stabilization of microsomal beta-glucuronidase)

Lysosomal beta-glucuronidase forms 71,5

50 kDa, pI 5,9, and 73kDa, pI 5,4 RCA-I-agarose RCA-I+ (binding of 13 or 37%, for normal or inbread strains, respectively; increased binding of Sialidase-treated enzymes) Carbohydr.: Sia Sialidase+ [1122]

Murine peritoneal macrophagues in culture beta-Glucuronidase with SU 63kDa (SDS-PAGE) ConA-Sepharose—Ab-precipitation ConA+ (complete binding; desorption of 90-95% with 0,5M MM) [1015] Murine P-388-D1 cells (CF) Extracellular beta-glucuronidase Asn-glycans of oligoMan-type [1123]

Human fibroblasts in culture Extracellular beta-glucuronidase RCA-I-agarose RCA-I+ (27 or 38%,for native or sialidase- treated enzyme, respect ively; desorption with 0,1M Gal) Carbohydr.: Sia Sialidase+ [1124] Lysosomal beta-glucuronidase RCA-I-agarose RCA-I+ (53 or 34%, for native or sialidase-treated enzyme, respectively) Carbohydr.: Sia Sialidase+ [1124]

Human liver Acid beta-glucuronidase Lectin-Sepharose4B ConA+ (binding of 97%; desorption of 90% with 1M MM), GSA-I+ (64%; desorption of 25% with 1M Gal), RCA-I+ (16%; desorption of 12% with 1M Gal), SBA+ (complete binding; desorption of 11% with 1M GalNAc), UEA-I+(5%; without desorption), WGA+ (complete binding; desorption of 62% with 1M GlcNAc) [707] Human lymphoblastoid cells transformed with Epstein-Barr virus from periferal lymphocytes of normal individuals and patients with I-cell disease beta-Glucuronidase RCA-I-agarose RCA-I+ (binding of 30 or 80%, for normal or I-cell- disease enzyme, respectively) [1043] Rat basophil leukemia tumor Lysosomal beta-glucuronidase 300kDa,

SU 75kDa, two forms pI 5,9 and 5,7 (including tumor-specific form) ConA-Sepharose**—SephadexG200— ConA+ (complete binding; DEAEC—CMC—Phosphocellulose desorption with 0,3M MM; 77%, 18-f) [1125] Rat liver Lysosomal acid purified beta-glucuronidase Lectin-Sepharose, Mb-bound hepatic ConA+ (almost complete lectin of enzyme elimination from binding), RCA-I-, blood Rabbit-serum-MBP+ (complete binding), Hepatic-lectin(endogenic)+ Enzyme treated with perjodate Binding to immobilized lectin Hepatic-lectin-, ConA+ (partial binding) [1050,1126,1127] Acid beta-glucuronidase 204kDa, SU 66+70kDa ConA-Sepharose4B**—HA—Sephadex- ConA+ (washing with 2,5% G200 sucrose; desorption with 1M MG; 80%,7,0-f) [1021] Lysosomal beta-glucuronidases 300 and 150kDa, SU 76kDa, >5 forms pI 4,3-4,7 ConA-Sepharose4B**—Blue-Sepharose ConA+ (desorption with 0,5 —Chromatofoc—DEAEC—Sepharose- M MG; 38%,15-f; partial CL6B—PreparPAGE inhibition of enzyme with MG) [1128] LIE ConA+ (3 forms), LCA+ or WGA+(2 forms), SBA+ or PHA+ (1 form), LTA+ [1128] Mixture of pronase Gp Lectin-agarose (sequential ConA+WGA-LCA-UEA-I+, chromatography) ConA2+LCA-WGA-, ConA-RCA-I+SBA+PHA-E+ (separation of Gp) Asn-glycans of oligoMan-type (43%), complex biantennary (40%), complex biantennary fucosylated (17%), hybrid and polylactosamine type (0,5% [1129]

Microsomal beta-glucuronidase LIE ConA3+, LCA2+, PSA+, RCA-I+, LTA-,WGA- [1128] Rat liver of dibutylphosphate-treated animals Lysosomal beta-glucuronidase Lectin-Sepharose ConA+ (binding of 53%; desorption of 47% with 0,75 M MG), RCA-I+ or WGA+ (low binding) Microsomal beta-glucuronidase 79kDa (SDS-PAGE) Lectin-Sepharose ConA+ (binding of 60%; desorption of 40% with 0,75 M MG), RCA+or WGA+ (low binding)

Golgi beta-glucuronidase Lectin-Sepharose RCA-I+ or WGA+ (strong binding; desorption with 0,5 M Gal or GlcNAc) [1130] Rat mammary regressing tumor induced by N-methylnitrourea beta-Glucuronidase cytosolic soluble and microsomal forms ConA-Sepharose ConA+ (desorption with gradient 0-1,5M MG; small differences between both forms) [1131] Rat serum beta-Glucuronidase 83kDa Ab(IgG)-Sepharose—IEF Lectin-Sepharose ConA+ (strong binding, Immobilization; partial desorption with 0,75M MG), RCA-I+ (binding of 83%;desorption of 65% with 0,5M Gal), WGA+ (binding of 45%; desorption of 26% with 0,5M GlcNAc), WGA+ (28%, for sialidase- treated enzyme) Carbohydr.: Sia Sialidase+ [1130]

3.2.1.33. Amylo-1,6-alpha-glucosidase Human fibroblasts in culture Cell enzyme ConA-agarose ConA+, ConA- (in the presence of 3mM MM; separation from the bound glucosidase form) [849] 3.2.1.35. Hyaluronoglucosaminidase

Viruses Temperate bacteriophage of group-A, type-49-streptococci (Streptococcus pyogenes)

Hyaluronidase 71kDa DEAEC—CMC**—LCA-Sepharose LCA+ (desorption with 0.2M MM; 71%, 1,1-f) Carbohydr.: 7.2% (Glc, Gal, GlcN) [1132]

Invertebrates Apis mellifera (honeybee lyophilized venom)

Hyaluronidase ConA-Sepharose ConA+ (complete binding; desorption with 0,5M MM) [1133] Mammals Bovine seminal plasma

Hyaluronodase aggregated forms DEAEC—SephadexG200—ConA- ConA+ (desorption with Sepharose4B** 0,2-0,3M MG using gradient 0-0,6M MG; separation from beta-glucuronidase and beta-N- acetyl-D-glucosaminidase) [1134] ConA-Sepharose**—Heparin- ConA+ (desorption with Sepharose—SephadexG200—Sephacryl- gradient 0-0,5M MM;80%, S200 7,5-f) [1135]

Bovine testis Crude hyaluronidase Type I (Sigma, USA) ConA-Sepharose6B (16mg ConA/ml gel) ConA+ (washing with 0,5M MG; desorption with 0,5M MG in the presence of 1M NaCl; 41%, 24-f) [1136] Human serum Hyaluronoglucosaminidase associated with hyaluronoglucuronidase (spreading factor) Glass-bead-column**—ConA- ConA+ (desorption with 50 Sepharose—DEAE-agarose—Heparin- mM Man; 67%, 2,5-f) [1137] agarose

Monkey testis (rhesus monkey, Macaca mulata) Hyaluronidase 62kDa, SU 15kDa, solubilized with TX100 SephadexG200—ConA-Sepharose ConA+ (complete binding; desorption with 0,25M MG using gradient 0-0,6M MG; 35%, 4,7-f, or 106%, 1,6-f) [1138,1139]

Rat liver Lysosomal hyaluronidase solubilized with TX100 ConA-agarose ConA+(desorption with sugar) [1126] 3.2.1.37. Xylan 1,4-beta-xylosidase

Bacteria Bacillus sp.(mesophilic strain, CF) beta-Xylanase 14,7kDa(SDS-PAGE), pI 6,2 PAS+ ConA-Sepharose4B ConA+(desorption with 0.1M MM;12%,4.1-f) Carbohydr.: 20% neutral hexoses [856]

Lower plants Cryptococcus albidus CCY 17-4-1 (yeast cell walls) beta-Xylosidase ConA-Sepharose ConA- (separation from the bound endo-1,4-beta-xylanase) [886] Emericella nidulans (fungal CF)

54 beta-Xylosidase 240kDa(gel-foltration), or 116kDa(SDS-PAGE), pI 3,25 BiogelP100**—DEAE-Sephadex—IEF Carbohydr.: 4% hexoses [1140]

Puccinia graminis f.sp.tritici Eriks and E.Henn, fungal race of inoculated leaves of wheat, Triticum aestivum L. beta-Xylosidase 6 forms pI 4-8, including 5 forms associated with arabinosidase activity 2-Dimential-PAGE—Blot—ConA ConA+ (all forms) [102]

Trichoderma viride (fungal CF) beta-Xylosidase 101kDa(SephadexG200), or 102kDa(SDS-PAGE), pI 4,45 DEAE-Sephadex—Column- electrophoresis—IEF Carbohydr.: 4,5% hexoses [1141]

Higher plants Triticum aestivum L. cv.Little Club (normal leaves,or leaves inoculated with the rust fungus, Puccinia graminis) beta-Xylosidase 6 forms pI 4-8, including 5 forms associated with arabinosidase activity 2-Dimential-PAGE—Blot—ConA ConA+ (all forms) [102]

Vigna radiata L.(Phaseolus aureus L.) (mung bean, mature seeds) beta-Xylosidase 42kDa SephacrylS200—ConA-Sepharose ConA+ (binding of 90%; desorption with 40mM MG using gradient 0-0,3M MG; separation from alpha- and beta-glycosidases) [955] Mammal Human liver Acid beta-xylosidase Lectin-Sepharose4B ConA+ (binding of 68%; low desorption), GSA-I+ (57%; desorption of 12% with 1M Gal), RCA-I+ (14%; without desorption), SBA-, UEA-I+ (12%), WGA+ (61%;desorption of 35% with 1M GlcNAc) [707] Neutral beta-xylosidase Lectin-Sepharose4B ConA-, GSA-I+(7%; without desorption), RCA-I-, SBA-, UEA-I+ (65%;desorption of 6% with Fuc), WGA+(24%; without desorption) [707] 3.2.1.38. beta-Fucosidase

