D-Glyceraldehyde-3-Phosphate

J. Gen. App!. Microbiol., 36, 19-32 (1990)

CHARACTERIZATION OF A HALOPHILIC GLYCER- ALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM THE ARCHAEBACTERIUM HALOARCULA VALLISMORTIS

GOMATHI KRISHNAN AND WIJAYA ALTEKAR*

Biochemistry Division, Bhabha Atomic Research Centre, Bombay-400 085, India

(Received December 12, 1989)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified to electrophoretic homogeneity from the halophilic archaebacterium Haloarcula (Halobacterium) vallismortis. The purification was achieved by (NH4)2SO4-mediated chromatography on Sepharose 4B and DEAE- cellulose, and hydrophobic and hydroxylapatite chromatography. In contrast to nonhalophilic archaebacteria, only a single NAD + -specific GAPDH was found in the halobacterium. However, it shares the property of insensitivity to the antibiotic pentalenolactone with the enzymes from other archaebacterial sources. Like all other GAPDHs the enzyme from H. vallismortis is a homomeric tetramer with catalytic properties comparable to the NAD +-specific enzymes characterized so far. The molecular mass of the subunit, deduced under denaturing conditions in the presence of a cationic detergent, was 40±2 kDa. The halobacterial GAPDH is a halophilic enzyme requiring high concentrations of salt for activation and stability and is an acidic protein. Immunological tests between the halobacterial enzyme and its counterparts from eubacterial, mammalian and nonhalophilic archaebacterial sources were negative.

D-Glyceraldehyde-3-phosphate: NAD + oxidoreductase, EC 1.2.1.12 (GA- PDH), a key enzyme of glycolysis, catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. The NAD +-dependent GAPDHs from a variety of sources have been isolated and characterized (1,8-11,14). The extensive studies on GAPDHs at the biochemical and molecular level in several eubacterial and eukaryotic sources have revealed strict homologies and sequence identity of 50%-60%. Furthermore each of the

* Address reprint requests to: Dr . W. Altekar, Biochemistry Division, Bhabha Atomic Research Centre, Bombay-400 085, India.

19 20 KRISHNAN and ALTEKAR VOL. 36 characterized NAD+-enzymes is a homomeric tetramer of molecular weight 144 kDa (23). Recently much work has been published on the GAPDHs from thermophilic (24,25) and methanogenic archaebacteria (17-20). But there is hardly any information available on GAPDH from halobacteria, the group placed in the third kingdom of life, the archaebacteria (21). Extremely halophilic bacteria are organisms that require high NaCI concentrations for growth and survival (30). Most halobacterial enzymes require high salt concentrations for activity and structural stability. Comparatively few halophilic enzymes have been purified, since maintenance of high salt concentrations during their purification procedures poses problems (30). In our studies on carbohydrate metabolism in halobacteria (40) we pointed out that GAPDH is one of the most active enzymes in halobacterial cells. The work described in this paper was undertaken to purify the GAPDH from Haloarculaa vallismortis, previously known as Halobacterium vallismortis (43), and to characterize its structural and biochemical properties for studying relationships with enzymes from archaebacteria, eubacteria and eukaryotes. In contrast to the enzyme from thermophilic and methanogenic archaebacteria, only a single NAD +-specific GAPDH was found. In addition to its halophilic property the halobacterial enzyme has structural and catalytic properties comparable to the NAD +-specific enzymes isolated so far.

