Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 3979-3983, August 1978 Medical Sciences AB variant of infantile GM2 : Deficiency of a factor necessary for stimulation of hexosaminidase A-catalyzed degradation of GM2 and GA2 ( storage disease/activator deficiency) E. CONZELMANN AND K. SANDHOFF Max-Planck-Institut fur Psychiatrie, Neurochemische Abteilung, Kraepelinstrasse 2, D-8000 Munchen 40, Federal Republic of Germany Communicated by Saul Roseman, May 24, 1978

ABSTRACT Human kidney extracts heated to 600 and de- the degradation of (10, 12). This paper presents void of hexosaminidase activity (2-acetamido-2-deoxy-13-Dglu- evidence that a component of an activating factor necessary for coside acetamidodeoxyglucohydrolase EC 3.2.1.30) stimulate the degradation of ganglioside GM2 and glycolipid GA2 cata- more than 20-fold the hexosaminidase A-catalyzed degradation of ganglioside GM2 and of glycolipid GA2, the neuronal storage lyzed by hexosaminidase A is defective in variant AB of in- compounds of GM2 gangliosidosis. The stimulating factor of this fantile GM2 gangliosidosis. extract, which is labile at temperatures above 60°, is also present in kidney extracts from patients with infantile GM2 gangliosi- MATERIALS AND METHODS dosis having a deficiency of hexosaminidase A (Tay-Sachs disease, variant B) and a deficiency of hexosaminidases A and Materials. 4-Methylumbelliferyl-2-acetamido-2-deoxy- B (variant 0). Evidence is presented that this factor is defective ,B-D-glucopyranoside (MUFGlcNAc) and N-acetyl-D-galac- in the AB-variant of infantile GM2 gangliosidosis which is tosamine (GalNAc) were from Koch-Light (England); sodium characterized by an accumulation of GM2 and GA taurodeoxycholate, trypsin, and trypsin inhibitor, from Sigma despite the fact that the degrading enzymes, hexosaminidases Chemical Company (St. Louis, MO); Pronase P (from Strep- A and B, retain normal activity levels. Thus, variant AB is an example of a fatal lipid storage disease that is caused not by a tomyces griseus), from Serva (Heidelberg). DEAE-cellulose defect of a degrading enzyme but rather by a defective factor (DE-52) was obtained from Whatman (Springfield Mill, Great necessary for the interaction of lipid substrates and the water- Britain), Bio-Gel P-200 from Bio-Rad Laboratories (Munich, soluble hydrolase. Federal Republic of Germany). Ganglioside GM2, tritiated in its GalNAc portion (2 ,uCi/,umol) (13), and glycolipid GA2 (190 Infantile GM2 gangliosidosis is a fatal inherited storage disease ,uCi/,umol), labeled by reduction of the double bond in the characterized by the accumulation of ganglioside GM2 and its moiety, were prepared as described (6). All other asialo derivative, GA2, in nervous tissue (1). Biochemically, three reagents were analytically pure or of the best grade available. variants can be distinguished, two of which could hitherto be Organs from patients with variant 0, B, or AB of infantile GM2 explained in terms of enzyme defects: patients with variant B gangliosidosis and from normals were deep frozen within 6 hr ("Tay-Sachs disease") lack hexosaminidase A (2-acetamido- after death and kept for 2-3 yr at -700 until used. 2-deoxy-f-D-glucoside acetamidodeoxyglucohydrolase, EC Preparation of Tissue Extracts. Frozen human tissues (0.5-1 3.2.1.30) (2, 3), the enzyme responsible for the further degra- g) were thawed, homogenized in 4 vol of distilled water with dation of the ganglioside GM2 (4), whereas in patients with an Ultra-Turrax homogenizer (Janke & Kunkel, Staufen, variant 0, major hexosaminidase isoenzymes A and B both are Federal Republic of Germany), and centrifuged at 13,000 X missing (5, 6), presumably due to the defect of a common g for 10 min. To obtain a preparation of the activating factor subunit (7). For the variant AB, however, no such enzyme de- virtually free of hexosaminidases, the extract was heated at 600 ficiency could be demonstrated. Patients having this variant for 1 hr, centrifuged, and the precipitated protein was dis- retain normal or show elevated levels of both hexosaminidases carded. Protein was