Invertebrates Euphausia superba L. (antarctic krill) beta-Fucosidase associated with glucosidase and galactosidase, forms I and II DEAEC**—ConA-Sepharose4B— ConA+ (for I; separation UltrogelAcA34—PreparPAGE from II), ConA- (15%, for II) [340] Littorina littorea L. (perwinkle) beta-Fucosidase associated with glucosidase and galactosidase activities SephadexG200—DEAEC**—GalN- agarose—ConA-Sepharose ConA+ (desorption with 0,5M Man; 37%, 2,8-f) [956] 3.2.1.41. alpha-Dextrin endo-1,6-alpha -glucosidase

Bacteria Clostridium thermohydrosulfuricum Z-21-109 Intracellular highly thermostable novel pullulanase 133kDa(Sephacryl- S200), or 140kDa(SDS-PAGE), pI 5,9, PAS+ solubilized with Candida cylindracea lipase DEAE-Sephacel—Octyl-Sepharose— Pullulan-Sepharose** [1142]

Clostridium thermohydrosulfuricum E-101-69 Pullulanase associated with PAS+ alpha-amylase, forms I (370kDa, SU 190kDa, pI 4,25), and II (330kDa, SU 180kDa, pI 4,25) Carbohydr.: 12 or 11% hexoses, for I or II, respectively [813]

3.2.1.45. Glucosylceramidase

Mammals Bovine brain Glucosylceramidase 138kDa (BiogelP200), SU 63+56kDa Decyl-agarose**—Octyl-Sepharose Enzyme—Lectin-peroxidase ConA+, DBA+, RCA-I-, SBA-, UEA-I+,WGA+ [1143] Human monoblast cell line U-937 Glucosylceramidase Asn-glycans of complex type important for efficient compartmentalization of enzyme into lysosomes [1144]

Human placenta

Lysosomal glucosylceramidase solubilized with taurocholate ConA-Sepharose—Phosphatidyl-Ser- ConA+ (desorption with

56 agarose** 0,1M MM; 80%, 7,7-f) [1145]

Lysosomal monomeric glucosylceramidase 67kDa Asn-glycans sialilated poliantennary of complex type [1146]

Human spleen Lysosomal acid glucocerebrosidase with SU 63+61,5+56kDa, Ab-precitated forms pI 5,5-6,5, and Ab-nonprecipit- ated form with SU 63kDa, pI 4,9 , solubilized with TX100 ConA-Sepharose4B ConA+ (desorption with 1M MG) [961] (3.2.1.45.) Protein activators of glucosylceramidase Mammals Guinea pig liver Lysosomal acid heat-stable activator with SU 6,5+8,5+10kDa (SDS-PAGE) DEAE-Sephacel—SephadexG75—ConA- ConA+ (separation of 3 Sepharose—Reverse-phase-C4-column forms) Carbohydr.: 11% (250 GlcN, 205 Man, PNGase+ ([6,5+8,5+10]—4kDa; 145 Gal, 40 Fuc, nM/mg protein); elimination of >90% carbohydr.) Asn-glycans: 2,5kDa in 6,5kDa, 4,5kDa In 8,5kDa, 6kDa in 10kDa [1147]

Human spleen (normal and of patients with Gaucher disease) Lysosomal activator 2 forms (PAGE) DEAE-Sephacel—DEAE-Sephacel—HA —Decyl-agarose—ConA-Sepharose ConA+ (102%,2-f, for normal form; 130%,2-f, for Gaucher disease enzyme) [1148] 3.2.1.46. Galactosylceramidase

Human liver Lactosylceramidase-I Lectin-Sepharose4B ConA+ (binding of 90%; desorption of 42% with 1M MM), GSA-I+ (96%; desorption of 31% with 1M Gal), RCA-I+ (8%; desorption of 7% with 1M Gal), SBA+ (13%;desorption of 9% with 1M GalNAc), UEA-I+ (46%; desorption of 15% with Fuc), WGA+ (88%; desorption of 22% with 1M GlcNAc) [707] Lactosylceramidase-II Lectin-Sepharose4B ConA+ (complete binding; desorption of 93% with 1M MM; 34-54-f), GSA-I+ (90%; desorption of 54% with 1M Gal), RCA-I+ (20%; desorption

of 6% with 1M Gal), SBA+ (68%; low desorption), UEA-I+ (8%), WGA+ (96%; desorption of 76% with 1M GlcNAc; 75-f) [707] Human lymphocytes Galactosylcerebrosidase 90kDa (SDS- PAGE,gel-filtration) Octyl-Sepharose—ConA-Sepharose**— ConA+ (desorption with 0,35 Phenyl-Sepharose—Phenyl-Sepharose— M MM using gradient 0-0,8M TSKgel-G-3000SW*—Protein-Pack-G- MM; 138%,12-f) Phenyl* [1149]

3.2.1.47. Galactoslgalactosylglucosyl- ceramidase Human placenta Ceramide trihexosidase ConA-Sepharose—Cellex-D—SP- ConA+ (desorption with 0.2 Sephadex—UltrogelAcA34—HA— M MM) Butyl-agarose Carbohydr.: Sia [1150]

3.2.1.48. Sucrose alpha-glucosidase

Mammals Human small intestinal cells in culture Sucrase-isomaltase mature form 245kDa (SU 145+130kDa) (I), form 212kDa with completely glycosylated core (II), and intermediate form 210kDa with Asn-glycans of oligoMan-type (III) Lectin-Sepharose HPA+ (for I, II, both SU of I; desorption with 10mM GalNAc), HPA- (for III), LCA+ (desorption with 50mM MM) [892] Rabbit small intestinal brush-border Mb Sucrase solubilized with TX100 ConA-Sepharose ConA+ (complete binding) [1151] Rat intestinal brush-border Mb Sucrase-isomaltase complex solubilized with papain SephadexG200 Binding to HTL-125I HTL+ [893]

Rat small proximal and distal intestine Sucrose alpha-glucosidase Lectin-Sepharose RCA-I+ (binding of 38 or 64%, for proximal or distal enzyme, respectively), WGA+ (55 or 28%) [530] 3.2.1.49. alpha-N-Acetylgalactosaminidase Porcine liver Dimeric enzyme 2 forms with SU 52kDa

DEAEC—ConA-Sepharose** ConA+ (desorption with 0,5M MM; 45%, 23-f) [1152] 3.2.1.50. alpha-N-Acetylglucosaminidase Mammals Human liver Acid enzyme Lectin-Sepharose4B ConA+ (binding of 75%; desorption of 62% with 1M MM), GSA-I+ (30%; low desorption) RCA-I-, SBA-, UEA-I-, WGA+ (69%;complete desorption with 1M GlcNAc) [707] Human urine Lysosomal-type enzyme 2 forms (with high or low clearance) identical in mol. mass, pI 3,3-6,0 ConA-Sepharose ConA+ (complete binding of both forms) [1153] 3.2.1.51. alpha- L-Fucosidase

Lower plants Fusarium oxysporum S-252 (fungal CF) alpha-L-Fucosidase 80kDa(SephadexG200) DEAE-Sephadex—HA—SephadexG150 —ConA-Sepharose4B** ConA+ (desorption with 50 mM MM; 50%, 9,0-f) [1154] Higher plant Vigna radiata L.(Phaseolus aureus) (mung bean, mature seeds) alpha-L-Fucosidase 90kDa SephacrylS200—ConA-Sepharose ConA+ (desorption of 95% with 40mM MG using sugar gradient; separation from alpha- and beta-glycosidases) [955] Mammals Hamster cells CHO (CF) alpha-L-Fucosidase 51kDa(SDS-PAGE) DEAE-Sepharose —L-FucN-agarose —ConA-Sepharose ConA+ (binding of 99%; desorption with 0,5M MM)

SDS-PAGE—Blot—ConA ConA+ [1155]

Human kidney lysosomes alpha-L-Fucosidase ConA-Sepharose—L-FucN-Sepharose— ConA+ (desorption with 0,75 UltrogelAcA34—SephacrylS200— M MM at pH 4,8) DEAE-Sepharose [1156]

Human liver Acid alpha-L-fucosidase forms including tetramer with SU 50kDa Lectin-Sepharose4B ConA+ (binding of 83%; desorption of 72% with 1M MM), GSA-I+ (15%;desorption of 8% with 1M Gal), RCA-I-,

SBA+ (34%;desorption of 26% with 1M GalNAc), UEA-I-, WGA+ (90%;desorption of 62% with 1M GlcNAc) [707]

ConA-Sepharose—FucN-agarose ConA+ (desorption with sugar) [1157] Carbohydr.: 3.7%; Asn-glycans of oligoMan-type (Man5-8) and complex LacNAc-type (3:1, M/M); biantennary sialylated of complex type [1157]

Human liver of patients with cystic fibrosis Purified alpha-L-fucosidase Binding to ConA ConA+, ConA- (in the presence of 33 or 13mM MM, for normal or abnormal enzyme, respectively) [1158] alpha-L-Fucosidase of extract Binding to ConA (8 cases) ConA- (in the presence of 13,2+3,4, or 5,6+0,4mM MM, for normal or abnormal enzyme, respectively) [1158] Human lymphoblastoid cells transformed with Epstein-Barr virus, from periferal lymphocytes of normal individualsand patients with I-cell disease alphaL-Fucosidase RCA-I-agarose RCA-I+ (binding of 50 or 80%,for normal or I-cell disease enzyme, respectively) [1043] Monkey brain alpha-L-Fucosidase ConA-Sepharose—Phosphomannan- ConA+ (desorption with MG Sepharose, ConA-Sepharose—RCA-I- or MM), RCA-I- (ConA+ RCA-I- Sepharose form) [742,1046]

Porcine kidney alpha-L-Fucosidase ConA-Sepharose ConA+ (complete binding; desorption of 38-50% with 0,5M MM; effectivity of MM >MG) [1159] 3.2.1.52. beta-N-Acetylhexosaminidase

Bacteria Aeromonas hydrophila subsp. anaerogenes A-52 (CF) Chitobiase 105kDa, pI 5,35 PAS+ CMC**—DEAE-Sephadex [899]

Mycobacterium leprae beta-D-2-Acetamido-2-deoxyglucosidase ConA-agarose (GlycosylexA, Sigma,USA) ConA+ (binding of 40%; desorption with MM)

[1119] Lower plants Aspergillus nidulans (Eidem) Wint (CECT- 2544) (fungal CF) beta-N-Acetylglucosaminidase 190kDa, pI 4,3 SephadexG200**—PAGE Carbohydr.: 19% (12,2% Man, 3,5% Xyl, 0,5% Glc, and 0,8% GlcN) [1160]