MATERIALS AND METHODS

Chemicals. DL-Glyceraldehyde-3-phosphate (diethyl acetal, barium salt), reduced glutathione, NAD+, fructose-l,6-diphosphate, iodoacetic acid, N- ethylmaleimide, 5, 5'-dithiobis(2-nitrobenzoic acid) and DEAE-cellulose were purchased from Sigma Chem. Co., U.S.A. Sepharose 4B, Sepharose CL-6B and Phenyl Sepharose were products of Pharmacia, Sweden. Hydroxylapatite was from Biorad, U.S.A. Freund's complete adjuvant was obtained from Difco laboratories, Detroit, U.S.A. Other chemicals used were of analytical grade. Growth of organism and preparation of cell extract. Haloarcula vallismortis (ATCC 34679) was grown in a medium containing 0.5% fructose, 25% NaCI and other salts. Other details of growth medium and conditions for growth, harvest and preparation of cell free extract by sonication were described previously (12). Enzyme assay. GAPDH activity was measured in a Varian DMS 200 spectrophotometer by following the reduction of NAD + at 340 nm (29). The reaction mixture in a volume of 1 ml contained 7.5 mM sodium pyrophosphate, 16 mM sodium arsenate, 5 mM BME, 0.5 mM NAD+, 2.5 M KCl-50 mM Tris-HC1 pH 8.6 and fractions containing hGAPDH. The reaction was started by adding 3 mM glyceraldehyde-3-phosphate solution. One unit of enzyme activity is expressed as the amount of enzyme that forms l umol NADH per min at 25°C. Protein assay. Protein was assayed by Lowry's method as modified by Peterson (38) with bovine serum albumin as standard. Enzyme purification. GAPDH from H. vallismortiswas purified to homogeneity. 1990 Halobacterial Glyceraldehyde-3-phosphate Dehydrogenase 21

Preparation of cell-free extract and (NH4)25O4 precipitation were conducted at 4°C. The remaining operations were performed at 25°C. A typical purification procedure ran as follows: Seventeen grams of H, vallismortis cells were suspended in 60 ml of 1 M KCl-1 M (NH4)25O45 mNt BME-50 mM Tris-HCI, pH 8.0. The suspension was sonicated for 5 min in a Vibracell sonicator, then 100, tg of de- oxyribonuclease and 0.1 mM phenylmethylsulfonyl fluoride was added to the sonicate. The suspension was stirred for 30 min, and centrifuged at 10,000 x g for 40 min. Of the supernatant obtained, 71 ml constituted the cell-free extract. (NH4) 2504 fractionation. To the cell-free extract were added simultaneously with stirring 1 volume of 100 mM Tris-HC1 pH 8.0 and solid (NH4)25O4 to obtain 80% saturation of the total suspension. The suspension was allowed to stand for 18 h at 4°C then centrifuged at 14,000 x g for 40 min, and the supernatant containing GAPDH was collected. (NH4)2SO4-mediated chromatography on Sepharose 4B. The supernatant was adsorbed on a Sepharose 4B column (20 x 4 cm) equilibrated with 3 M (NH4)25O45 mM BME-50 mM Tris-HCI, pH 8.0 (buffer 1). The column was washed with 1.51 of buffer 1 and developed with a linear gradient consisting 600 ml each of 3 M (NH4)2SO45 mM BME-50 mM Tris-HCI, pH 8.0, and 1 M (NH4)25O45 mM BME-50 mM Tris-HCI, pH 8.0. Fractions 8 ml each were collected at the rate of 60 ml/h. The active fractions were pooled. DEAE-cellulose chromatography. The pooled fractions of the previous step was loaded on a DEAE-cellulose column (10 x 2.5 cm) equilibrated with buffer 1. After the column was washed with the equilibrating buffer, 1 column volume of 1.4 M (NH4)25O45 mM BME-50 mM Tris-HCI, pH 8.0, was passed. GAPDH activity was eluted with a linear gradient of increasing KCl (0 to 1 M) concentration in the buffer, 1.4 M (NH4)25O45 mM BME-50 mM Tris-HCI, pH 8.0, the total volume of the gradient being 500 ml. The pooled active fractions were dialyzed against 15 volumes of 4 M KCl-5 mM BME-50 mM Tris-HCI, pH 8.0 (buffer 2) for 18h. Phenyl sepharose chromatography. The dialyzed fraction was applied on a Phenyl Sepharose column (15 x 2.5 cm) equilibrated with buffer 2. After washing the column with the same buffer the chromatogram was developed by batchwise elution with 5 mM BME-50 mM Tris-HCI, pH 8.0 buffers containing decreasing concentrations of KCI. The KCl concentration was varied from 4 M to 1 M. GAPDH activity was eluted in 2.5 M KCl-5 mM BME-50 mM Tris-HCI, pH 8.0 (buffer 3). Hydroxylapatite chromatography. The pooled fractions of GAPDH were loaded on a hydroxylapatite column ('8x 2 cm) equilibrated with buffer 3. The column was washed sequentially with 5 volumes of buffers each containing 50 mM-, 100 mM- and 150 mM-K2HPO4/KH2PO4 in 2.5 M KCl-5 mM BME, pH 8.0. This resulted in separation of the remaining impurities from hGAPDH which was eluted in the buffer containing 150 mM phosphate. SDS-PAGE. SDS-PAGE was performed using 7.5% gels according to the method of Weber and Osborn (46), after desalting the protein. Bovine serum albumin 22 KRISHNAN and ALTEKAR VOL. 36