determined according to Lowry et al. (14) in all organs examined (3, 6), yet the ganglioside GM2 never- with crystalline bovine serum albumin as standard. theless accumulates in the brain tissue at the same rate as in the Ion Exchange Chromatography. Heated tissue extract (1.2 B and 0 variants, and the clinical symptoms and course of the g of protein in 230 ml) prepared as described above was di- disease are the same as in these variants. Biochemical and im- alyzed against 10 mM phosphate buffer (pH 6.5) and loaded munological comparison of an AB patient's hexosaminidase A onto a DEAE-cellulose column (Whatman DE-52; volume, 300 with the enzyme from normal subjects revealed no difference ml) that had been equilibrated with the same buffer. After a (8). In particular, the patient's storage compounds, ganglioside washing with 2 vol of buffer, the column was eluted with a GM2 and glycolipid GA2, were split by the patient's hexosami- nidase A in the presence of detergent at a normal rate, thus linear NaCl gradient, 0-0.5 M in 1 liter of 10 mM phosphate practically ruling out the possibility of a mutant hexosaminidase buffer (pH 6.5). Fractions (25 ml) were collected and assayed A. for protein and stimulating factor as described below. Recent publications have demonstrated the necessity for the Proteolytic Digestion. Components a and f (50,l each) presence of a nonenzymic protein to bring about the interaction obtained after gel filtration of the stimulating factor were in- of some water-soluble hydrolases (9-11) with their substrates. Abbreviations: MUFGIcNAc, 4-methylumbelliferyl-2-acetamido-2- Such activator proteins have also been reported for deoxy-fl-D-glucopyranoside; GaINAc, N-acetyl-D-galactosamine; NeuAc, N-acetylneuraminic acid; Cer, ; The costs of publication of this article were defrayed in part by the GA2, gangliotriaosylceramide (GgOse3Cer; GalNAc31-4Gal- payment of page charges. This article must therefore be hereby marked f1-4GlI31-1Cer); ganglioside GM2, II3NeuAc-GgOse3Cer (GalN- "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Acf11-4Gal(3. -2aNeuAc)31-4Glfl-BCer); ganglioside GM3, this fact. NeuAca2-3Gal31-4Glcll-lCer. 3979 Downloaded by guest on September 29, 2021 3980 Medical Sciences: Conzelmann and Sandhoff Proc. Natl. Acad. Sci. USA 75 (1978) cubated with Pronase P (0.5 mg) at 370 (pH 6.5, 10 hr). Under in all three. The solvent systems were: (i) chloroform/metha- these conditions, both components were inactivated by more nol/water, 14:6:1 (vol/vol); (if) chloroform/methanol/con- than 90%. Controls were run under the same conditions except centrated NH4OH, 65:35:4 (vol/vol); and (hi) chloroform/ that the protease was added only after incubation to show that methanol/0.25% NaCl in water, 62.38:8 (vol/vol). were the subsequent assay for the stimulating components is not visualized with acetic acid/sulfuric acid/anisaldehyde, 50:1:0.2 disturbed by the protease which is inactive at acidic pH. (vol/vol) 15 min at 1400. The distribution of Preparation of Purified Hexosaminidase A. Hexosamin- radioactivity on idase A was isolated from normal human liver as described (4). the plates was measured with a , camera LB 290B (Berthold, Bovine serum albumin (0.1 mg/ml) was added and the enzyme Wildbad, Federal Republic of Germany). solution was stored at -70°. Enzyme Assays. Hexosaminidase activity was tested as de- RESULTS scribed (4), with MUFGlcNAc as substrate; 1 unit of hexo- Stimulation of Enzymic Hydrolysis of Glycolipids by saminidase activity releases 1 1mol of 4-methylumbelliferone Heated Extracts of Organs. Tissue extracts whose hexosami- per min at 37°. nidase activity had been destroyed by heating to 600 for 1 hr Ganglioside Gm2 N-acetyl-ft-D-galactosaminidase was es- stimulated the degradation of ganglioside Gm catalyzed by sentially determined according to O'Brien et al. (13). Gangli- purified hexosaminidase A (Fig. 1). Kidney extracts activated oside GM2 (10 nmol), labeled in its N-acetyl-D-galactosamine the enzymic hydrolysis of ganghoside more than 18-fold lin- portion (2 ACi/timol) (12), in chloroform/methanol, 2:1 (vol/ early with the amount of extract protein added, whereas ex- vol), was pipetted into the reaction vessel and dried under a tracts fromh brain and liver showed only a slight stream of nitrogen. Citrate buffer (pH 4.2; 4 Mmol) and water activation at were added and the mixture was sonicated for 5 sec. As indi- low protein concentrations (3- to 4-fold) and this diminished cated in the legends of figures, extract, supernatant of heat- with higher concentrations of protein. Corresponding obser- inactivated extract, or detergent and purified hexosaminidase vations were obtained for the enzymic degradation of glycolipid were added to give a final volume of 40 Al. The mixture-was GsA. Because kidney samples were available from variant AB vigorously shaken and incubated at 370 for 2-4 hr. The reaction as well as from other variants of infantile GM2 gangliosidosis, was stopped by the addition of 750 ll of CHC13/CH30H/2.5 further investigations on this activating factor were carried out M aqueous NH&, 80:201 (vol/vol), followed by 0.5 ml of 1 mM with these different kidney extracts. The activating factor was N-acetyl-D-galactosamine in water. After thorough shaking, stable up to 600 but labile at higher temperatures (Figs. 2 and the phases were separated by centrifugation. The upper phase 3). Because of this stability at 600, it is possible to prepare kidney was collected and loaded onto a small column (1 ml) of extracts rich in activating factor but essentially free of hexo- DEAE-cellulose that had previously been washed with distilled saminidase activity, so that these extracts degrade neither water. The column was rinsed twice with 850-,ul portions of 1 ganglioside Gm (Fig. 1) nor glycolipid GA2 without the addition mM N-acetyl-D-galactosamine to elute the enzymically liber- of hexosaminidase A. ated GalNAc, the effluents were combined, and their radio- Unheated extracts from normal kidneys hydrolyzed gan- activity was assayed in a liquid scintillation counter. Degradation of glycolipid GA2 (190 ;&Ci/,gmol) was essen- tially performed as described (4). The 200-,ul mixtures con- tained 0.2 unit of hexosaminidase A and were incubated for 2 hr.

Product Identification. [3H]Ganglioside GM2 labeled in its A GalNAc moiety was incubated with purified hexosaminidase A in the presence of heat-inactivated extracts (1 hr, 600) of normal kidneys or of those from variant 0 or B for 5 hr. In this case, all quantities were 10 times those described above. Ali- S- quots were analyzed in two different thin layer systems. (i) Adsorbent was cellulose plates (Merck, Darmstadt, Federal Republic of Germany); development was by two runs with the nonaqueous phase of the mixture ethyl acetate/pyridine/water, 0 2:1:2 (vol/vol); sugars were visualized with silver nitrate. (i) E25 Adsorbent was silica gel plates (Merck, Darmstadt, Federal C) Republic of Germany); the solvent system was n-butanol/eth- '4: anol/water, 2:1:2 (vol/vol); spots were visualized with acetic acid/sulfuric acid/anisaldehyde, 50:1:0.2 (vol/vol), 15 min at -0 1400. Distribution of radioactivity on the thin layer plates was determined with a,8-camera LB 290B (Berthold, Wildbad, 50 100 Federal Republic of Germany). Besides unreacted [3Hlsubstrate Supernatant added, jug protein/assay the only tritiated product found comigrated with authentic N-acetyl-D-galactosamine in both systems. FIG. 1. Stimulation of the hexosaminidase A-catalyzed degra- For the identification of lipophilic reaction products [3H]- dation of ganglioside GM2 by extracts from normal human tissues. ganglioside GM2 (4) (16 uCi/,umol) and glycosphingolipid GA2 Tissues were extracted, heated for 60 min at 600, and centrifuged; (6) (1901ACi/Amol) labeled in their sphingosine portions were aliquots of each supernatant were assayed. The incubation mixtures, used as substrates (with 10-fold greater quantities than as de- containing increasing amounts of the supernatants, 30 milliunits of purified hexosaminidase A, and 10 nmol of [3Hjganglioside GM2, were scribed above). Besides unreacted 3H-labeled substrates, the analyzed for [3H]GalNAc formed. Controls lacking hexosaminidase only tritiated products found comigrated with authentic gan- A were run for each value and subtracted. A, Kidney; 0, brain; 0, glioside GM3 andGalf14Glc1, lCer, respectively, in three liver; X, control incubations with heated kidney extract without ad- different thin-layer systems. The adsorbent was silica gel plates ditional hexosaminidase A. Downloaded by guest on September 29, 2021 Medical Sciences: Conzelmann and Sandhoff Proc. Nati. Acad. SC{. USA 75 (1978) 3981