Aspergillus niger beta-N-Acetylglucosaminidase 149kDa, pI 4,4 DEAEC—HA—SephadexG150**—SP- Sephadex Carbohydr.: 12,6% (7,0% Man, 1,2% Gal, 0,13% Fuc, and 1,9% Glc) [1161]

Aspergillus oryzae beta-N-Acetylglucosaminidase DEAEC—ConA-Sepharose beta-N-Acetylglucosaminidase 140kDa, ConA+,(desorption with from Taka-diastase (Sankyo Co., Japan) 0,2M MM; separation from - unbound endo-beta-N-acetyl- Carbohydr.: 3.8% hexoses, 0.9% GlcN glucosaminiodase [966]

Botrytis cinerea (fungus pathogenic for grape, CF) beta-N-Acetylglucosaminidase 70kDa Affinity to ConA ConA+ (strong binding, immobilization) [1162] Cephalosporium acremonium 237 (mold CF) beta-N-Acetylglucosaminidase 240kDa, pI 4,96 DEAEC—Lectin-agarose ConA+ (desorption with 0,1 M maltose followed by 0,2 M MM), LCA+ (desorption with 30mM followed by 0,4M MG), PHA-, WGA+ (slight retardation of elution without sugar; activation of enzyme) [968] Geotrichum candidum (fungal CF) Partially purified beta-N-acetylglucosaminidase ConA-Co3+-Spheron ConA+ (10-f) [786]

Gliocladium virens RV14 (fungal CF) beta-N-acetylhexosaminidase forms I (70kDa, SDS-PAGE) and II (70+68+60kDa) Chromatofoc—DEAE-MemSep*(**)— CM-MemSep* Asn-glycans: 9kDa in 68kDa,5kDa in EndoH+ or 70kDa or 60kDa PNGaseF+ (70—

67kDa; [70+68+60]— [65+59+55] kDa) [1163] Mucor fragilis IFO 6449 (fungal CF) beta-N-Acetylhexosaminidase 125kDa, SU 70kDa DEAEC—HA**—SephadexG150 —ConA- ConA+ (desorption with 0,5 Sepharose4B M MM; 41%, 5,8-f) [1164] Penicillium oxalicum IFO 5748 (fungal CF) beta-N-Acetylhexosaminidase 142kDa, SU 68kDa DEAEC—HA**—SephadexG15—ConA- ConA+ (desorption with Sepharose4B gradient 0-0,5M MM; 60%, 1,1-f) Carbohydr.: 9,1% hexoses, 1,0% aminosugars [1165]

Higher plants Acer pseudoplatanus L. (sycamore cell protoplasts in culture) beta-N-Acetylglucosaminidase ConA-Ultrogel ConA+ (desorption of 63% with MG; separation from mannosidase) [1024] Artemisia annua L.(sweet wormwood) beta-N-Acetylglucosaminidase forms CMC—ToyopearlHW55—Lectin-agarose ConA+ (separation from beta-galactosidase), LCA+ (retardation of elurion; separation from beta-glucosidase), HPA-, RCA-I-, WGA- [952] Precipitation with lectins ConA+ or PSA+(separation of forms) [952] Activation and/or stabilization with lectins PNA+, SBA+, WGA+ [952]

Ficus glabrata L. (ficus, latex) beta-N-Acetylhexosaminidase ConA-Sepharose ConA+ (desorption with gradient 50-500mM MM; 70%, 110-f) [1166] Glycine max L. (soybean, seeds) beta-N-Acetylhexosaminidase 133kDa Phenyl-Sepharose—DEAEC—ConA- ConA+ (binding at pH 5,0; Sepharose —Superose6(*)**, desorption with 0,5M SBA-Sepharose NaCl), ConA- (at pH 8), SBA- (pH 5-8) [1167] Gossypium hirsutum L. Im-216 (cotton hypocotyles of 2-day seedlings) beta-N-Acetylhexosaminidase 125kDa (SephadexG200) CM-Sephadex**—DEAE-Sephadex—Sephadex- G200—SephadexG200—ConA-Sepharose ConA+ (desorption with 0,1 M MM; 73%, 2,2-f [1168] Lupinus luteus L.(lupine seeds and

62 nodules) beta-N-Acetylhexosaminidase forms A (66kDa, SDS-PAGE), and B1 with SU 66kDa, from seeds ConA-Affigel—DEAE-Sepharose ConA+ (for both forms; desorption with 0,5M MM) Carbohydr.: 6,8 or 12,5% hexoses, for A or B1, respectively [1169] Other multiple forms from seeds and nodules, 4 types of SU ConA-Affigel—SDS-PAGE ConA+ (for 4 or 2 types of SU, for seed or nodule enzyme, respectively) [795] Malus domestica Borhk cv.Golden delicious (apple at climacteric stage of ripeness) beta-N-Acetylglucosaminidase 236kDa (Superose12*), SU 29kDa (SDS-PAGE) MonoQ(*)**—ConA-Sepharose4B ConA+ (washing with 3mM MM; desorption with 0,3M MM; 20%, 4,3-f; separation from the bound alpha-mannosidase) [1030] Pisum sativum L. cv.Burpeeana (pea mature dry seed protein bodies, wet and germinating seeds) beta-N-Acetylhexosaminidase 210kDa, forms I and II ConA-Sepharose**—Zn2+-chelating- ConA+ (for both forms; agarose—Leu-Sepharose(I+II)—p-Amino- 238-, 250-, or 137-f, for phenyl-thio- -GlcNAc-Affigel dry, wet, or germinating seeds, respectively) [1170] Pisum sativum L.(pea seedling cotyledons) beta-N-Acetylglucosaminidase ConA-Sepharose**(instead of DEAEC)— ConA+ (complete binding; SephacrylS200 desorption with 0,3M MG; 23%, 70-f) [1031] Prunus serotina (L.) Ehrh.(black cherry mature seeds) beta-N-Acetylhexosaminidase SephadexG25—DEAEC—ConA-Sepharose** ConA+(desorption with —GlcNAc-agarose gradient 0-25mM MG; 73%, 19-f; partial separation from amigdalin hydrolase with higher affinity to ConA) [1171] Trigonella foenum-graecum L.(fenugreek germinating seeds) beta-N-Acetylhexosaminidase forms I (84kDa, SU 28kDa, pI 6,78), II (72kDa, SU 24kDa, pI6,3), III (180kDa, SU 30kDa, pI 4,9), and IV (150kDa,SU 30kDa, pI 4,65) ConA-Sepharose4B ConA+ (for all forms; desorption with sugar; 3-10-f, for I, II, or IV)

Carbohydr.(M/M): 4,5 Man, 2 GlcNAc, 0,8 Fuc, (I); 6,2 Man, 2 GlcNAc, 1,1 Fuc (II); 9,1 Man, 2 GlcNAc, 1,1 Fuc (III); 8,0 Man, 2 GlcNAc, 0,8 Fuc (IV) [1172]

Triticum aestivum L.(wheat leaves of 8-day seedlings) beta-N-Acetylhexosaminidase major forms pI 5,13+5,18, and minor forms pI 5,23+ 5,31+5,36 TSKgel-G-2000SW(*)**—TSK-535-CM —ConA-Sepharose ConA+ (desorption with 0,4M MM; 69%, 4,1- [1173] Triticum aestivum L.(wheat resting seed aleurone layers) beta-N-Acetylglucosaminidase 88kDa, pI 4,5 WGA-Sepharose**—DEAEC—Sephadex- WGA+ (desorption with G150—PAGE GlcNAc; 21%, 34-f) [1174]

Affinity to WGA-125I WGA+ [1054]

Protozoans Tetrahymena pyriformis HSM (CF) beta-N-Acetylhexosaminidase forms A (170 kDa),and B (93kDa) SephadexG75—DEAEC—CMC—ConA- ConA+ (complete binding Sepharose of A; desorption with MM; partial separation from B) [1175] Invertebrates Allolobophora caliginosa (annelid) beta-N-Acetylhexosaminidase forms A and B ConA-Sepharose**—Chromatofoc ConA+ (desorption with gradient 50-500mM MM; 63%, 11-f) [1176]

SephadexG200—ConA-Sepharose**— ConA+ (90%, 8,9-f) Chromatofoc [1177]

Arion rufus L.(slug digestive gland) beta-N-AcetylhexosaminidaseA 150kDa, pI 4,5 SephacrylS200**—DEAEC—ConA- ConA+ (desorption of up Sepharose—GlcNAc-Sepharose to 75% with 1M MG; 67%, 3,7-f; MG better than MM) [1178] beta-N-AcetylhexosaminidaseB (320kDa, pI 5,5) SephacrylS200**—DEAEC— - ConA+ (desorption with Sepharose 1M MM; 71%, 1,4-f) [1178]

Artemia nauplii (brine shrimp) beta-N-Acetylhexosaminidase forms I (83kDa, pI 5,4), II-1 (110kDa, pI 5,9), and II-2 (56kDa, pI 5,9) DEAE-SepharoseCL6B**(I+II)—Gel- filtration(II-1+II-2)—ConA-Sepharose ConA+ (desorption with

0,4M MM; 48%, 3,2-f, for I; 41%, 4,5-f, for II-1; 71%, 6,0-f, for II-2) [1179] Dugesia lugubris L. (planaria, normal and regenerating) beta-N-Acetylhexosaminidase ConA-Sepharose ConA+ (desorption with 0,5M MM; 63%, 8,0-f, or 26%, 6,0f, for normal or regenerating enzyme, respectively) [1180] Hirudo medicinalis L. (annelid) beta-N-Acetylhexosaminidase SephadexG200—ConA-Sepharose**— ConA+ (80%, 6,4-f) Chromatofoc [1177]

Paracentrotus lividus (sea urchin spermatozoa) Mb-bound beta-N-acetylglucosaminidase 160kDa solubilized with nonionic detergent Gel-filtration-sorbent—Ion-exchange- sorbent—ConA-Sepharose ConA+ [1181]

Spirographis spallanzanii (annelid) beta-N-Acetylhexosaminidase SephadexG200**—ConA-Sepharose— ConA+ (82%, 4,5-f) Chromatofoc [1177]