(66 kDa), egg albumin (45 kDa), rabbit muscle GAPDH subunit (36 kDa), carbonic anhydrase from bovine erythrocyte (29 kDa), trypsinogen from ox (24 kDa), trypsin inhibitor (20 kDa) and a-lactalbumin (14 kDa) were the standard protein markers. CTAB-PAGE. Polyacrylamide gel electrophoresis in the presence of CTAB was performed by the method of Eley et al. (16). The 7.5% acrylamide gels and running buffer containing 0.1 % CTAB were used. The electrophoresis was conducted by reversing the electrodes. The CTAB gels were stained as described (16) and destained by the method used for SDS gels. Molecular weight. The molecular weight of hGAPDH was determined by analytical gel filtration (5) on a column of Sepharose CL-6B (100 x 1 cm), developed with 3 M KCl-50 mM Tris-HCI, pH 8.0. The molecular weight markers used were, A-bovine thyroglobulin (669 kDa), B-horse spleen apoferritin (443 kDa), C-pig heart fumarase (194 kDa), D-rabbit muscle GAPDH (144 kDa), E-bovine serum albumin (66 kDa), F-egg albumin (45 kDa), G-bovine erythrocyte carbonic anhydrase (29 kDa) and H-horse heart cytochrome c (12.5 kDa). Thermal stability. The thermal inactivation profile of GAPDH was obtained as follows: screwcapped tubes contained 800 ul of enzyme (100 µg/ml) in basic in- cubation buffers of 1 M KCI, 1 M (NH4)2SO4 or 4 M KCl prepared in 50 mM Tris- HCI, pH 8.0. The tubes with the different additions were incubated at the temperatures indicated in Fig. 5. At 5-min intervals a sample was taken from each tube, which was quickly reclosed. The samples were cooled in ice and immediately assayed for activity. Effect of pentalenolactone. 100 µg of pentalenolactone dissolved in 100,u1 of 2.5 M KCl-50 mM Tris-HCI, pH 8.0 was used for testing the effect on the hGAPDH activity. Amino acid analysis. Purified enzyme (1 mg), desalted on a Sephadex G-25 column (5 x 1.5 cm), was freeze dried. The sample was dissolved in performic acid, oxidized and evaporated (36). After addition of 1 ml of 6 N HCl the samle was divided into two ampoules. These were evacuated and sealed, then one ampoule was hydrolyzed for 24 h and the other for 48 h. They were analyzed with a Beckmann 119 CL automatic amino acid analyzer. Identification of COOH- and NH2-terminated residues. The COON-terminal residues of hGAPDH were identified by digestion with carboxypeptidase A (4). GGAPDH (3 mg) was desalted and lyophilized then treated with carboxypeptidase A in 0.4 M NaHCO3, pH 7.6 to obtain an enzyme: substrate molar ratio of 1: 400, in a volume of 0.3 ml. Samples (60,u1)were removed at time intervals ranging from 0 to 3 h and added to 5 ,ul of glacial acetic acid. The amino acids released in the supernatant were identified using thin layer chromatography. Reaction mixtures containing only carboxypeptidase A were treated in a similar manner to correct for any autodigestion. The NH2-terminal was determined by dansylation (22), using 500ug enzyme. The dansylated amino acid was identified by using thin layer chromatography developed in water-90% formate (200:3) and benzene-pyridine-acetic acid 1990 Halobacterial Glyceraldehyde-3-phosphate Dehydrogenase 23

