The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.     

Purification and characterization of inositol 1,4,5-trisphosphate 3-kinase from pig aortic smooth muscle.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase, which phosphorylates InsP3 to form inositol 1,3,4,5-tetrakisphosphate, was purified to apparent homogeneity by (NH4)2SO4 fractionation and sequential chromatographic steps on DEAE-sepharose, calmodulin-Affi-Gel and DEAE-5PW h.p.l.c. The purified enzyme had a specific activity of 24.4 nmol of inositol tetrakisphosphate formed/min per mg of protein, which represented a purification of approx. 195-fold with a 0.29% recovery, compared with the cytosol fraction of the muscle. SDS/polyacrylamide-gel electrophoresis showed a single protein-staining band of Mr 93,000. Moreover, the major protein peak, of Mr 84,000, was detected by TSK gel G3000SW gel-permeation chromatography of the purified sample. As this value was approximately consistent with the Mr determined by SDS/polyacrylamide-gel-electrophoretic analysis, the InsP3 3-kinase might be a monomeric enzyme. The purified enzyme had a Km for InsP3 of 0.4 microM, with an optimum pH range of 5.8-7.7. The enzyme was maximally activated by calmodulin, with a stoichiometry of 1:1.     

Quaternary structure of erythrocruorin from the nematode Ascaris suum. Evidence for unsaturated haem-binding sites.

The quaternary structure of erythrocruorin from the nematode Ascaris suum was studied. The native protein had a sedimentation coefficient, at a protein concentration of 1 mg/ml, of 11.6 +/- 0.3 S and an Mr, as determined by sedimentation equilibrium, of 332,000 +/- 17,000. SDS/polyacrylamide-gel electrophoresis gave one band with a mobility corresponding to an Mr of 43,000 +/- 2000. The Mr of the polypeptide chain was determined to be 41,600 +/- 1,500 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. Cross-linking with glutaraldehyde followed by SDS/polyacrylamide-gel electrophoresis yielded a maximal number of eight bands. The haem content of Ascaris erythrocruorin was observed to vary from one preparation to another. This finding was shown to be due to non-realization of the full binding capacity for haem. By titration with haemin, the haem content was found to attain a maximal value of 2.86 +/- 0.14%, corresponding to a minimal Mr per haem group of 21,000 +/- 1,000. Our findings indicate that Ascaris suum erythrocruorin is composed of eight identical polypeptide chains, carrying two haem sites each.     

Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein.

The N-terminal fragment (G1-G2) of cartilage proteoglycan protein core contains two globular domains, binding region (G1) and a second globular domain (G2), G1-G2 was isolated after mild trypsin digestion of purified proteoglycan aggregates followed by chromatography first on Sepharose CL-2B under associative conditions and then on a TSK-4000 column in 4 M-guanidinium chloride. It migrated as a single band (apparent Mr 150,000) on SDS/polyacrylamide-gel electrophoresis. G2 was isolated by V8-proteinase digestion of G1-G2 followed by aggregation of the G1-containing fragments with hyaluronate and chromatography on TSK-4000. It migrated as a single band on SDS/polyacrylamide-gel electrophoresis of apparent Mr 66,000 after digestion with keratanase. G2 did not interact with proteoglycan monomer, hyaluronate, link protein or other extractable cartilage matrix proteins. A polyclonal antibody raised against G2 did not cross-react with G1 or link protein. These data show that, despite a high degree of sequence similarity, G1 and G2 do not share any functional properties nor have major antigenic sites in common.     