Trichinella spiralis L. (parasitical nematode, muscle-stage larvae) beta-N-Acetylhexosaminidase 100kDa (SDS-PAGE) DEAEC(filtration)—LCA-Sepharose4B— LCA+ (desorption with SephacrylS200 0,5M MM; 73%, 10-f)

SDS-PAGE—Blot—ConA-biotin ConA+ [1182]

Turbo cornutus L. (gastropod) beta-N-Acetylhexosaminidase 100kDa, forms A and B DEAE-Sephadex—CMC—ConA-Sepharose4B ConA+(desorrption with —SephadexG200 0,1M MM; 31%, 3.1-f, for A; 56%, 3,4-f, for B) [1183] Fishes Cyprinus carpi (carp erythrocytes) Neutral beta-N-acetylglucosaminidase SephadexG200—DEAE-Sepharose ConA- (separation from the bound acid enzyme) [1184] Birds Hen brain Acid beta-N-acetylhexosaminidase forms I, IIa, IIb, and neutral enzyme III DEAEC—ConA-Sepharose ConA+ (I, IIa, IIb; desorption with 1M MG), ConA- (III), ConA+ (partial binding of

Sialidase-treated III) Carbohydr.(III): Sia Sialidase+ [1185]

Hen embrional lung and skin (1-,7-,9-, 11-,and 14-day embryos) Acid beta-N-acetylhexosaminidase forms I (maximum at 14-day),and II (maximum at 9-day), and neutral enzyme III (maximum at 14-day) DEAEC(I+II+III)—ConA-Sepharose ConA+ (complete binding of I, II; desorption with MM), ConA- (III) [1186] Reptiles Dasypus sp. (armadillo liver) beta-N-Acetylglucosaminidase ConA-agarose (Glycosylex-A,Sigma,USA) ConA+ (binding of 80%; desorption with MM) [1119] Mammals Baboon kidney (Papio ursinus) beta-N-Acetylglucosaminidase 82kDa(PAGE), or 52kDa(SDS-PAGE),or 89kDa(SDS-PAGE, for reduced protein), forms A1(pI 4,97), A2 and B ConA-Sepharose**—DEAE-Trsacryl—HA ConA+ (desorption of all —PreparPAGE forms with 0,3M MM; 66%, 32-f) Carbohydr.: 31% hexoses, 6% Sia (A1); 17% hexoses, 1% Sia (A2) [1187]

Bovine brain Lysosomal acid beta-N-acetylglucosaminidase forms A, B, and I, SU 50+53kDa ConA-Sepharose—GalNAc-Sepharose**— ConA+ (desorption of all forms DEAE-Sephacel—SP-Sephadex(for B) with 0,5M MM; 69%, 19-f)

Carbohydr. (mkg sugar/100 mg protein): 2,70 Man, 1,24 Gal, 2,06 GlcNAc, 0,18 NeuAc (A); 1,72 Man, 0,82 Gal, 1,33 GlcNAc, 0,18 NeuAc (B); 3,41 Man, 1,25 Gal, 2,47 GlcNAc, 0,11 NeuAc (I) [1188]

Bovine mammary gland beta-N-Acetylglucosaminidase forms A (118kDa, SU 55+25kDa), and B (234kDa, SU 55+25kDa) DEAEC—ConA-Sepharose**—SephadexG200 ConA+ (desorption with —IEF 1M MG; 91%, 4,8-f, for A; 92%, 7,6-f, for B) [1189] Bovine testis beta-N-Acetylhexosaminidase 61-62kDa ConA-Sepharose**—DEAEC ConA+ (desorption with 0,1M MM in the presence or 50% ethyleneglycol; 47%, 9,2-f) [1190] Human brain and thymus Acid beta -N-acetylhexosaminidase forms A (115kDa), and B

ConA-Sepharose ConA+ (for both forms; desorption with 0,2-0,4M MG or 0,5M MM) [1191,1192] Neutral beta-N-acetylhexosaminidaseC ConA-Sepharose ConA- (separation from the bound A and B) Carbohydr.(for A): 27% hexoses (mainly Man,Glc,and Gal), 2,6% hexosamine, Sia [1191]

Human colon beta-N-Acetylhexosaminidase ConA-Sepharose4B ConA+ (desorption with 0,5M MM; 56%, 10-f) [1180] Human colon tumor beta-N-Acetylhexosaminidase ConA-Sepharose ConA+ (desorption with 0,5M MM; 73%, 6,8-f) [1180] Human cordial muscle Lysosomal acid beta-N-acetylglucosaminidase forms A (110kDa) and B (130kDa) Blue-Sepharose—ConA-Sepharose**— ConA+(desorption with GalNAc-Sepharose—DEAE-Sephadex 0,5M MG; 65%, 15-f) [1193] Human cultural fibroblasts of patients with I-cell disease (CF and cells) Extracellular (I) and lysosomal acid (II) beta-N-acetylglucosaminidase forms RCA-I-Sepharose RCA-I+ (binding of mainly I than II) [1194] Human kidney cortex beta-N-Acetylhexosaminidase 100kDa (ultracentrifugion), forms A (SU 18+40kDa, pI5,4), and B (SU 18kDa, pI 7,5) For A: DEAEC—SephadexG200—Ecteola- Cellulose; For B: DEAEC—CMC—SephadexG200; ConA-Sepharose ConA+ (for both forms; desorption with 0,1M MM; 60%, 13-f) [1195] Human liver beta-N-Acetylglucosaminidase in complex with endogenic hepatic Mb-bound Man-6-P- specific receptor lectin Lectin+ (desorption of 50% using 10mM Man-6-P) [1196] Human liver of patients with I-cell disease beta-N-Acetylglucosaminidase in complex with endogenic hepatic Mb-bound lectin(s) Lectin(s)+(dissociation of complex in the presence of Fuc, Man, or GlcNAc) [1196] Human placenta Lysosomal beta-N-acetylhexosaminidase forms A (119kDa), and B (112kDa) For A: DEAEC—QAE-Sephadex—Sephadex- G200—QAE-Sephadex—PreparPAGE—QAE-

Sephadex For B: DEAEC—HA—SephadexG200— QAE-Sephadex—CMC; ConA-Sepharose—ConA-Sepharose(for ConA+ (for A and B; ConA--form) desorption with 0,5M MM; 57%, 63-f) [1197] beta-N-AcetylhexosaminidaseA ConA-Sepharose**—SephadexG200— ConA+ (desorption with DEAEC—IEF—Ecteola-Cellulose 0,1M MM; 56%, 16-f) [1198]

ConA-Sepharose—DEAEC—SephacrylS200 ConA+ (desorption with —CMC sugar) [1199]

Carbohydr.: 2,5% hexoses, 2,5% GlcNAc (for A); 5,4% hexoses, 3,4% GlcNAc (for B) [1197] beta-N-Acetylhexosaminidase-I 29kDa(SDS-PAGE) ConA-Sepharose—Ab(IgG)-agarose— ConA+ SephadexG200—DEAEC Carbohydr.: Sia; Sialidase+ Asn-glycans of oligoMan-type EndoH+ [1200]

Acid beta-acetylhexosaminidase tetrameric forms A (beta2 beta2), B (alpha2 beta2), S (alpha2 alpha2), I-1 and I-2; 25kDa ConA-Sepharose**—GlcNAc-Sephacryl- ConA+ (for all forms; S200—DEAE-SepharoseCL6B(separation of desorption with sugar; forms) 75%, 45-f) [1201] IEF—Lectin-125I Lectin- (for alpha-chain), ConA+, RCA-I+, or WGA+ (for beta-chain) [1202] Mixture of Gp from trypsin- and chymo- trypsin-treated beta-chain Reverse-phase-sorbent*—ConA-Sepharose ConA+ (separation of Gp) [1202,1203] Asn-glycans: Man3GlcNAc2 - b- chain); Man5-7GlcNAc2 b-chain) [1203]

Lysosomal acid beta-N-acetylglucosaminidase solubilized with Triton WR1339 ConA-Sepharose ConA+ (desorption with 1M MM; 80%, 119-f) [960] Human saliva during a day beta-N-Acetylhexosaminidase ConA-Sepharose ConA+(varying during a day) [1204] Human seminal plasma beta-N-AcetylhexosaminidaseA 195kDa, SU 50kDa DEAEC—ConA-Sepharose**—Sepharose6B ConA+ (desorption with —SephadexG200 0,5M MM; 66%, 8,6-f) [1205] beta-N-Acetylhexosaminidase-B 210kDa, SU 50kDa DEAEC—ConA-Sepharose**—CMC—

Sepharose6B—SephadexG200 ConA+ (desorption with 0,5M MM; 89%, 13-f) [1205] Human skin fibroblasts, acute leukaemic lymphoblasts CCRF/CEM, and nonmalignant lymphoblasts SM1

Acid beta-N-acetylhexosaminidase mature and precursor forms Lectin-Sepharose, Serotonin-Sepharose ConA+ (without differences (as Sia-binding), in combination with between fibroblast and glycosidase-treatments lymphoblast enzyme), RCA-I-, Serotonin+ (increased binding of hypersialated enzyme of cells CCRF/CEM) [1206] Enzyme Gp Lectin-agarose (in combination with ConA+, RCA-I+, WGA+ glycosidase-treatments) (separation of Gp) Carbohydr.: Sia; Sialidase+ Asn-glycans EndoF+ [1207]

Human spleen of patients with hairy-cell leukaemia beta-N-Acetylhexosaminidase forms A (pI around 5,0), B (pI >5,0), and malignant form with intermediate pI (15% total activity) ConA-Sepharose**—Phenyl-boronate- ConA+ (all forms; agarose(Chromatofoc) desorption with gradient 0-0,5M MM; 58%, 40-f) [1208] Human tissue fibroblasts of healthy adults, and lymphoblasts of patients with I-cell disease Acid beta-N-acetylhexosaminidase RCA-I-agarose RCA-I+ (binding of 20-65% or 69-95%, for fibroblast or lymphoblast enzyme, respectively) [1043,1209] Human tissue fibroblasts (beta-galactosidase- deficient) in culture (CF, lysosomes) Extracellular acid beta-N-acetylhexosaminidase RCA-I-agarose RCA-I+ (binding of 19 or 44%, for native, or Sialidase-treated enzyme, respectively; desorption with 0,1M Gal) Carbohydr.: Sia Sialidase+ [1124] Lysosomal acid beta-N-acetylhexosaminidase RCA-I-agarose RCA-I+ (binding of 38 or 27%, for native or Sialidase-treated enzyme, respectively; desorption with 0,1M Gal) Carbohydr.: Sia Sialidase+ [1124]