(80:20:2). Immunological cross reactivity. Antiserum to hGAPDH was developed in a rabbit by subcutaneously injecting 500 pg of desalted protein in complete Freund's adjuvant. After a fortnight, the injection was repeated. Blood samples were collected two weeks later, and the antisera obtained were stored at - 20°C. Ouchterlony double diffusion tests were performed at 25°C, in 1% agar gels in 10 mM Tris-HCI, pH 7.4 (37). Crude extracts and pure enzyme preparations containing 0.2 to 1 U of GAPDH were used against varied amounts of antiserum (0, 8, 12 and 15 #1). After the precipitin lines were formed, the gels were washed, with many changes of normal saline, over a period of 18 h„ The gels were fixed and stained in Coumassie blue. hGAPDH as a coupling enzyme. The halophilic GAPDH was tested as coupling enzyme in the spectrophotometric assay of halophilic aldolase. The reaction mixture (1 ml) contained 2.5 M KC1--75mM sodium pyrophosphate-16 mM sodium arsenate-5 mM BME-1 mM NAD +-2 mM reduced glutathione-50 mM Tris- HCI, pH 8.0 and halobacterial aldolase. The aldolase reaction was started by adding 5 mM FDP. After 5 min, 1-2 units of hGAPDH was added to the reaction mixture and the NADH formed was measured spectrophotometrically at 25°C. hAldolase activity was also measured colorimetrically by the Sibley and Lehninger (42) method modified by D'Souza and Altekar (13).

RESULTS

Purification and molecular weight By using the purification procedure described here, 43.6% of the original GAPDH activity present in the crude extract of H. vallismortis was recovered (Table 1). Although hGAPDH is a NAD+-specific enzyme, affinity chromatography on NAD +-Sepharose did not show any advantages over the steps described (unpublished data). Polyacrylamide gel electrophoresis was used to determine the purity and the subunit size of hGAPDH. That the purified enzyme was homogenous was apparent from SDS and CTAB-PAGE patterns (Fig. 1). During SDS gel electrophoresis, acidic proteins may exhibit reduced mobilities compared to marker proteins, apparently due to the binding of less SDS. Hence the molecular weight Table 1. Purificationof GAPDHfrom H. vallismortiscells. 24 KRISHNAN and ALTEKAR VOL. 36

Fig. L SDS- and CTAB-PAGE. Lanes 1, 2 and 1 +2, represent hGAPDH, rabbit muscle GAPDH and their coelectrophoresis respectively, in presence of SDS. Lanes 3 and 4 represent rabbit muscle GADPH and hGAPDH; lane 3+4 represents coelectrophoresis of the two when SDS was replaced by CTAB. of the enzyme subunit may be overestimated (15, 26). Therefore polyacrylamide gel electrophoresis in the presence of a cationic detergent, CTAB, was considered essential for ascertaining the subunit molecular mass of the enzyme. Figure 1 shows that the mobility of hGAPDH on SDS-PAGE (lanes 1, 2 and 1 + 2) was much slower than the rabbit muscle enzyme, and the estimated molecular weight of the halobacterial enzyme was 52±2 kDa as against 36 kDa for the mammalian enzyme. Though the movement of hGAPDH on CTAB-PAGE (lanes 3, 4 and 3+4) was certainly slower than that of the rabbit enzyme, the difference between their mobilities was not so striking as on SDS-PAGE and the molecular weight of hGAPDH was 40 + 2 kDa. From the coelectrophoretic pattern of the halobacterial and mammalian GAPDHs it is evident that hGAPDH is the larger of the two. Figure 2 shows the gel filtration pattern of hGAPDH on Sepharose CL-6B. From the elution volumes, the molecular weight of the native enzyme correspond to 190 ± 10 kDa. The electrophoresis data indicates that hGAPDH is a homomeric tetramer.