~70 070- 0 0 0 5 1

Z 50- Z50- E LEo t 1 * .i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... _ 4-crJ K e 0 2 30- 230

iid Ie 10

0 5 10 15 20 0 5 10 15 20 60 70 Kidney, extract, Al Kidney, extract,Jaid Temperature, 0C FIG. 4. Ganglioside Gm2-N-acety1-(l-D-galaCtosaminidase activity FIG. 2. Thermal inactivation of human kidney hexosaminidase of normal and pathological human kidney extracts. Frozen, thawed and of the factor activating the hexosaminidase A-catalyzed degra- human kidneys were extracted as described in the text, centrifuged, dation of ganglioside GM2. Normal human kidney was extracted, and aliquots of each supernatant assayed for hydrolysis of [3H]gan- heated for 60 min at the indicated temperature, and centrifuged; 20-AI glioside GM2 Incubation mixtures containing increasRingamounts of aliquots of each supernatant were assayed. For hexosaminidase (A), kidney extract (13 mg of protein per ml) and 10 nmol of [3H]gangli- the substrate was 4-MUFGIcNAc (4). The supernatant activity (un- oside GM2 as substrate were incubated for 2 hr and analyzed for heated) was 0.18 units/mg ofprotein; this value = 100%. For activating [3HJGalNAc liberated. 0, Kidney extract, normal case 1; +, kidney factor (0), the stimulation of [3H]ganglioside GM2 hydrolysis by 65 extract, normal case 2; X, kidney extract, variant 0; A, kidney extract, milliunits of purified hexosaminidase A was measured. Controls variant B (Tay-Sachs disease); o3, kidney extract, variant AB;@0, lacking hexosaminidase A were subtracted from each value. The 100% mixture (1:1, vol/vol) of extracts from normal kidney (case 1) and value represents the stimulating activity ofunheated supernatant (224 variant AB kidney. (Left) Without sodium taurocholate. (Right) With pmol of [3H]GalNAc formed/hr per mg of supernatant protein). 1.85 nmol of sodium taurodeoxycholate per Ag of protein.

glioside GM2 at rates of 0.1-0.2 nmol/hr per mg of protein, control cases 1 and 2) found in these extracts. Mixing experi- whereas unheated extracts of kidney tissues from patients with ments excluded the presence of an inhibitor for the ganglioside variant 0 or B yielded only much lower values (Fig. 4 left). GM2 degradation in the kidney extracts of variant AB. Fur- However, in the case of unheated kidney extracts from the thermore, the addition to the incubation mixtures of sodium patient having variant AB no significant degradation of gan- taurodeoxycholate, which is known to stimulate the enzymic glioside Gmg (Fig. 4 left) or glycolipid GA2 (not shown) was degradation of glycolipids (4), resulted in an almost normal observed, despite the normal levels of hexosaminidase A and degradation rate of ganglioside Gm2 (Fig. 4 right) and gly- B (0.09 unit/mg of protein compared to 0. 1-0.15 unit/mg in colipid GA2 (not shown), indicating that the hexosaminidases of extracts from AB variant are able to attack the lipids stored in this disease. The minor acitivity of the extracts from variants 1001 B and 0 in the presence of detergent is probably due to the presence of hexosaminidase B and S, respectively, in these ex- tracts (4, 15). Defective Activating Factor in Kidney Extract from Var- iant AB. The findings presented in Figs. 1-4 raised the possi- bility that an activating factor that is present in normal tissue and that can be replaced in vitro by sodium taurodeoxycholate may be defective in the variant AB tissue. Therefore, heat- inactivated (600, 1 hr) extracts of normal and pathological kidneys were compared for their ability to stimulate the en- zymic hydrolysis of glycolipids. Fig. 5 shows the