Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase

Antibodies against the alpha (Mr 67,000) and beta (Mr 60,000) subunits of wheat seedling Fru-2,6-P2-stimulated pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) were used to probe the subunit structures of several partially purified plant PFPs after tryptic digestion. Antisera to the alpha and beta subunits of wheat seedling PFP cross-reacted with the corresponding alpha and beta subunits of PFP preparations from wheat germ, potato tubers, and lettuce leaves. With the mung bean PFP, both antisera reacted with a protein band of Mr 60,000. A protein band corresponding to the Mr 67,000 alpha subunit was not detected in the mung bean PFP preparation. Tryptic digestion of wheat seedling and potato tuber PFPs resulted in the preferential cleavage of the alpha subunit. The trypsinized PFP retained most of its Fru-2,6-P2-stimulated activity but not its basal activity. The proteolyzed enzyme also exhibited a 2-fold increase in Ka for Fru-2,6-P2. Studies with the mung bean enzyme revealed that the anti-alpha immunoreactive component was more sensitive to trypsinization than the anti-beta immunoreactive component of the Mr 60,000 protein band. Thus, the Mr 60,000 protein band of the mung bean PFP appears to be heterogeneous and contains both alpha and beta-like proteins. The above observations indicate that the alpha and beta subunits of PFP are two distinct polypeptides and that alpha acts as a regulatory protein in regulating both the catalytic activity and the Fru-2,6-P2-binding affinity of the beta subunit.     

Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12.

Acetohydroxyacid synthase I from Escherichia coli K-12 has been purified to near homogeneity. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two polypeptides, one with a molecular weight of 60,000 and one with a molecular weight of 9,500. These two polypeptides were present in constant proportion to each other and to enzyme activity. The molar ratio of the two polypeptides (Mr 9,500:60,000), estimated from stained polyacrylamide gels, was 1. Antisera raised against the 60,000 Mr polypeptide precipitated both the 60,000 and the 9,500 Mr polypeptides from extracts of cells labeled with [35S]methionine. The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide, and only the larger one was recovered. The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species has a molecular weight of about 200,000; this species contains both the 60,000- and 9,500-molecular-weight polypeptides.     

Molecular Comparison of Pyrophosphate- and ATP-Dependent Fructose 6-Phosphate 1-Phosphotransferases from Potato Tuber

The aim of this work was to compare the molecular properties of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) and ATP:fructose 6-phosphate 1-phosphotransferase (PFK). Both enzymes were purified to apparent homogeneity from potato tubers (Solanum tuberosum cv Record). Neither PFP nor PFK preparations contained detectable activity of the other enzyme. PFP was composed of two polypeptides of apparent molecular weight 58,000 and 55,700 whereas PFK contained four polypeptides of apparent molecular weight between 46,300 and 53,300. Chemical cleavage of individual PFP and PFK polypeptides gave a different set of fragments for each polypeptide. On Western blots antisera against PFP failed to cross-react with any of the four PFK polypeptides, and antibodies against PFK failed to bind to either of the PFP polypeptides. Antibodies that immunoprecipitate PFP activity had no effect on PFK activity. Conversely, antibodies against the four PFK polypeptides precipitated the activity of PFK, but not that of PFP. This work shows that potato tuber PFP and PFK are composed of distinct, unrelated polypeptides and indicate that interconversion between PFP and PFK is unlikely.     

Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells.

PP(i)-dependent phosphofructokinase (PFP) activity, measured in the forward direction, increased approximately 19-fold when suspension cell cultures of black mustard (Brassica nigra) were subjected to 18 days of P(i) deprivation. Fructose 2,6-bisphosphate (2 microM) elicited a 10-fold activation of PFP from P(i)-deficient cells, compared to only a 2-fold activation of the enzyme from nutrient-sufficient cells. Also, PFP from P(i)-starved cells exhibited a greater affinity for the activator (Ka = 0.09 microM) than the enzyme from nutrient-sufficient cells (Ka = 0.32 microM). Western blots of extracts from P(i)-deficient cells were probed with rabbit anti-(potato tuber PFP) immune serum and revealed equal intensity staining immunoreactive polypeptides of M(r) 66,000 (alpha-subunit) and 60,000 (beta-subunit) that co-migrated with the alpha- and beta-subunits of homogeneous potato tuber PFP. By contrast, only the M(r) 60,000 beta-subunit was observed on immunoblots of extracts prepared from nutrient-sufficient cells. Quantification of immunoblots indicated that in black mustard cells experiencing transition from P(i) sufficiency to deficiency or vice versa, the relative amount of immunoreactive alpha-subunit correlated with the degree of activation of PFP by fructose 2,6-bisphosphate. These observations provide additional evidence that (i) plant PFP is an adaptive enzyme that may function in glycolysis during P(i) deprivation, and (ii) the alpha-subunit acts as a regulatory protein in controlling the catalytic activity of the beta-subunit and its regulation by fructose 2,6-bisphosphate.     