Catalytic properties The effect of various salts on hGAPDH activity are shown in Fig. 3. The activity was enhanced by increasing concentrations of RbCI, KCI, NaCI, and NH4C1. Optimum enzyme activity occurred in 3 M RbCI. Since RbCI is not a physiological salt, KCl was considered the best salt activator in these studies. The enzyme activity was only partially enhanced by LiCI, and (NH4)2SO4 inhibited at 1 M (Fig. 3). hGAPDH was very stable in 4 M KCl but was inactivated within 12 h in 0.5 M KCl-50 mM Tris-HCI, pH 8.0, at 25°C. Adding 40 mM K2HPO4 to the enzyme in 0.5 M KCl buffer lowered the inactivation and 70% activity remained after 72 h. The loss of 50% hGAPDH activity in 1.5 M KCl-50 mM Tris-HCI, pH 8.0 could 1990 Halobacterial Glyceraldehyde-3-phosphate Dehydrogenase 25

Fig. 2. Molecular weights determined by gel filtration on Sepharose CL-6B. The molecular weight markers used were: A, thyroglobulin (669 kDa); B, apoferritin (443 kDa); C, fumarase (194 kDa); D, rabbit muscle GAPDH (l44 kDa); E, bovine serum albumin (66 kDa); F, egg albumin (45 kDa); G, carbonic anhydrase (29 kDa) and H, cytochrome c (12 kDa). X, the molecular weight of hGAPDH was calculated from its elution volume.

Fig. 3. Salt-dependent activity of /iGAPDH. Protein concentration in assay 4 pg. The standard assay medium was used in the presence of different salts: (U) RbCI; (•) KCI; (0) NaCI; (o) NH4C1; (X) LiCI; (o) (NH4)2SO4. 26 KRISHNAN and ALTEKAR VOL. 36

Fig. 4. Effect of salt on enzyme stability. The enzyme was incubated in 50 mM Tris- HCI, pH 8.0 buffers containing different salts. Protein concentration 50 ug/ml. (•) 1.5 M KCl-1 M(NH4)2SO4; (O)0.5M KCl-40 mM P043-; (X) l.5 M KCI; (A) 1 MKCI; (U)0.5M KCI.

Fig. 5. Salt-dependent thermal inactivation. (0) 1 M KCI; (o) 1 M (NH4)2SO4; (o) 4 M KCI.

be prevented by adding 1 M (NH4)2SO4 to the buffer containing 1.5 M KCl (Fig. 4). Figure 5 shows the salt-dependent thermal inactivation of hGAPDH at the end of 5 min. The temperature of 50% enzyme inactivation was increased by 15°C, when KCl concentration was increased from 1 M to 4 M or when 1 M KCl was replaced by 1 M (NH4)2SO4. The optimum pH for hGAPDH in Tris-HC1 buffers was 8.6. The Km value of hGAPDH for NAD + was 0.25 mM with 4 mM glyceraldehyde-3-phosphate. In the "active," dehydrated form of the aldehyde (44) with 0.25 mM NAD +, the Km for glyceraldehyde-3-phosphate was 53 µM. We examined the effect of sulfhydryl reagents or compounds and heavy metal ions on hGAPDH activity. It was completely inhibited in 5 mM iodoacetamide or p-chloromercuribenzoate or in 1 mM HgClz or CuC12. hGAPDH activity depended on sulfhydryl compounds, such as BME or reduced glutathione, and the activity was maximal at 5 mM of the former.

Sensitivity to pentalenolactone Pentalenolactone, an antibiotic, is a potent inhibitor of glycolytic and gluconeogenetic pathways in prokaryotes and eukaryotes (33) due to the irreversible inactivation of GAPDHs. The concentration of pentalenolactone was determined by first testing the inhibition of rabbit muscle GAPDH (34) in 1 ml of standard assay medium devoid of salt. 1990 Halobacterial Glyceraldehyde-3-phosphate Dehydrogenase 27

Table 2. Amino acid composition of GAPDH from H. vallismortis, M. fervidus (16) and B. stearothermophilus (45).

hGAPDH was insensitive to pentalenolactone even when 100,ug of the antibiotic was added to the assay system, while the rabbit muscle enzyme was inhibited at 12 ng concentrations.