activation (corrected for the "endogeneous" hexosaminidase activity of the extracts) of the hexosaminidase A-catalyzed hydrolysis of ganglioside GM2 and glycolipid GA2. The activating capacity exhibited by the extracts from two 20 60 120 variants of Gm2 gangliosidosis was equal (variant B) or even Inactivation time, min superior (variant 0) to that from normal controls, whereas the extract FIG. 3. Kinetics of thermal inactivation (60°) of normal human from variant AB kidney showed no activating effect. kidney hexosaminidase and of the factor activating the hexosamin- A mixture (1:1, vol/vol) of variant AB extract with normal ex- idase A-catalyzed degradation of ganglioside GM2. Human kidney was tract had about half the activating capacity of the pure normal extracted, heated for the times indicated at 600, and centrifuged; 20-,ul extract, indicating that deficiency of an activating factor rather aliquots ofeach supernatant were assayed. For hexosaminidase (A), than presence of an inhibitor was responsible for the lack of the substrate was 4-MUFGlcNAc (4). The supernatant activity (un- activation. Furthermore, as shown in Fig. 6 this activating factor heated) was = 0.18 unit/mg protein; this value 100%. For activating was factor (0), the stimulation of [3H]ganglioside GM2 hydrolysis by 65 also absent from unheated kidney extracts of variant AB, milliunits of purified hexosaminidase A was measured. The 100% ruling out the existence of a more heat labile factor in this value represents the stimulating activity ofunheated supernatant (224 variant. pmol of [3H]GalNAc formed/hr per mg of supernatant protein). Characterization of the Activating Factor. Te activating Downloaded by guest on September 29, 2021 3982 Medical Sciences: Conzelmann and Sandhoff Proc. Nati. Acad. sci. usA 75 (1,978)

, 0.5 a p 8a0 '04 "I .~~~~~. 4.-S 0.4- Z 1.5 XE ZE 00~~~~~~~ x~~~~~~~~ E ,m 0.3

0 .c -E 5~~~~~~~ CO~ ~ ~ ~ ~~~ IS 3060 9 12 0 4 030 5 0 L90 ._ 0. 0. 5 4'2.

E 0 W 30 60 90 120' 'O 150 300 450 600 + Extract added, ug protein Extract added, Mg protein 10 15 20 25 Fraction FIG. 5. Activation of hexosaminidase A-catalyzed degradation FIG. 7. Resolution of the stimulating factor into two components, ofglycolipids GM2 (Left) and GA2 (Right) by normal and pathological a and (3, by gel filtration. Fractions containing the stimulating factor human kidney extracts. Human kidneys of normal or pathological from the DEAE-cellulose column were combined, dialyzed against origin were extracted, heated for 1 hr at 600, and centrifuged. Aliquots distilled water, lyophilizedandtaken up in 20 mM phosphate buffer, of the supernatants containing increasing amounts of protein were pH 6.5/25 mM NaCl to give a concentration ofabout 20 mg ofprotein assayed. (Left) Activation of hexosaminidase A-catalyzed degradation per ml. A 6-ml sample was applied to a 150-ml column of Bio-Gel of ganglioside GM2. Standard incubation mixtures containing 65 P-200 equilibrated with 20 mM phosphate buffer, pH 6.5/25 mM milliunits ofpurified hexosaminidase A and 10 nmol of [3Hjganglio- NaCl. The column was eluted with the same buffer at a flow rate of were for protein (. side GM2 were analyzed for [3H]GaINAc formed. Blanks were run for 12 ml/hr. Fractions (5 ml) collected and assayed ); each value without added hexosaminidase A and subtracted. (Right) 10 MI of each fraction was assayed for ability to stimulate the enzymic Activation of hexosaminidase A-catalyzed degradation of glycos- breakdown of ganglioside GM2 by hexosaminidase A at 0.1 unit per assay The same was also tested in the presence of an ad- phingolipid GA2. Incubation mixtures containing 200 milliunits pu- (A). ability Ml a or hexosaminidase A and 10 nmol glycosphingolipid as sub- ditional 10 of fraction 10, conting component (-), fraction rified Gs2 void volume strate were analyzed for product (Gal(31-4G1c31-1[3HjCer) formed 15, containing component ft (0). Arrows indicate (V0) as with blue dextran 2000 and (4). For symbols, see legend of Fig. 4. and salt volume (Vt) determined [3H]leucine, respectively. a, component a; (3, component (3.