Human urine beta-N-AcetylhexosaminidaseA PAS+

DEAE-Sephadex—DEAE-Sephadex—ConA- ConA+ (desorption with -Sephadex; 0,5M MM; 63%, 14-f), RCA-I-agarose RCA-I+ (binding of 25%) ConA-precipitation ConA+ (83%) Carbohydr.: Man and Gal (3,5:1,0, M/M) [1210,1211]

Human urine of patients with mucolipidosis-III beta-N-AcetylhexosaminidaseA RCA-I-agarose RCA-I+ (binding of 71%) ConA-precipitation ConA+ (65 or 50%, for native or EndoH-treated enzyme, respectively) Carbohydr.: Man and Gal (2,1:1,0, M/M); Asn-glycans of oligoMan-type EndoH+ [1211]

Monkey brain (bonnet monkey, Macaca radiata L.) beta-N-Acetylhexosaminidase ConA-Sepharose—Phosphomannan-Sepharose; ConA+ (desorption with MG ConA-Sepharose—RCA-I-Sepharose or MM), RCA-I- (>95%, ConA+RCA-I- form) [742,1046] beta-N-AcetylhexosaminidaseA 100kDa SephadexG200—ConA-Sepharose**— ConA+ (desorption with DEAE-Sephadex—CM-Sephadex 0,5M MM; 82%, 15-f) Carbohydr.: 27% hexoses (Man, Glc, Gal), 2.6% hexosamines, Sia [1191]

Monkey fibroblasts of healthy donors and cultural fibroblasts ML-II from donors with mucolipidosis-II (bonnet monkey, Macaca radiata L.) beta-N-Acetylhexosaminidase Precipitation with lectins RCA-I-(100%,for native enzyme), RCA-I+(binding of 96 or 10%, for Sialidase-treated enzyme of cells ML-II or normal fibroblasts, respectively), ConA+ (binding of up to 100%) Carbohydr.: Sia-alpha-Gal-beta- Sialidase+ [1212]

Porcine brain beta-N-AcetylhexosaminidaseA 160kDa (SephadexG200) DEAEC—IEF—ConA-Sepharose ConA+ (desorption of 68% with 1M MG) [1213] Porcine gastric mucosa beta-N-Acetylglucosaminidase forms A and B ConA-Sepharose—DEAEC(A+B) ConA+ (desorption of both forms with sugar) [1214] Rabbit seminal plasma beta-N-AcetylhexosaminidaseII HA—SephadexG200—ConA-Sepharose** ConA+ (complete binding; desorption with 0-0,5M

MM; 84%, 8,3-f) [1215] Rat brain (1- or 2-week neonatal rats) Neutral and acid beta-N-acetylhexosaminidases ConA-Sepharose—DEAEC—HA—GalNAc- ConA- (neutral enzyme), Sepharose ConA+ (acid enzyme; desorption with sugar) [1216,1217] Rat liver Lysosomal acid beta-N-acetylhexosaminidase solubilized with TX100 Lectin-agarose ConA+ (desorption with sugar), RCA-I+ (11%), Rabbit-serum-MBP+ (80%) [1050] Lysosomal acid chitobiase 43kDa (SDS-PAGE) CMC—UltrogelAcA54—CMC—ConA- ConA+ (desorption with Sepharose**—Phenyl-SPW-column* 0,2M MM; 17%, 6,0-f), ConA- (25-75%) [1218] beta-N-Acetylglucosaminidase ConA-Sepharose—LIE ConA+ (3 forms), LCA+(2 forms), PHA+or SBA+ (1 form), WGA+ (2 forms), HPA- [1128] Rat macrophagues Acid beta-N-acetylglucosaminidase Immobilized rat brain ligatin Ligatin+ (complete binding; desorption of 99% with 0,5mM Glc-1-P) [1219] Rat neonatal ileal enterocytes Acid beta-N-acetylglucosaminidase 225kDa, SU 125+115kDa Immobilized rat brain ligatin (endogenic lectin) Ligatin+ (complete binding; desorption of 90-100% with 40mM Man-6-P) [1219] Enzyme-Ligatin complex solubilized from the ileal Mb with 10mM CaCl2 [1220]

Rat spleen cell cytosol Neutral hexosaminidase (O-GlcNAc-selective beta-N-acetylglucosaminidase) 106kDa (ultracentrifugion), SU 54+51kDa DEAEC**—ConA-Sepharose4B—Phenyl- ConA- (117%, 1,4-f; Sepharose—Blue-agarose—HA— separation from acid Superose12* hexosaminidase) [1221]

Acid hexosaminidase ConA-Sepharose ConA+ (desorption with 0,5M MM; separation from neutral hexosaminidase) [1221] Rat urine Acid beta-N-acetylhexosaminidase forms A (pI 6,4+7,5), B (pI 7,6), and I (pI 7,5) DEAEC(A+B+I)—ConA-Sepharose** ConA+ (desorption with — GlcNAc-Sepharose 1M MG; 78%, 19-f) [1222] Sheep brain

Lysosomal acid beta-N-acetylhexosaminidase Precipitation with ConA ConA+ (26-32%, 20-31-f) [1223,1224] 3.2.1.53. beta-N-Acetylgalactosaminidase Acremonium sp. (fungal CF) Monomeric enzyme 55kDa DEAE-Sephadex**—HA—SephadexG150— ConA-Sepharose4B ConA+ (desorption with 5mM MM; 44%, 1,1-f) [1225] 3.2.1.55. alpha-N-Arabinofuranosidase

Bacteria Ruminococcus albus 8 (CF) Enzyme 305-310kDa, SU 75kDa PAS+ 6% carbohydr. Composition (% of total sugars): 42% Man, 32% Gal, 17% GlcN, 8% Fuc [1226]

Lower plants Aspergillus niger (fungus) alpha-L-Arabinofuranosidase 61kDa, from commercial vrude powder marketed as Hemicellulase REG-2 (Gist-Brocades, Seclin, France) UltrogelAcA44 —DEAE-SepharoseCL6B**— ConA-UltrogelAcA22 ConA+ (desorption with gradient 0-0,1M MM; 19%, 1,9-f; partial separation of 3 forms; separation from gluco sidase and rhamnosidase) [1227] Dichomitus squales (Karst) Reid CBS 432.34 (basidiomycete, CF) alpha-L-Arabinofuranosidase 60kDa, pI 5,1 Chromatofoc**—UltrogelAcA54— Chromatofoc—ConA-Ultrogel ConA+ (complete binding; desorption with 10mM MM using gradient 0-50mM MM; 46%, 1,5-f) [1228] Puccinia graminis f.sp.tritici Eriks, and E.Henn.,inoculated into wheat leaves (race of rust fungus) Soluble alpha-L-arabinofuranosidase 5 forms pI 4-8, associated with beta-xylosidase, from intercellular fluid of leaves 2-Dimentional-PAGE—Blot—ConA ConA+ (all forms) [102]

Higher plants Daucus carota L. cv.Kintoki (carrot cells in culture, CF) alpha-L-Arabinofuranosidase 110kDa (SephadexG100), or 94kDa (Na-laurylsilfate-PAGE) DEAE-Sephadex**—SephadexG150—ConA- ConA+ (desorption with Sepharose4B—CM-Sephadex—PreparPAGE 0,75M MM; separation from exopolygalacturonase) [1229]

Raphanus sativus L. var.hortensis cv.Aokubi (radish seeds) alpha-L-Arabinofuranosidase 64kDa (SDS-PAGE), or 63kDa (SephadexG150), or 57kDa(TSKgel-G-3000SW*) DEAE-Sepharose—CM-Sepharose**— SephacrylS200—HA—Octyl-Sepharose— ConA-Sepharose ConA+ (complete binding; desorption with 0,5M Glc or 0,2M MM) [1230] Triticum aestivum L. (wheat leaves inoculated with rust fungus, Puccinia graminis) Intracellular alpha-L-arabinosidase 5 forms associated with beta-xylosidase 2-Dimentional-PAGE—Blot—ConA ConA+ (all forms) [102]

Vigna radiata L.(Phaseolus aureus) (mung bean, mature seeds) alpha-L-Arabinofuranosidase 162kDa SephacrylS200—ConA-Sepharose ConA+ (binding of 96%; desorption with 0-0,3M MG; separation from other alpha- and beta-glycosidases) [955] Mammals Human liver alpha-L-Arabinofuranosidase Lectin-Sepharose4B ConA+ (binding of 88%; desorption of 66% with 1M MM; 35-54-f), GSA-I+ (78%; desorption of 20% with 1M Gal), RCA-I+ (20%), SBA+ (21%), UEA-I+ (26%;desorption of 6% with 1M Fuc), WGA+ (83%; desorption of 38% with 1M GlcNAc) [707] 3.2.1.58. Glucan 1,3-beta-glucosidase

Lower plants Candida utilis CECT 1061 (yeast cell-free extract) Exo-beta-glucanase 36kDa(ultracentrifugion), or 20kDa(SephadexG100), pI 4,1 DEAE-Sephadex**—SephadexG100— SephadexG200—SephadexG50—BiogelP100 —ConA-Sepharose4B ConA- (17%) Precipitation with ConA ConA+ (beta-glucosidase- treated enzyme) Carbohydr.: 68% hexoses (mainly Glc, beta-Glucosidase+ minor Man) [1231]

Saccharomyces cerevisiae (baker's yeast CF, spores) Exo-beta-1,3-glucanase 58,8kDa (43,0kDa,

73 for deglycosylated enzyme from tunicamycin- treated cells) DEAE-Sephadex—DEAE-Biogel**—ConA- ConA+ (desorption with Sepharose 0,37M MM; 62%, 1,2-f) [1233] Precipitation with ConA ConA+ (100%, for 58,8kDa; 5-16%, for 43,0kDa) [1232-1234] Carbohydr.: approx. 20% Composition (% of total sugars): 70% Man, 25% Glc, 5% GlcN [1233]

Exo-beta-glucanase forms I (56kDa), and II (83kDa) as EXG1-gene products, from yeast strains X14 and TD28 Carbohydr.: 11% (I), 40% (II); Asn-glycans of oligoMan-type EndoH+(I and II) [885] Sporulation-specific exo-beta-glucanase 44,5-45,0kDa, pI 4,9, from spores DEAE-Biogel(filtration)—ConA- ConA- (115%, 4,8-f) Sepharose—SephacrylS200** [1235]