Amino acid composition Table 2 shows the amino acid composition of hGAPDH. It was not determined whether glutamine or asparagine were present. Tryptophan and tyrosineval- ues were determined by the spectrophotometric method of Bencze and Schmid (6).

COOH- and NH2-terminal residues In carboxypeptidase digestion, leucine was the first amino acid to be released, followed by phenylalanine. The third amino acid liberated, at the end of 30 min, was identified as alanine. Hence the COON-terminal sequence was -Ala-Phe- Leu-COOH. The NH2-terminal was identified as methionine.

Immunological cross reactivity Antiserum raised against the hGAPDH from H. vallismortis, were tested for cross reaction with enzymes from Escherichia coli (NCIM 2065), Lactobacillus casei 28 KRISHNAN and ALTEKAR VOL. 36

(ATCC 7469), Staphylococcus aureus (ATCC 12600), rabbit muscle and a few other halobacteria. The antiserum did not cross react with the nonhalobacterial GAPDHs. Precipitin lines of identity were formed with the enzyme from Halobacterium marismortui (Ginsburg's strain), while a non-identical reaction was observed with the GAPDH from Halobacterium saccharovorum (ATCC 29252). Antisera raised to GAPDHs from methanogenic and thermophilic archaebacteria did not cross react with the hGAPDH.

Halophilic GAPDH as a coupling enzyme One of the spectrophotometric assay methods for FDP aldolase involves coupling this activity to that of GAPDH (7). We tried to couple the aldolase activity (crude extract) from H. vallismortis with hGAPDH in a similar assay under halophilic conditions, and the results obtained were in good agreement with the colorimetric assay of aldolase (data not shown).

DISCUSSION

The foregoing data gives the first description of a halobacterial GAPDH from H. vallismortis thus allowing further insights into the enzyme's differentiation from the archaebacterial urkingdom. Only one type of enzyme requiring NAD + as cofactor has been reported so far for all eubacteria and animals (23). They are homomeric tetramers of molecular weight 144 kDa. However the GAPDHs from plant chloroplasts and blue green alga are also active with NADP+. The hGAPDH was NAD+-specific. Our earlier work (40) has shown that in halobacteria there is no production of an NADP +-dependent GAPDH when growth conditions were altered. This was true of Haloferax mediterranei in which we have demonstrated the presence of CO2 fixation enzymes of the Calvin cycle under heterotrophic growth conditions (3,41). GAPDH from Methanothermus fervidus grown autotrophically could use both NAD + and NADP + as cofactors and its amino acid sequence was reported to be homologous with the eubacterial and eukaryotic enzymes (18). Whereas in another archaebacterium, Thermoproteus tenax (24) two different GADPHs, one specific for NAD+ and other for NADP+ were present when grown under heterotrophic or autotrophic (Calvin cycle absent) conditions, and the NADP + enzyme showed significant homologies with GAPDHs from eubacteria and eukaryotes. Nonetheless hGAPDH like the GAPDHs from these two different groups of archaebacteria is not inhibited by pentalenolactone. The NAD + enzymes from nonarchaebacterial sources are usually inhibited by this antibiotic (33). The relative mass of native hGAPDH was found by gel filtration to be 190,000. The relative mobilities of hGAPDH subunit in SDS and CTAB gave different values for the molecular weight, viz 52 + 2 and 40 + 2 kDa respectively. That the values for the molecular weights were different on the cationic and anionic gels is indicative of the acidic nature of hGAPDH. Since acidic proteins exhibit anomalous migration on SDS gels (15, 26), the value of 40 kDa may be considered 1990 Halobacterial Glyceraldehyde-3-phosphate Dehydrogenase 29

Table 3. Comparison of amino acid content of acidic and basic residues of GAPDHs from various sources.