Release of dog polymorphonuclear leukocyte cathepsin G, normally and in endotoxin and pancreatitic shock. Isolation and partial characterization of dog polymorphonuclear leukocyte cathepsin G

Dog polymorphonuclear leukocyte cathepsin G was isolated from a granule extract using a two-step procedure including affinity chromatography on a Trasylol-Sepharose gel and ion-exchange chromatography on a CM 52 column. 22 of the first 24 N-terminal amino acids were determined and showed 83% and 71% identity to those of human and rat cathepsin G, respectively. Total amino-acid composition demonstrated the basic nature of the protein. In an SDS/polyacrylamide-gel electrophoresis the protein showed an Mr of 29,400 compared to the Mr of 26,800 calculated from the total amino-acid composition. The enzyme was shown to form complexes with alpha 1 alpha 2-macroglobulin and alpha 1-proteinase inhibitor. A specific enzyme-linked immunosorbent assay was developed for the determination of cathepsin G/alpha 1-proteinase inhibitor complex in dog plasma and tissue fluids. The mean concentration of cathepsin G in normal dog plasma was determined to be 38 micrograms/l, measured as cathepsin G/alpha 1-proteinase inhibitor complex. When active dog cathepsin G was added to normal dog plasma in vitro, approximately 56% could be measured by the assay. Slow intravenous infusion of a lethal dose of endotoxin in dogs was followed by a marked drop in white blood cell count and thrombocytes and a simultaneous rapid increase in plasma cathepsin G concentration, reaching a maximum level of 150 micrograms/l. Bile-induced experimental pancreatitis in dogs was accompanied by successive increase in cathepsin G levels in plasma as well as in peritoneal exudates, reaching a maximum level of about 300 micrograms/l in plasma and 18 mg/l in the exudates during the late stages of disease.     

Purification and characterization of a second form of acid lipase in human liver.

We describe here the purification and characterization of a form of acid lipase from human liver (designated ALII), which differed from the more abundant Mr-29000 form (ALI). ALII was solubilized from frozen human liver with Triton X-100 and purified 8500-fold by chromatography over concanavalin A-sepharose, CM-cellulose and finally h.p.l.c. over a Mono S column. ALII migrated as a single band on polyacrylamide-gel electrophoresis in both the presence and the absence of SDS. The Mr of ALII was estimated to be 58,500 by SDS/polyacrylamide-gel electrophoresis. Gel filtration on Sephacryl S-200 gave an apparent Mr of 69,000. 4-Methylumbelliferyl (4MU) palmitate, cholesterol oleate and triolein were substrates for ALII, with apparent Vmax values of 5000, 1100 and 2500 nmol/min per mg respectively and Km values of 1.0, 1.5 and 1.8 mM respectively. Cholesterol oleate and triolein were hydrolysed optimally by ALII at pH 4.5, whereas 4MU palmitate was hydrolysed optimally at pH 5.3. Antisera were raised against ALI and ALII and, on immunoblot analysis, no antigenic similarity was observed between ALI and ALII. Cellulose acetate electrophoresis followed by reaction with 4MU palmitate revealed two forms of lipase, corresponding to ALI and ALII. The two enzymes were also separated by hydrophobic chromatography. The activity of ALII was stimulated by several proteins and was partially inhibited by millimolar concentrations of NaCl, CaCl2 and MgSO4.     

Partial purification and characterization of a formylmethionine deformylase from rat small intestine.

A formylmethionine deformylase from rat small-intestinal mucosa has been isolated, characterized and partially purified. The enzyme catalyses the release of equimolar amounts of formate and the free amino acid. The deformylase was active against formylmethionine (Km 7.1 mM) and formylnorleucine, but showed reduced activity against formyl-leucine. It was inactive against a range of other polar and nonpolar formyl-amino acids and against formyl di- and tri-peptides. The Mr of the native enzyme was between 45,000 and 66,000, as determined by h.p.l.c. gel permeation. Further purification of the enzyme either by h.p.l.c. ion-exchange chromatography and concanavalin A-Sepharose or by isoelectric focusing yielded a preparation with one predominant band of Mr 50,000 on SDS/polyacrylamide-gel electrophoresis. Bacteria in the intestine present the host with substantial amounts of formylmethionine (fMet) from proteinase and carboxypeptidase digestion of bacterial formyl-peptides in the intestinal lumen. fMet (0.01-1.0 mM) inhibited translation of a test RNA from brome mosaic virus in vitro, indicating that it could have adverse effects on cellular metabolism. Gut epithelial fMet deformylase may be required for deformylation of this exogenous (bacterial) and also endogenous (mitochondrial) fMet.     