the molecular weights are approximately 25,000-for component completely to DEAE- factor from normal kidney adsorbed (8 and 60,000-80,000 for component a. Both components were cellulose at pH 6.5 and was eluted by a NaCi gradient at a digested by Pronase P. indicating their protein nature. Substi- concentration of 0.15 M.This activating factor was resolved by tution experiments with components a and jB, respectively, filtration on Bio-Gel P-200 into two components, a and ,, each of which produced only negligible stimulation when assayed showed that extracts prepared from kidney tissue with variant AB contained component a but were deficient in component alone (Fig. 7). For sigiant stimulation of the hexosaminidase (Table 1). A-catalyzed degradation of ganglioside Gm, both components ft were required. As estimated from gel filtration experiments, DISCUSSION The interaction of water-soluble lysosomal hydrolases with their amphiphilic sphingolipid substrates is in vitro strongly dependent on anionic detergents (e.g., bile salts). In the absence 'C of detergentsrthese enzymes show only very low activity toward E their sphingolipid substrates. Their activity in vivo was there- 0 fore puzzling until the discovery of the "activators" (9-11) %4. /01,+ -water-suble proteins that facilitate the interaction between z the lipid substrates and their degrading enzymes. The most 'as 00 extensively studied example is probably the activator for the others have been re- E degradation of sulfate (9); 0. .010~ ~ .00-a Table 1. Stimulation of hexosaminidase A-catalyzed degradation ._ of ganglioside GM2 by mixtures of various kidney extracts and activator components. -0 0 0 I 300 Enzyme activity* with addition of 100 20 kidney extract from Kidney extract, Mg protein Variant Variant FIG. 6. Absence of activating factor from unheated kidney extract H20 AB B Normal of variant AB. Stimulation of the hexosaminidase A-catalyzed hy- drolysis of [3Hjganglioside GM2 was measured in the presence of ex- Enzyme 0.06 0.07 0.38 0.34 tracts (13 mg of protein per ml) prepared from human kidneys. In- Enzyme + a (5 gi) 0.09 0.09 0.39 0.37 cubation mixtures containing increasing amounts of kidney extracts, 0.52 10 nmol of [3H]ganglioside GM2, and 65 milliunits of purified hexo- Enzyme + * (10 Ml) 0.11 0.44 0.74 saminidase A were analyzed for [5H]GaINAc formed. Blanks were run Kidney extracts (6 mg of protein per ml) were heated to 600 for 1 for each value without additional hexosaminidase A and subtracted. hr; activator components a and ft were obtained by gel filtration (see 0, Kidney extract, normal case 1; +, kidney extract, variant 0; A, kidney extract, variant B (Tay-Sachs disease); 0, kidney extract, Fig. 7). variant AB; *, mixture (1:1, vol/vol) of extracts from normal kidney * nmol of ganglioside GM2 cleaved per hr per unit of hexosaminidase (case 1) and variant AB kidney. A. Downloaded by guest on September 29, 2021 Medical Sciences: Conzelmann and Sandhoff Proc. Nati. Acad. Sci. USA 75 (1978) 3983

ported for the degradation of glucocerebroside (11) and gan- tissue extracts from such patients are able to hydrolyze the gliosides (10, 12). storage compounds in the presence of sodium taurodeoxycho- This paper demonstrates the presence of an activating factor, late. Furthermore, this variant emphasizes the importance of composed of two components (a and 0), for the hexosaminidase such activators in the catabolism of lipids by demonstrating that A-catalyzed hydrolysis of glycolipids Gm and GA in extracts the defect of this activating factor has the same clinical conse- of various organs. Among those tissues tested, the factor appears quences as a deficiency of the enzyme. to be most active in kidney extracts. Both components together The nature of the glycolipids stored also allows for some are necessary for the stimulation of the enzymic degradation important conclusions concerning the often-raised question of of lipid substrates by hexosaminidase A but do not affect the the specificity of the lipid hydrolase activators with respect to hexosaminidase A-catalyzed hydrolysis of