3.2.1.62. Glycosylceramidase Lumbricus terrestris L. (gutted earthworm) Phlorizin hydrolase BiogelA0.5m—Octyl-Sepharose—ConA- ConA- Sepharose [1236]

3.2.1.67. Galacturan 1,4- -galacturonase

Lower plants Botrytis cinerea (fungal CF) Exopolygalacturonase monomeric forms 65kDa, pI 4,9, and 70kDa, pI 3,5 Column-IEF—Ion-exchange-sorbent—ConA- ConA+ sorbent [908]

Geotrichum lactis ATCC 58590 (fungal CF) Exopolygalacturonase 53kDa(gel-filtration) SephadexG100**—DEAE-Sephadex, ConA-Sepharose ConA+ (binding of 85 or 20%, for native or EndoH-treated enzyme, respectively), ConA+ (for native enzyme in the presence of 0,1M MM) Asn-glycans of oligoMan-type EndoH+ [1237]

Higher plants Daucus carota L.(carrot cultural cells, CF) Exopolygalacturonase DEAE-Sephadex—SephadexG100—ConA- ConA- Sepharose [1229]

3.2.1.68. Isoamylase Flavobacterium sp.(bacterial CF) Enzyme from commercial liophillized CF

(British Drug Houses Ltd., Bole, England) DEAEC—Glycogen-ConA-Sepharose4B— (Glycogen-ConA)+ BiogelP60 [1238]

3.2.1.74. Glucan 1,4-beta-glucosidase

Lower plants Schizophyllum commune (white-rot fungal CF) Exo-beta-1,4-glucanase forms 59,3 and 58,2kDa ConA-Sepharose4B ConA- [872]

Streptomyces flavogriseus IAF 45-CD (ATCC 33331) (CF) Monomeric exo-beta-glucanase (avicellase) 46kDa (SDS-PAGE) DEAE-Biogel—BiogelP60—IEF—ConA- ConA- (45%) Sepharose4B [1239]

3.2.1.75. Glucan endo-1,6-beta-glucosidase Neurospora crassa IFO 6068 (fungal CF) Endo-beta-1,6-glucanase 47kDa CM-Cellulofine—ConA-Sepharose4B— ConA+(desorption with SepharoseCL6B—Glycogen-PAGE**— 0,5M MM; 65%, 4,5-f) Glycogen-PAGE [1240]

3.2.1.76. L-Iduronidase

Mammals Human liver Lysosomal acid alpha-L-iduronidase, SU 65kDa (ConA-Sepharose—Blue-agarose)**—(CM- ConA+Blue- (desorption Sepharose—Biogel-HT)—Cu2+-chelating- with 0,75M MM; 95%, Sepharose 380-f) [1241]

Human lung alpha-L-Iduronidase forms 68+70+82+85kDa DEAEC—ConA-Sepharose**—HA— ConA+ (desorption of 86% BiogelP100 total activity with 1M MM; 540-f, for 68+70kDa; 52-f, for 82+85kDa; separation from hemoglobin) [1242] Human urine alpha-L-Iduronidase forms I (87kDa), and II (68kDa) SephadexG200—Heparin-Sepharose— Lectin-Sepharose ConA+ (binding of 96 or 91%, for I or II, respectively; desorption with 0 5M MM), RCA-I+(42 or 7%) [1243] Porcine liver Lysosomal acid alpha-L-iduronidase 70kDa (SephadexG100), or 70+62kDa (SDS-PAGE) Phosphocellulose—SephacrylS200—ConA- ConA+ (desorption with Sepharose4B**—Heparin-Sepharose4B— 0,2M MM; 83%, 8,2-f) Toyopearl-HW55—SephadexG100—Cu2+- Chelating-Sepharose6B [1244]

3.2.1.78. Mannan endo-1,4-beta-mannanase Aspergillus tamarii IP 1017-10 (fungal CF) Monomeric beta-mannanase 53kDa(SDS-PAGE) HA**—DEAEC, ConA-Ultrogel ConA+ (desorption with 0,2M MM) Carbohydr.(M/M): 6 Man, 1.5 Gal, 1 GlcN [967]

3.2.1.91.Cellulose 1,4-beta-cellobiohydrolase

Lower plants Coniophora puteana (Schum ex Fr.) Karsten, strain EMPA62 (brown-rot fungus) Exocellobiohydrolase forms I (65kDa PAS+(forms I and II) [TSKgel-G-3000SW*], or 52kDa [SDS-PAGE], pI 3,6), and II (60kDa [TSKgel-G-3000SW*], or 50kDa [SDS-PAGE], pI,55) Q-Sepharose*—Superose12*—Fractogel-TSK- DEAE-650S Carbohydr.: 12,5% (I), 6% (II), Asn-glycans of oligoMan-type: 6,5kDa (I), EndoH+ 2,5kDA (II) [1245]

Sclerotium rolfsii CPC 142 (fungal CF) Monomeric beta-1,4-glucan-cellobiohydrolase 41,5kDa, pI 4,34 SephadexG75—DEAE-Sephadex—PreparPAGE; ConA-Sepharose ConA+ (complete binding; desorption with 50mM MM) Carbohydr.: 7% hexoses (Perjodate-fuchsin)+ [1246]

Trichoderma reesei VTT-D-80133 (filamentous fungus, CF) CellobiohydrolaseII 58kDa, pI 6,3 Ab-Sepharose4B—ConA-Sepharose(instead ConA+ (desorption with of SephacrylS200 or Chromatofoc) 10mM MM) Carbohydr.: 8% hexoses [1247,1248]

Cellobiohydrolase-I 71-75kDa Mixture of pronase-treated Gp ConA-Sepharose ConA+ (desorption of 34 or 66% with 5mM or 0,2M MM, respectively) [1249] Asn-glycans including Man5-9GlcNAc2; Ser/Thr-glycans [1250,1251]

3.2.1.96. Mannosyl-glycoprotein endo-beta-N- acetylglucosaminidase

Lower plants Aspergillus oryzae (fungus) Endo-beta-N-acetylglucosaminidase 31kDa (Sephadex100) DEAEC**—ConA-Sepharose—CM-Sephadex ConA-(66%, 4,4-f; partial —SephadexG100 separation from beta- galactosidase and beta-N-

acetylglucosaminidase) [966] Mucor hiemalis f.hiemalis strain 314 (fungal CF) Endo-beta-N-acetylglucosaminidase 95kDa (gel-filtration) DEAE-SepharoseCL6B**—SephadexG200— HA—TSKgel-G-HW65F*—ConA-Sepharose ConA+ (desorption with 50mM MM; 65%, 1,2-f) [1252] Streptomyces plicatus (CF) EndoH {(Saccharomyces cerevisiae invertase-Gp)- (Gp-ConA)+ (desorption ConA-SepharoseCL4B}**—Superose12* with 0 5M MM; 34-52%, 965-1,070-f) ConA-Sepharose ConA-(>90%) [1253]

Higher plants Ficus glabrata L. (fig latex) Endo-beta-N-acetylglucosaminidase forms I (52kDa, gel-filtration), and II (17,5kDa, gel-filtration) For I: SephadexG100—DEAE-Sephadex— SephadexG100—CM-Sephadex For II: SephadexG100—DEAE-Sephadex— SephadexG50—ConA-Sepharose ConA- [1254]

Mammals Human kidney Endo-beta-N-acetylglucosaminidase forms I and II DEAE-Sephadex—Octyl-Sepharose—ConA- ConA+ (for II; desorption Sepharose—MonoS*—MonoS* with 0,5M MM; 50%, 4,7-f), ConA- (for I) [1255] Rat liver Cytoplasmic endo-beta-N-acetylglucosaminidase ConA-Sepharose—CM-Sephadex—BiogelP150 ConA- (87%) [1256]

3.2.1.97. Glycopeptide alpha-N-acetylgalactosaminidase Alcaligenes sp. F-1906 (bacterial CF) Endo-alpha-N-acetylgalactosaminidase 160kDa DEAE-Sephadex—SephadexG200—ConA- ConA- Sepharose [1257]

3.2.1.102-3. Endo-beta-galactosidase Human urine Enzyme SephadexG200—DEAE-Sephadex—ConA- ConA- Sepharose [1258]

3.2.1.108. Lactase Mammals Human small intestine Lactase 290-320kDa, SU 160kDa, amphiphilic (I) and hydrophilic (II) forms solubilized with TX100 followed by trypsin- or papain- treatment

SDS-PAGE—Blot—ConA-biotin ConA+ (for I and II) [1259] Human small intestinal mucosal brush- border Mb Lactase-phlorizin hydrolase 126kDa solubilized with TX100 Lectin-Sepharose—Ab-precipitation— HPA+ and LCA+ SDS-PAGE (separation of forms)

Mixture of Gp from pronase-treated enzyme Lectin-agarose (6 lectins in sequential ConA- (83% as O-glycan- chromatography) containing Gp) Carbohydr.: 8kDa in 126kDa; TFMS+ (126—118kDa) mainly Ser/Thr-glycans; Asn-glycans PNGaseF+ [1260]

Rat small intestinal brush-border Mb Lactase-phlorizin hydrolase forms 220 and 130kDa (SDS-PAGE), from adult and suckling animals SDS-PAGE—Blot—Lectin-peroxidase ConA+, LPA+, PNA+, or UEA-I+ (suckling rat enzyme), GSA-I+, SBA+, WGA+ [1261] Carbohydr.: Sia; Sialidase+ Asn- and Ser/Thr-glycans [1261-1263]

3.2.1.113. Mannosyl-oligosaccharide 1,2-alpha- mannosidase

Higher plants Vigna radiata L.(Phaseolus aureus) (mung bean, seed hypocotyls or 2-3-day seedlings) Microsomal mannosidase-I (GP) Mannan-Sepharose, ManN-Sepharose [1264]

Insects Spodoptera frugiperda (lepidopteran insect cells IPLB-SF21AE infected with baculovirus) Recombinant Golgi-Mb-bound alpha-1,2- mannosidaseI 63kDa(SDS-PAGE) solubilized wth TX100 DEAEC—Phosphocellulose—HA**, ConA-Sepharose ConA- [1265]