Heterogeneity of mammalian DNA ligase detected on activity and DNA sequencing gels.

A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed. After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP. Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography. Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity. Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells. The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000. Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity. Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000.     

Biosynthesis of Rauscher leukemia viral proteins

Antisera to disrupted Rauscher leukemia virus (RLV) or to the purified Rauscher viral 30,000 dalton polypeptide were used to specifically precipitate newly synthesized intracellular viral polypeptides from extracts of infected NIH Swiss mouse cells (JLS-V16). Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from cells pulse-labeled for 10–20 min with ³⁵S-methionine showed that immune precipitates contained none of the nonglycosylated internal structural polypeptides of mature viruses. The major viral-specific polypeptides labeled in 10 min included polypeptides of 180,000, 140,000, 110,000, 80,000, and 60,000 daltons with minor polypeptides of 65,000, 50,000, and 40,000 daltons. Labeling the intracellular virus-specific polypeptides with ¹⁴C-glucosamine indicated that the 180,000, 110,000, 80,000, and 60,000 dalton polypeptides were glycosylated, and all but the 110,000 dalton polypeptides are contained in the mature virions. Based on pulse-chase experiments, it appears that at least 3 of the large polypeptides (140,000, 65,000, and 50,000 daltons) are precursors to the three major internal structural polypeptides of the mature virions.     

Immunological comparison of purified DNA polymerase alpha from embryos of Drosophila melanogaster with forms of the enzyme present in vivo

Specific antisera to purified DNA polymerase alpha from embryos of Drosophila melanogaster and to two of the four constituent subunits (alpha, beta, gamma, and delta) were prepared. These antibodies have revealed the following features of the enzyme. (i) The Mr = 148,000 alpha subunit is very likely derived by in vitro proteolysis from polypeptides with molecular weights of 185,000 and 166,000 that are present in vivo. (ii) The Mr = 60,000 beta subunit occurs in rapidly replicating embryos as both an 85,000- and a 60,000-dalton form, but predominantly as a 60,000-dalton form in more slowly replicating cultured cells. (iii) There is no detectable immunologic cross-reactivity between the four subunits. (iv) There is an abundance of antigenic material in embryos that co-migrates with the delta subunit of the purified enzyme during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Comparison of adipose glycerol kinase of hyperglycemic obese mice and lean litter-mates

Summary Glycerol kinase activity found in the epididymal adipose tissue of lean litter-mates of hyperglycemic obese mice exhibits two distinct Km values. The Km(s) obtained graphically by a Hoftsee plot are 40 and 637 m. The glycerol kinase of obese mice showed 29 times more total activity per fat pad or 9 times more activity per mg of protein as compared to that of the lean controls. The increased glycerol kinase activity found in the obese mice predominantly exhibited low Km value. Increase in the activity with the high Km value was minimal. The apparent molecular weight of adipose glycerol kinase is approximately 60,000–65,000. A higher activity of glycerol kinase in the epididymal adipose tissue of the obese mice (ob/ob) has been reported by Treble and Mayer ¹. The significance of this increased enzyme in obesity² has been investigated by many investigators^3–6, using either whole epididymal fat pad or isolated fat cells as the source of glycerol kinase. However, little is known regarding the properties of this enzyme, and even the characterization of the liver glycerol kinase is not yet complete^{7, 8}.We have recently shown that adipose glycerol kinase of Sprague-Dawly rat and of Swiss mice exhibit two Km values, and the apparent molecular weight is approximately 55,000–60,000⁹. The present study was undertaken in order to establish whether the enzyme in ob/+ mice also exhibits two Km values and if so, whether the activity associated with both Km(s) is increased. We found that the specific conditions for enzyme activity determination and the basis on which the activity is expressed are quantitatively important in clarifying the difference in glycerol kinase activity of ob/ob and ob/+ mice. The increased activity in ob/ob mice is predominantly the low Km enzyme, and the apparent molecular weight is approximately 60,000–65,000.Supported in part by the Juvenile Diabetes Foundation. We thank Mr. CHARLES ROSENTHAL for technical assistance.     