synthetic, water- substrate as well as enzyme. The deficiency of component # of soluble MUFGlcNAc (data not shown). The extent of stimula- the factor described here leads to the storage of only two, closely tion (up to >2 nmol of ganglioside GM cleaved per hr per en- related, glycolipids (GM2 and GA2) and the severity of the dis- zyme unit) obtained even with crude extracts, and far from ease clearly indicates that none of the other activators can take saturation conditions, clearly differentiates components a and over the task of the defective protein to any significant ex- i from the activators described by Li and Li (10) and Fischer tent. and Jatzkewitz more than (9). Despite being purified 1000-fold, We are indebted to Dr. A. D. Patrick and Dr. R. Ellis, London, for those activators stimulate only up to 0.2 and 1.8 nmol of gan- providing deep-frozen organs of a patient with variant AB of infantile glioside GM cleaved per hr per unit of hexosaminidase A, re- GM gangliodos and to Mr H. Nehrkom for her excellent asiance spectively (4). Furthermore, they are not resolved into two in performing the experiments with glycolipid GA2. We thank Dr. Anzil components by gel filtration. On the other hand, the activator for his help with the manuscript. This work was supported in part by purified by Hechtman (12, 16) about 100-fold stimulates the the Deutsche Forschungsgemeinschaft (Grant Sa-257/4). hexosaminidase A-catalyzed degradation to a much lesser GM2 1. extent = per per Sandhoff, K. (1977) Angew. Chem. Int. Ed. Engl. 16, 273- [V., 0.1 nmol of ganglioside GM cleaved hr 285. unit of hexosaminidase A (16)] but is equally thermolabile and 2. Okada, S. & O'Brien, J. S. (1969) Science 165,698-700. may be similar or identical to one of the two components de- 3. Sandhoff, K. (1969) FEBS Lett. 4,351454. scribed here (probably the lower molecular weight component, 4. Sandhoff, K., Conzelmann, E. & Nehrkorn, H. (1977) Hoppe- Seyler's Z. Physiol. Chem. 358,779-787. It was demonstrated that extracts heated to 600 as well as 5. Sandhoff, K., Andreae, U. & Jatzkewitz, H. (1968) Pathol. Eur. unheated extracts from the kidney of a patient who died from 3,278-285. the very rare variant AB of infantile Gm gangliosidosis com- 6. Sandhoff, K., Harzer, K., Wissle, W. & Jatzkewitz, H. (1971)1. pletely fail to enhance the enzymic breakdown of the glycoli- Neurochem. 18, 2469-2489. pids GM2 and This is certainly not due to the presence of 7. Geiger, B. & Arnon, R. (1976) 15,3484-3493. GA2. 8. some stored lipids that might act as inhibitors because other Conzelmann, E., Sandhoff, K., Nehrkorn, H., Geiger, B. & Arnon, R. (1978) Eur. J. Biochem. 84,27-33. variants of this disease exhibit a normal or even increased ac- 9. Fischer, G. & Jatzkewitz, H. (1975) Hoppe-Seyler's Z. Physiol. tivating effect, nor can it be attributed to the presence of a Chem. 356,605-613. specific inhibitor (this is ruled out by the results obtained with 10. Li. S. C. & Li, Y. T. (1976) J. Biol. Chem. 254, 1159-1163. mixtures of normal and AB variant extracts). 11. Ho, M. W. & O'Brien, J. S. (1971) Proc. Nati. Acad. Sci. USA 68, From these data and from substitution experiments with 2810-2813. components a and # it may be concluded that there is a defi- 12. Hechtman, P. (1977) Can. J. Biochem. 55,315-324. ciency of the (B component of the natural activator for the hy- 13. O'Brien, J. S., Norden, A. G. W., Miller, A. L., Forst, R. G. & drolysis of glycolipids GM2 and GA by hexosaminidase A. This Kelly, T. (1977) ClIn. Genet. 11, 171-183. explains satisfactorily why patients having variant AB of in- 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. fantile GM2 gangliosidosis exhibit and die from the storage of (1951) J. Biol. Chem. 193,265-275. 15. Geiger, B., Arnon, R. & Sandhoff, K. (1977) Am. J. Hum. Genet., gangliosides Gm and GA in their nervous tissues, the same way 29,508-522. patients with deficient hexosaminidase A do, although their 16. Hechtman, P. & LeBlanc, D. (1977) Biochem. J. 167, 693- hexosaminidases appear to be normal (6, 8). It also explains why 701. Downloaded by guest on September 29, 2021