Mammals Bovine liver microsomes Processing neutral mannosidase-I two forms (SU 56kDa) splitting Man9GlcNAc2, solubilized with lubrol-PX followed by TX100 N-5-Carboxypentyl-1-deoxymannojirimycin- Sepharose4B**—ConA-Sepharose—DEAE- ConA- (64%, 1,1-f; Sephacel separation from the bound processing mannosidase and aryl-

mannosidase), ConA+ (<30%) [1266] Porcine liver Microsomal trimming Man9-mannosidase (mannosidase-I) as alpha-1,2-mannosidase 65kDa (SDS-PAGE) solubilized with LubrolPX and TX100 CM-Sepharose—N5-Carboxypentyl-1-deoxymannojirimycin-Sepharose4B**—ConA- ConA- (92%, 1,1-f; Sepharose—DEAE-Sephacel separation from the bound residual pNP-alpha- mannosidase activity) [1038] Rabbit liver Microsomal phospholipid-dependent alpha-1,2-mannosidase solubilized with TX100 DEAE-sorbent—HA—DEAE-sorbent—ConA- ConA+ (desorption with Sepharose** 0,75M MM; 29%, 6,0-f) [1267] Monomeric microsomal Ca2+-dependent processing alpha-1,2-mannosidase 52kDa solubilized with NP40, splitting glycans of PHA-P DEAEC—ConA-Sepharose4B**—TSKgel- ConA+ (complete binding; GHW55—Blue-agarose—TSKgel-GHW50 desorption with 0,75M —TSKgel-GHW50 MM; 77%, 27-f) Carbohydr.(M/M): 2,6 GlcN EndoH-, EndoF- [1268]

Rat brain Microsomal alpha-mannosidase splitting Man8GlcNAc, solubilized with TX100 Phosphocellulose—DEAEC—ConA- ConA+ (desorption with Sepharose- gradient 10-500mM K-phosphate) [1269] Rat liver Cytosolic mannosidase-I ConA-Sepharose ConA- (separation from the bound lysosomal mannosidase) [1052] Golgi mannosidase-I SephacrylS300—Phosphocellulose**— DEAEC—HA, ConA-Sepharose ConA- (separation from the bound mannosidase-II) [1270] Golgi Mannosidase-Ia 230kDa,SU 57+58kDa, splitting Man9GlcNAc into Man5GlcNAc Lectin-Sepharose ConA+ (binding of 70%, for 16-hour contact; desorption of 50% with 1M MM), ConA- (EndoH- treated enzyme) Carbohydr.: 0,88% hexoses; Asn-glycans of oligoMan-type EndoH+ [1271] alpha-Mannosidase from endoplasmic reticulum, preferentially splitting Man9GlcNAc, solubilized with DOC SM2-Biobead—ConA-Sepharose—ConA- ConA- (separation from Sepharose the bound GP) [1270]

3.2.1.114. Mannosyl-oligosaccharide 1,3-1,6-alpha-mannosyltransferase

Higher plants Vigna radiata L.(Phaseolus aureus) (mung bean, seed hypocotyls or 2-3-day seedlings) Microsomal mannosidaseII 125kDa (SDS-PAGE) ConA-Sepharose—PreparPAGE ConA+ [1264] DEAEC—DEAEC—HA**—ConA-Sepharose ConA+ (desorption with (20mgConA/ml gel)—SephacrylS300 0,3M MM; 32%, 2,4-f) [1272] Mammals Rat brain Microsomal alpha-mannosidase-B splitting Man5GlcNAc, solubilized with TX100 Phosphocellulose—DEAEC—ConA- ConA+ (desorption with Sepharose4B—HA gradient 10-500mM K-phosphate) [1269] Rat liver Golgi mannosidase-II ConA-Sepharose ConA+ (separation from mannosidase-I) [1270] 3.2.1.117. Amygdalin beta-glucosidase Prunus serotina Rhrh. (black cherry mature seeds) Monomeric amygdalin hydrolase forms I (60kDa, pI 6,6), and II (60kDa, pI 6,5) ConA-Sepharose4B—CMC—HA—IEF ConA+ (desorption with 0,2M MG) [1273] Monomeric cyanogenic amygdalin beta- glucosidase 52kDa, forms pI 6.4+6.2+6.0+5.8 PAS+(all forms) ConA-Sepharose4B**—CMC—Chromatofoc ConA+ (for all forms; desorption with 0,2M MG; 28%, 53-f) [1274] 3.2.1.118. Prunasine beta-glucosidase Prunus serotina EhRh.(black cherry mature seeds) Prunasine hydrolase forms I (monomeric, 68kDa), IIa (140kDa, SU 69,5kDa), and IIb (monomeric, 68kDa), splitting prunasine into Glc and mandelonitrile ConA-Sepharose**—HA—SephacrylS200 ConA+ (desorption with —SephacrylS200 0,3M MG; 107%, 24-f) [1275] ConA-Sepharose4B**—DEAEC—Red-120- ConA+ (desorption with agarose 0,2M MG; 35%, 20-f) [1274] 3.2.1.-. Linustatin beta-glucosidase Linum ussitatissimum L. (flax seeds) Cyanogenic linustatin beta-glucosidase dimers (39+19kDa) Red-agarose—ConA-agarose** ConA+ (desorption with —Chromatofoc 0,1M MG; 64%, 32-f) [1276] 3.2.1.-. Linamarin beta-glucosidase

High plants Linum ussitatissimum L. (flax seeds) Cyanogenic linamarin beta-glucosidase decamers (62 or 65kDa, for beta- or alpha-chain, respectively) ConA-agarose**—DEAEC—SepharoseCL6B ConA+ (desorption with 0,1M MG; 32%, 44-f) SDS-PAGE—Blot—ConA-peroxidase ConA+ [1276]

Trifolium repens L. (white clover) Homodimeric cyanogenic linamarin beta- glucosidase mannosylated form (SU 62kDa), and nonglycosylated form (SU 59kDa, from tunicamycin-treated cells) Carbohydr.(for 62kDa): 4,8% [1277]

3.2.1.-. Dhurrin beta-glucosidase Sorghum bicolor (L.) Moench (sorghum shoots of seedlings) Cyanogenic dhurrin beta-glucosidase forms Ia and Ib (200-240kDa, SU 57kDa), from seedlings growthed in dark HA—SephadexG25—DEAEC**—Red-agarose —ConA-Sepharose—UltrogelAcA ConA- (106%, 1,1-f; for both forms) [1278] Cyanogenic beta-glucosidase (dhurrinase2) forms 2a and 2b (250-300kDa, SU 61kDa), from seedlings growthed in light HA—SephadexG25—Red-agarose—Ultrogel- AcA34**—ConA-Sepharose ConA- (91%, 1,3-f, for 2a) [1278] 3.2.1.-. Glucosidase-I

Lower plants Saccharomyces cerevisiae (yeast microsomes) Trimming glucosidase-I, SU 95kDa, solubilized with Lubrol-PX DEAE-Sephacel**—N5-Carboxypentyl-1- deoxyjirimycin-sorbent—ConA-Sepharose ConA+ (desorption with 1M MM; 79%, 4,5-f) [1279] Higher plants Vigna radiata L. (Phaseolus aureus) (mung bean, seedling cell microsomes)

GlucosidaseI 316kDa (SephacrylS300) DEAEC—HA—SephadexG200—Dextran- Sepharose—ConA-Sepharose ConA+ (desorption with 0,3M MM; 25%, 3,0-f) [1280] Mammals Bovine liver microsomal Mb Neutral processing glucosidase-I, SU83kDa, solubilized with Lubrol-PX followed by TX100 N5-Carboxypentyl-1-deoxyjirimycin- sorbent—ConA-Sepharose ConA+ (complete strong binding; partial

separation from unbound processing mannosidase) [1266,1281] Porcine liver microsomes Trimming glucosidaseI 85kDa (polypeptides 69+45kDa, SDS-PAGE) N5-Carboxypentyl-1-deoxyjirimycin- Sepharose4B, ConA-Sepharose ConA+ (without desorption by MM), ConA- (in the presence of 0,8M MM) [1282] SDS-PAGE—Blot—ConA—Ab(anti-ConA)- ConA+ alkaline-phosphatase Asn-glycans of oligoMan-type: 3kDa in EndoH+ (69—66kDa, 69kDa; 3kDa in 45kDa 45—42kDa) [1282]

Rat liver microsomes alpha-1,3(4)-Glucosidase splitting Glc1-2Man9GlcNAc2 (glucosidase-I) solubilized with NP40 ConA-Sepharose—IEF ConA+ (desorption with 0,1M MM; unstability of the bound enzyme) [1283] Rat mammary endoplasmic reticulum luminal Mb GlucosidaseI 85kDa (SDS-PAGE) with a luminal catalytic domain 39kDa ConA-Sepharose ConA+ SDS-PAGE—Blot—ConA-biotin ConA+, ConA- (for EndoH- treated enzyme) Asn-glycans of oligoMan-type: 2kDa in EndoH+(85—83kDa) 85kDa [1284]

3.2.1.-. Glucosidase-II

Higher plants Vigna radiata L.(Phaseolus aureus) (mung bean, seedling microsomes) Enzyme splitting Glc2Man9GlcNAc, 220kDa, SU 110kDa, solubilized with detergent DEAEC**—HA—DEAEC—ConA-Sepharose ConA+(desorption with —SephacrylS300 0,3M MM; 73%, 9,7-f) Asn-glycans of oligoMan-type EndoH+, EndoF+ [1285]

Mammals Bovine lactating mammary gland cell microsomes Processing glucosidase-II 290kDa, SU 63 kDa, solubilized with TX100 ConA-Sepharose4B**—HA— ConA+(desorption with DEAE-Sephacel 0,2M MM; 79%, 7,7-f) Asn-glycans of oligoMan-type EndoH+, EndoF+ [1286,1287]