The dephosphorylation of lens alpha-crystallin A chain

The present communication reports the presence of a phosphoprotein phosphatase activity in bovine lens preparations which dephosphorylates alpha Ap, the phosphorylated form of alpha A, one of the alpha-crystallin polypeptides, in a Ca2+/calmodulin dependent manner. The activity was found in soluble preparations from epithelial cells but it could not be detected in similar preparations from fiber cells. A 60,000 Mr calmodulin binding polypeptide and a 15,000 Mr polypeptide found in the epithelial cell preparations comigrated in SDS-PAGE with the A and B subunits of bovine brain calcineurin (phosphoprotein phosphatase 2B) respectively. The 15,000 Mr was specifically recognized by an anti-bovine brain calcineurin antiserum. Bovine brain calcineurin was as effective in dephosphorylating alpha Ap as the lens preparations. Thus, it is likely that the activity present in the lens is related to this enzyme. The results indicate that the lens specific polypeptide alpha A may be subject to metabolic control through phosphorylation and dephosphorylation pathways regulated by cAMP and calcium respectively. Changes in the activities of these pathways appear to occur during differentiation of the lens epithelial cell and may be related to gene regulation during the differentiation process.     

Large polypeptides of 10S DNA polymerase alpha from calf thymus: rapid isolation using monoclonal antibody and tryptic peptide mapping analysis

The polypeptides recognized by a monoclonal antibody against calf thymus DNA polymerase alpha (secreted from a hybridoma CL22 -2- 42B , Nucleic Acids Res. (1982) 10, 4703-4713) were identified by the immunoblot method as the large polypeptides of the partially-purified 10S DNA polymerase alpha fraction. Using an immunoprecipitation technique with the monoclonal antibody, a rapid immunological isolation of the polypeptides has been achieved. By this method, the large polypeptides with Mr = 140,000, 145,000, and 150,000 were isolated from a partially-purified preparation of 10S DNA polymerase alpha. On the other hand, the polypeptides with Mr = 150,000, 180,000, and 240,000 were obtained from a crude extract of calf thymus. Tryptic peptide maps showed that the large polypeptides with Mr = 150,000, and 180,000 were very similar in primary structure and that the structures of Mr = 180,000 and 240,000 polypeptides contained partially common sequences. Among these polypeptides, the Mr = 150,000 polypeptide was shown to correlate with the enzyme activity. These results suggest that the large polypeptide of 10S DNA polymerase alpha is initially synthesized as Mr = 180,000 or larger polypeptide, then converted to the form with Mr = 150,000. The Mr = 140,000 and 145,000 polypeptides in the purified preparation may be artificial products formed during purification.     

Separation and characterisation of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum.

The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.     

Immunological Characterization of the Pyrophosphate Dependent Fructose-6-Phosphate Phosphotransferase

The pyrophosphate dependent phosphofructokinase (PFP, EC 2.7.1.90 [EC] )was purified from potato tubers, bean seeds and cucumber seeds.The PFP of all three species appears to contain two subunitswith a molecular weight of approximately 60,000 and 66,000 dalton.The purified proteins were used as the antigens to produce polyclonalantibodies in rabbits. Two of the obtained sera (anti-potatoPFP and anti-cucumber PFP) proved to be monospecific for thePFP polypeptides on protein blots. The antipotato serum crossreacts with the PFP from all the tested higher plant specieson protein blots, but no cross reaction with the PFP of Propionibacteriumsharmanii was found. This shows that the PFP subunits from thehigher plant species have similar antigenic determinants inthe primary structure but differes largely from that of thePropionibacterium. The differences observed in the efficiencyof the sera to inactivate the PFP from the different species,however, indicate that the surface antigenic determinants onthe native PFP enzymes differ between the higher plant speciesand even within the Cucurbitaceae. (Received June 15, 1987; Accepted November 20, 1987)     

The identification of specific Rhesus-polypeptide-blood-group-ABH-active-glycoprotein complexes in the human red-cell membrane.