closer to the enzyme subunit size. From the co-electrophoresis patterns of the halobacterial and mammalian GAPDH it is evident that hGAPDH is the larger of the two, though the difference is not great. But the subunit size of hGAPDH falls within the range 37 to 49 kDa, as also reported for its counterparts from the other two archaebacterial groups (17, 18, 24). However it would be better to use alternate methods to determine hGAPDH molecular weight. The lack of immunological cross reaction of the hGAPDH antiserum with the GAPDHs from rabbit muscle, E, coli, or L. casei suggested large evolutionary distances between them. Whether hGAPDH shares homology with other characterized enzymes requires amino acid sequence information about the halobacterial protein. No immunological cross reactivity could be detected between the GAPDH from H. vallismortis and the antisera to GAPDHs from other nonhalophilic archaebacteria viz, M. fervidus, Met hanobacterium formicicum, Methanobacterium bryantii and T. tenax. There was no antigenic homology between the GAPDHs from M. fervidus and T. tenax (17). The available information on the nature of halobacterial proteins in comparison to their nonhalophilic counterparts shows that the former usually have more acidic residues than the latter (31). The acidic residue generally present on the surface enhance the hydration properties and stability of the protein at high KCl concentrations (39). Thus the preponderance of acidic residues over basic ones is a characteristic of halophilic proteins (28, 31, 32, 35). Table 3 shows a comparison of ratios of acidic/basic amino acids of hGAPDH with GAPDHs from a variety of sources: E. coli (9), yeast (1), lobster (9), pig (1) and M. fervidus (17), the last one being an archaebacterium. The value of 3.9 obtained for the H. vallismortis enzyme is much higher than the range of 1.5 to 2.2 obtained for other GAPDHs. The N-terminal of hGAPDH was identified as methionine which is similar to that reported for eukaryotes and methanogenic bacteria (18). Eubacterial GAPDHs have a leucine residue at the C-terminal end, and the enzyme from H. vallismortis also had leucine at the C-terminal position. The catalytic properties of hGAPDH were more or less similar to those reported for the NAD + -specific enzyme from other sources with respect to optimum pH, 30 KRISHNAN and ALTEKAR VOL. 36 requirement of free-SH groups and Kmfor glyceraldehyde-3-phosphate. As expected for a halobacterial enzyme hGAPDH was halophilic. The enzyme also required high KCl concentrations for stability. Halophilic enzymes exhibit greater stability in the presence of salting out type salts (2). Thus P043 - was highly effective in reducing the time-dependent inactivation of hGAPDH in moderate KCl concentration. hGAPDH was stable for several months in 2.5 M KC1-200 mM K2HP04, pH 8.0. A parallelism between halophilicity and thermophilicity has often been observed in halophilic proteins (27). However hGAPDH did not exhibit any thermophilic character since it was inactivated within 4 min at 56°C in 2.5 M KCI. The temperature of 50% enzyme inactivation was raised when the hGAPDH was exposed to higher KCl concentration or when KCl was replaced by a salting out type salt. Since NAD+ does not stabilize the enzyme any further, it appeared that conditions that favour the hydrophobic interactions are responsible for the thermostability of the hGAPDH. The enhanced stabilization at higher temperatures in the presence of phosphate also occurs in GAPDHs from thermophilic and methanogenic bacteria (17, 24). Many members of the Methanobacteriaceae have elevated levels of KCl but it is not known whether M. fervidus also has equally high internal K } concentration (17). Use of commercially available enzymes in coupled assays poses many problems since most commercial enzymes are not active at the high salt concentrations essential for the activity of a halophilic enzyme. We were successful in using the halophilic GAPDH as a coupling enzyme. It may be worthwhile to purify other halobacterial enzymes to use as analytical reagents. Further studies on the amino acid sequence would help to understand the features that contribute to its halophilic nature and help comparisons among enzymes from the three archaebacteria) groups and other sources. The authorsare gratefulto Dr. G. Zaccai(France) for usefulsuggestions during the course of this work.Pentalenolactone was kindly given by Prof. D. Meckeand Dr. M. Weidmann(FRG). Antisera to GAPDHsfrom thermophilicarchaebacteria and methanogenswere kindlygiven by Dr. R. Hensel (FRG).

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