Isolation and characterization of latherin, a surface-active protein from horse sweat.

A protein, latherin, with unusual surface activity was isolated from horse sweat by gel filtration and ion-exchange chromatography. The protein has a Stokes radius, determined by gel filtration, of 2.47 nm, and in the ultracentrifuge sediments as a single species with S20,W 2.05 S, indicating an Mr of 24,400. On SDS/polyacrylamide-gel electrophoresis the molecule behaves as a single peptide chain of apparent Mr 20,000. Latherin contains a high proportion of hydrophobic amino acids (37.2%), and the leucine content (24.5%) is exceptionally high. The unusual composition of the protein may account for apparent anomalies in the Mr of latherin determined by empirical methods. Evidence indicating that latherin is responsible for much of the surface activity of horse sweat was obtained by a simple assay for surface tension and by contact-angle measurements. Latherin adsorbs very readily at hydrophobic surfaces, rendering them wettable. A possible role for latherin in thermoregulation is proposed.     

Purification and characterization of a labile rat glutathione transferase of the Mu class.

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.     

Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii.

We have isolated and characterized argininosuccinate lyase (ASL; EC 4.3.2.1) from the photosynthetic green alga, Chlamydomonas reinhardtii. The general properties of Chlamydomonas ASL are very similar to those described previously for ASLs from phylogenetically diverse organisms. The algal ASL has a native Mr, determined by gel-filtration chromatography, of 218,000 +/- 25,000, and a pI of 5.4-5.6. The Km for argininosuccinate at 37 degrees C and pH 7.5 is 0.26 mM. The subunit Mr of Chlamydomonas ASL is approx. 50,000, determined by SDS/polyacrylamide-gel electrophoresis, in contrast with a previously reported value of 39,000. Rabbit antisera prepared against the Mr-50,000 protein completely abolished ASL activity in vitro. In contrast, serum prepared against the Mr-39,000 protein was ineffective in inhibiting ASL activity. Despite the general similarity of the physical properties of Chlamydomonas ASL and those of other ASLs, antiserum raised against the algal ASL did not cross-react with ASL preparations from Escherichia coli, Saccharomyces cerevisiae or bovine liver.     

Purification and characterization of beta-N-acetylhexosaminidase I2 from human liver.

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.     

Purification and characterization of biotin-binding protein II from chicken oocytes.

BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-I activity separated from BBP-II activity during elution from the first DEAE-cellulose column. Purified BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.     

Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134.

Dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134 was purified to homogeneity. The enzyme has an Mr of about 270,000 as determined by gel filtration and consists of six to eight subunits of identical Mr 40,000 as determined by SDS/PAGE. Mn2+ ions as well as thiol groups are required for activity. A high Km value of about 4 mM for cis,cis-muconate explains the reported low activity with this compound. Relatively high Km values were also calculated for monochloro-substituted cis,cis-muconates (300-500 microM), in contrast with the low Km value of 20 microM for 2,4-dichloro-cis,cis-muconate. The catalytic constant of the pure enzyme was 3820 min-1 when measured with 2,4-dichloro-cis,cis-muconate.     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.     

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing.

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.     

Purification and properties of beta-galactosidase from chicken seminal plasma

1. beta-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its Km was 0.65 mM with p-nitrophenyl-beta-D-galactoside as substrate. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl- ions. 4. The enzyme had pI value of 4.0. 5. The active enzyme had Mr approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of beta-mercaptoethanol showed four bands corresponding to Mr of approx. 90,000, 75,000, 65,000 and 13,000.     

High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis.

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.