Porcine kidney microsomes Glucosidase-II ConA-Sepharose**—DEAE-Sephacel, ConA+ (desorption with Lectin-agarose (including LCA- 0,2M MM; 57%, 9,5-f), Sepharose4B, WGA-Sepharose6MB) SBA+ (without desorption

by GalNAc), GSA-I-, LCA-, LPA-, PNA-, RCA-I-, UEA-I-, WGA- [1288] Rat liver microsomes Glucosidase-II (alpha-1,2-glucosidase splitting Glc3Man9GlcNAc2) ConA-Sepharose—IEF ConA+ [1283] alpha-Glucosidase-II eliminating Glc from Glc2Man9GlcNAc2, forms A and B SephadexG150—PAGE(pH 8,9), ConA-Sepharose4B ConA+ (separation from unbound alpha-glucosidase splitting glycogen) [1289] 3.2.1.-. Rhamnogalacturonase Saccharomyces cerevisiae (yeasts) plus Aspergillus aculeatus KSM510 (fungus) system Fungal enzyme expressed in yeasts, forms A (62kDa, pI 4,5), and B (55kDa, pI 5,2) SDS-PAGE—Blot—Lectin GNA+ or PWM+ (for A), GNA- or PWM- (for B) Carbohydr.: 11kDa in 55kDa (including Asn350-glycan); 3kDa-overglycosylation in 62kDa [1290]

3.2.1.132. Chitosanase Bacillus circulans MH-K1 (bacterial CF) Enzyme 27kDa(HPLC), or 32kDa PAS+ (SDS-PAGE), pI 9,2 [1291]

3.2.1.-. Endoamylopullulanase Thermoanaerobacter B-6A (bacterial CF) Enzyme 450kDa (SU 220kDa), pI 4,5 PAS+ [1292]

3.2.1.-. Endo-beta-glucuronidase (saponin- hydrolase) Aspergillus oryzae KO-2 (fungal CF) Soybean-saponin-hydrolase 158kDa, SU 35+45kDa PAS+ SephadexG200**—SephadexG200 [1293]

3.2.2.5. NAD+ nucleosidase (NADase)

Bovine seminal plasma NADase 36,3kDa ConA-Sepharose**—SephadexG100 ConA+ (desorption with 0,2 M MG; 80%, 19-f) Carbohydr.: 10,4% hexoses [1294]

Bovine thyroid gland plasma Mb NAD-glycohydrolase solubilized with TX100 and Na-DOC SephadexG200—LCA-Sepharose4B LCA+ (desorption with MM) [1295]

Mb-bound enzyme Inhibition with lectins (0,1mg/ml) WGA+ (6%), ConA-, LCA- [474]

3.2.2.11. beta-Aspartyl-N-acetylglucosaminidase

Human liver Enzyme with SU 24+18+17kDa ConA-agarose—BiogelP100—Chromatofoc ConA+ —Ion-exchanger*—Reverse-phase-sorbent* [1296]

Lysosomal beta-aspartyl-N-acetylglucosaminidase native form 56kDa (SDS-PAGE), or denatured form with polypeptides 24,6+18,4+17,4kDa (SDS-PAGE) ConA-Sepharose**—DEAE-Sepharose— ConA+ (desorption with SP-Sephadex—HA—SephadexG150—HA gradient 0-0,8M MM; 86%, 32-f) Asn-glycans: 1,6kDa in 24,6kDa; EndoF+ (24,6—23,0kDa, 2,6kDa in 18,4kDa 18,4—15,8kDa, 1,6kDa in 17,4kDa 17,4—15,8kDa) [1297]

Human tissue Aspartylglucosaminidase ConA-Sepharos—SDS-PAGE ConA+ [1163]

3.2.2.22. rRNA N-glycosidase High plants Luffa cylindrica L. (sponge gourd seeds) Luffin "a" (RIP, ribosome-inactivating protein), 27kDa, 248 aar Asn28,33,77,84,206,227-glycans [1298]

Momordica charantia L.(bitter pear melon seeds) Momordin "a" (RIP) 30kDa 227 XylMan(+Fuc)GlcNAc2-Asn [1299]

Ricinus communis L. (castor bean, seeds) Ricin A-chain associated with RNAse activity, forms 30 and 32kDa Affinity to ConA ConA+ (heterogenic picture) Carbohydr.(M/M):1 Fuc, 1 Xyl (also Man and GlcNAc) [1300]

3.2.3.1. beta-Thioglucosidase Higher plants Arabidopsis thaliana L. (seedlings) Myrosinase ConA-Sepharose4B ConA+ (separation from unbound thiohydroximate beta-glucosyltransferase) [299] Brassica napus L. cv.Niklas (cabbage seeds) beta-Thioglucosidase 154kDa, SU 77kDa, forms Ca (pI 5,0),Cb (pI 4,96),and Cc (pI 4,94)

ConA-Sepharose**—DEAEC—MonoQ* ConA+ (complete binding; desorption with 0,5M MM; 89%, 8,4-f) Carbohydr.: 13,1% Man, 2,6% Fuc, 3,2% GlcNAc (Ca); 8,7% Man, 1,5% Fuc, 1,5% GlcNAc (Cb); 5,9% Man, 1,4% Fuc, 2,3% GlcNAc (Cc) [1301]

Sinapsis alba L. (white mustard seeds) Myrosinase ConA-Sepharose ConA+ (33-f) [1302]

Abbreviations ac acetyl(ated) aar amino acid residues AH aminohexyl approx. approximately Ara arabinose (m)Ab antibodies(monoclonal) blot blotting carbohydr. carbohydrates CF cultural fluid CHAPS 3-[3-(cholamidopropyl)dimethylammonoium]-1-propanesulfonate Chromatofoc chromatophocusing CMC carboxymethylcellulose DEAEC diethylaminoethylcellulose DOC deoxycholate DTAB dodecylmethylammonium bromide DTT dithiotreitol EDTA-Na ethylenediaminetetracetate-Na ELBA enzyme-lectin binding assay ELISA enzyme-linked immunosorbent assay EndoD,F,H,M endoglycosidases D, F, H, M f fold FF Fast Flow FITC fluoresceine isothiocyanate FPLC fast protein liquid chromatography Fuc L-fucose FucN L-fucosamine Gal D-galactose GalN D-galactosamine GalNAc N-acety-D-lgalactosamine Galp D-galactopyranoside Glc D-glucose Glc-1-P D-glucose-1-phosphate GlcN D-glucosamine GlcNAc N-acetyl-D-glucosamine GP glycoprotein(s) Gp glycopeptide(s)

85 hsc high speed (ultra) centrifugion HA hydroxyapatite HPLC high pressure liquid chromatography HPLAC high pressure liquid affinity chromatography IEF isoelectric focusing Ig(A,E,G,M) immunoglobulins A, E, G, M IGF insulin growth factor IL interleukin(s) Lac D-lactose LAE lectin-affinity electrophoresis LIE lectin-immunoelectrophoresis LPS lipopolysaccharide(s) Mal D-maltose Man D-mannose ManN D-mannosaminyl Man-6-P D-mannose-6-phosphate Mb membrane(s) ME 2-mercaptoethanol (alpha-ME) Mel D-melibiose MG methyl-alpha-D-glucopyranoside MM methyl-alpha-D-mannopyranoside mol molecular MUF methyl-umbelliferyl NeuAc N-acetyl-D-neuraminic acid Neu5Ac N-acetyl-5-O-acetyl-D-neuraminic acid NP nitrophenyl pNP para-NP NP40 nonidet P-40 OG n-octylglucoside PAGE polyacrylamide gel electrophoresis PMSF p-phenylmethylsulfonyl ftoride PNGase protein N-glycanase prepar. preparative PS polysaccharide(s) Rha L-rhamnose SDS sodium dodecyl sulfate Sia sialic acid STI soybean trypsin inhibitor SU subunit(s) TEAEC tetraethylaminoethylcellulose TFMSA trifluoromethane-sulfonic acid TX100 Triton X-100 Xyl D-xylose

Abbreviations and simplified specificities of lectins Abbreviations, Full name and sources Specificities used in table

BBL bovine brain lectin (Bos taurus) Man-6-P

CJA Crotalaria juncea ag. Gal- > GalNAc- CJA-I isole. of CJA Gal- > GalNAc- ConA ConcanavalinA (Canavalia ensiformis) Man-alpha-; Glc-alpha- sConA succinilated ConA(dimeric) Man-alpha-; Glc-alpha- ConG ConcanavalinG (Canavalia gladiata) Man-alpha-; Glc-alpha- DSA Datura stramonium ag. (GlcNAc-beta-4-)2-4 GNA Galantus nivalis Man-alpha-; oligoMan- GSA-I Griffonia simplicifolia ag. (isole. I) Gal-alpha-1,3Gal- HTL heat labile lectin (Escherichia coli) beta-gal-; complex HPA Helix pomatia ag. GalNAc-alpha-3GalNAc- LCA Lens culinaris ag. Man-alpha-; Fuc-alpha-6 LCA-B isolectin B of LCA Man-alpha-; Fuc-alpha-6 Ligatin mammals Glc-1-P LPA Limulus polyphemus ag. Sia-alpha- LTA(see TPA) Lotus tetragonolobus ag. Fuc-alpha-2Gal- MBP mammalian mannan-binding protein Man-alpha-; Man-6-P PHA phytohemag. (Phaseolus vulgaris) complex PHA-E, E-PHA erythroag.of PHA complex PHA-E4 homotetrameric PHA-E complex PHA-L, L-PHA leukoag.of PHA complex PHA-M mucopolysaccharide form of PHA complex PHA-P protein form of PHA(PHA-E+PHA-L) complex PNA peanut ag. (Arachis hypogaea) Gal- beta-1,3-GalNAc- POA Pleurotus ostreatus ag. Fuc-alpha- ; Lac PSA Pisum sativum ag. Man-alpha-; Fuc-alpha-6- PVA Psathyrella velutina ag. GlcNAc PWM pokeweed mitogen (Phytolacca americana) Gal-beta-4(3)GlcNAc- RCA Ricinus communis ag. Gal-beta-3(4)GlcNAc- RCA-I tetrameric RCA Gal-beta-3(4)GlcNAc- RCA-II heterodimeric RCA GalNAc RSA Raphanus sativus ag. (horseradish) complex SBA soybean ag. (Glycine max) GalNAc-1,3-Gal >Gal-beta STA Solanum tuberosum ag. (GlcNAc-beta-4)2-3 TPA(see LTA) Tetragonolobus purpuratus ag. L-Fuc-alpha- UEA Ulex europaeus ag. complex UEA-I le.-I of UEA Fuc-alpha-2Gal- VEA Vicia ervilia ag. complex VVA Vicia villosa ag. complex WGA wheat germ ag. (Triticum vulgare) (GlcNAc-beta-)2-3 >Sia-alpha-