1. RhD,c and E immune complexes isolated from 3H- and 125I-surface-radiolabelled and unlabelled intact human red cells were analysed by SDS/polyacrylamide-gel electrophoresis. 2. Apparent Mr values of 31,900 for RhD polypeptide and 33,100 for Rhc,E polypeptide were obtained under both reducing and non-reducing conditions. Glycosylation of RhD,c and E polypeptides was not detected. 3. RhD,c and E immune complexes also contain a glycoprotein component. RhD glycoprotein (apparent Mr 45,000-100,000) is distinct from Rhc,E glycoprotein(s) (apparent Mr 35,000-65,000). Rh (Rhesus) glycoprotein carbohydrate moieties are susceptible to endo-beta-galactosidase digestion and carry blood-group-ABH determinants. This suggests the presence of polylactosaminoglycan-type structures. 4. Rh glycoproteins are not present in Rh immune complexes as a result of non-specific adsorption of membrane glycoproteins during the membrane-solubilization phase of immune-complex isolation because RhD immune complexes isolated from a 1:1 (v/v) mixture of Acde/cde and OcDE/cDE red cells do not contain blood-group-A-active glycoprotein. 5. Blood-group-A immune complexes isolated from group-A red cells of the appropriate Rh phenotypes contain the 31,900- and 33,100-apparent-Mr Rh polypeptides. 6. It was concluded from the above evidence that non-covalent Rh-glycoprotein-Rh-polypeptide complexes exist in the native red-cell membrane. 7. The 31,900- and 33,100-apparent-Mr Rh polypeptides are absent from blood-group-A immune complexes isolated from regulator type Rhnull cells (donor A.L.), but are replaced by a 33,800-apparent-Mr Rhnull-specific polypeptide (Rhnull polypeptide). It is suggested that Rhnull polypeptide is an aberrant product of the Rh gene complex.     

Quaternary structure of pyruvate carboxylase from Pseudomonas citronellolis.

Physical-chemical studies of pyruvate carboxylase from Pseudomonas citronellolis demonstrate that the enzyme has an alpha 4 beta 4 structure. The individual polypeptides, alpha (Mr = 65,000) and beta (Mr = 54,000), were separated and isolated by preparative gel electrophoresis. Analysis of the relationship between Coomassie blue staining and protein quantity for each polypeptide indicated that the alpha and beta subunits are present in a 1:1 stoichiometry in the native enzyme. Determinations of the molecular weight of the protein by sedimentation equilibrium (Mr = 454,000), gel filtration analysis (Mr = 510,000), disc gel electrophoresis (Mr = 530,000), and mass measurement from the Scanning Transmission Electron Microscope (Mr = 530,000) are consistent with the proposed alpha 4 beta 4 structure. Disc gel electrophoresis studies revealed that under certain circumstances the enzyme may dissociate to a smaller molecular weight species (Mr = 228,000). This dissociation phenomenon could explain the earlier reported observation of Taylor et al. ((1972) J. Biol. Chem 22, 7388-8390) that the enzyme had a molecular weight of 265,000. Evidence from electron microscopic studies shows that the three-dimensional structure of this enzyme is quite distinct from other species of pyruvate carboxylase. The enzyme does not show the typical rhombic appearance which has been noted for chicken liver, sheep liver, and yeast pyruvate carboxylase.     

The moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome cL. In this study, four polypeptides of Mr 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement of the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the Mr-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the Mr-20,000 polypeptide, was identified as mature cytochrome cL, and the product of moxI, the Mr-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the Mr-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the Mr-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the Mr-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the Mr-60,000 MeDH polypeptide. Our data suggest that both the Mr-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.     

Regulation of lysosomal alpha-mannosidase-1 synthesis during development in Dictyostelium discoideum

The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development.     

Acetyl-coenzyme-A carboxylase from rat liver. Subunit structure and proteolytic modification.

The subunit structure of rat liver acetyl-coenzyme-A carboxylase has been studied by polyacrylamide gel electrophoresis in the presence of dodecylsulfate. A number of individual preparations of the enzyme purified by the same procedures exhibited three different types of electrophoretic patterns as follows: first, a single slow-moving protein bands (Mr 230000); secondly, two adjacent fast-moving protein band (M4 124000 and 118 000); finally, all three protein bands. With the use of the [14C]biotin-labelled enzyme, the biotinyl prosthetic group was shown to be associated with the polypeptide of 230000 Mr as well as with that of 124000 Mr, but not with the polypeptide of 118000 Mr. Studies were next made with the labelled enzyme to examine the possibility that the two light polypeptides might have been formed by proteolytic modification of the heavy polypeptide during the procedures used for the purification of the enzyme. Treatment of the enzyme with trypsin or chymotrypsin resulted in cleavage of the heavy polypeptide into two nonidentical polypeptides with molecular weights of approximately 120000. Incubation of the enzyme with proteases derived from rat liver converted the heavy polypeptide into lighter polypeptides of 80000-130000 Mr. Acetyl-CoA carboxylase isolated from crude rat liver extracts by means of immunoprecipitation with specific antibody invariably showed only the heavy polypeptide. The biotin content of the enzyme was found to be 1 mol per 237000 g protein. These results indicate that rat liver acetyl-CoA carboxylase, unlike bacterial and plant biotin enzymes, has only one kind of subunit, which has a molecular weight of 230000 and contains one molecular of biotin. Thus, the mammalian enzyme exhibits a highly integrated subunit structure.     

Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12

Hydrogenase isoenzyme 1 from the membrane fraction of anaerobically grown Escherichia coli has been purified to near homogeneity. The preparation involved dispersion of the membrane fraction with deoxycholate followed by ammonium sulphate precipitation, ion-exchange, hydroxyapatite and gel filtration chromatography steps. The enzyme was assayed by quantification of the H2:benzyl viologen oxidoreductase activity immunoprecipitated by a non-inhibitory antiserum specific for the enzyme. The enzyme constituted about 8% of the hydrogenase activity found in the detergent-dispersed membranes, the remainder being attributable to hydrogenase isoenzyme 2. Isoenzyme 1 was purified 130-fold and the specific activity of the final preparation was 10.6 mumol benzyl viologen reduced min-1 (mg protein)-1 (H2:benzyl viologen oxidoreductase). The final preparation contained polypeptides of apparent Mr 64,000, 31,000 and 29,000. Antibodies were raised both to the final preparation and to immunoprecipitation arcs containing hydrogenase isoenzyme 1, excised from crossed immunoelectrophoresis plates. The former cross-reacted with all three polypeptides in the enzyme preparation but the latter recognised only the Mr-64,000 polypeptide. Immunological analysis revealed that the polypeptides of apparent Mr 31,000 and 29,000 are fragments of a single polypeptide of Mr 35,000 which is present in the detergent-dispersed membranes. The fragmentation of the Mr-35,000 polypeptide during the preparation correlates with a change in the electrophoretic mobility of the enzyme. A similar electrophoretic mobility change was observed, accompanied by cleavage of the Mr-35,000 polypeptide to one of 32,000 when the enzyme was analysed after exposure of detergent-dispersed membranes to trypsin. The enzyme in the detergent-dispersed membranes consists minimally of two subunits of Mr 64,000 and two subunits of Mr 35,000. It contained 12.2 mol Fe and 9.1 mol acid-labile S2-/200,000 g enzyme. The enzyme, purified from bacteria grown in the presence of 63Ni, was found to contain 0.64 (+/- 0.20) mol Ni/200,000 g enzyme. A constant ratio of 63Ni immunoprecipitated to hydrogenase isoenzyme 1 activity immunoprecipitated by antiserum specific for the enzyme was observed during the preparation, consistent with Ni being part of the enzyme. The enzyme has a low Km for H2 (2.0 microM) in the H2:benzyl viologen oxidoreductase assay. It catalyses H2 evolution employing reduced methyl viologen as electron donor. It is inhibited reversibly by CO and irreversibly by N-bromosuccinimide.