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.     

Purification of two forms of kanamycin acetyltransferase from Escherichia coli

Kanamycin acetyltransferase acylates aminoglycoside antibiotics using acetyl-CoA, and thereby conveys bacterial resistance to several clinically important antibiotics, notably amikacin. The enzyme was quantitatively and reproducibly released from Escherichia coli W677 harboring plasmid pMH67 by a modified osmotic shock procedure (bacterial cells are incubated overnight in sucrose and again without sucrose before onset of osmotic shock). The enzyme was purified by dye-ligand chromatography on Affi-Gel Blue in addition to antibiotic affinity chromatography on neomycin-Sepharose-4B. The activity did not increase with subsequent chromatography on ion-exchange, hydrophobic, or molecular-exclusion gels. However, both dye-ligand and molecular-exclusion chromatography, as well as disc-gel electrophoresis, separated the purified enzyme equally into two active protein fractions. Based on the more active of the two forms, the purification was 112-fold with a specific activity of 1.9 IU/mg. The less-active form has an unusual absorbance spectrum, with a maximum near 255 nm, which cannot be explained by the amino acid composition. Chromatography of this form alone regenerated both forms, suggesting that the enzyme is noncovalently conjugated to an uncharged chromophore, such as a lipid. The purified enzyme has a very sharp pH optimum at 5.5 with a plateau on the alkaline side, but is most stable between pH 8.5 and 9.5. Data from electrophoresis in the presence of sodium dodecyl sulfate and gel-filtration on Ultrogel AcA 44 are consistent with a tetrameric protein of 60-70,000 Da.     

Purification of the membrane-bound hydrogenase of Escherichia coli.

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.     

Dihydroorotase from Escherichia coli. Purification and characterization

Dihydroorotase (4,5-L-dihydroorotate amidohydrolase (EC 3.5.2.3], which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to dihydro-L-orotate, has been purified to homogeneity from an over-producing strain of Escherichia coli. Treatment of 70 g of frozen cell paste produces about 7 mg of pure enzyme, a yield of about 35%. The native molecular weight, determined by equilibrium sedimentation, is 80,900 +/- 4,300. The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 38,400 +/- 2,600, and by amino acid analysis is 41,000. The enzyme is thus a dimer and contains 0.95 +/- 0.08 tightly bound zinc atoms per subunit when isolated by the described procedure, which would remove any loosely bound metal ions. Isoelectric focusing under native conditions yields a major species at isoelectric point 4.97 +/- 0.27 and a minor species at 5.26 +/- 0.27; dihydroorotase activity is proportionately associated with both bands. The enzyme has a partial specific volume of 0.737 ml/g calculated from the amino acid composition and a specific absorption at 278 nm of 0.638 for a 1 mg/ml solution. At 30 degrees C, the Michaelis constant and kcat for dihydro-DL-orotate (at pH 8.0) are 0.0756 mM and 127 s-1, respectively; for N-carbamyl-DL-aspartate (at pH 5.80), they are 1.07 mM and 195 s-1.     

Purification and characterization of two types of fumarase from Escherichia coli

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.     

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli.

The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.     

Fructose bisphosphatase from Escherichia coli. Purification and characterization

Escherichia coli fructose-1,6-bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme. The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration. The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations. Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (Km of approximately 5 microM), shows inhibition by substrate above 0.05 mM, requires Mg2+ for catalysis, and has a maximum of activity around pH 7.5. The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 microM). Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in vivo. Fructose 2,6-bisphosphate did not affect the rate of reaction.     

Protease II from Escherichia coli. Purification and characterization.

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.     

Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli

Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.     

Purification and characterization of phosphopantetheine adenylyltransferase from Escherichia coli.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4'-phosphopantetheine yielding 3'-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3'-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Purification and characterization of 3-dehydroquinase from Escherichia coli.

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.     

Purification and characterization of a DNA-dependent ATPase from Escherichia coli.

A DNA-dependent ATPase has been isolated and purified from an Escherichia coli cell-free extract. The ATPase has the following characteristics: preferential dependence on single-stranded DNA, specificity for ATP hydrolysis, Km value of 1.4 X 10-4 M for ATP, and molecular weight of approximately 69,000. The ATPase can be shown to bind to single stranded DNA. The resemblance between this ATPase and that isolated from vaccinia cores is discussed.     

Purification and characterization of DNA-dependent ATPase II from Escherichia coli.

A new DNA-dependent ATPase was isolated and purified from soluble extracts of Escherichia coli. This enzyme, called ATPase II, has a molecular weight of 86,000 and exists in a monomeric state. It degrades ATP (or dATP) to ADP (or dADP) and Pi in the presence of magnesium and requires a double-stranded polynucleotide as cofactor. A correlation between the efficiency as cofactor and the melting point of the polynucleotide has been found; the lower the melting temperature, the higher the stimulation of ATPase II. The enzyme binds to single-stranded DNA and poly[d(A-T)] copolymer, but not to the double-stranded circular DNA (Form I) of simian virus 40.     

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.     

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.     

Escherichia coli enterotoxin. Purification and partial characterization.

Enterotoxin, a diarrheagenic protein elaborated by pathogenic Escherichia coli strains has been isolated from the supernatant of fermenter cultures of E. coli strain P263, a porcine enteropathogen. Purification steps involving Bio-Gel agarose A-5m, Sephadex G-75 chromatography, and preparative isotachophoresis were used in the isolation. The resulting product appears to be pure according to immunoelectrophoretic, disc electrophoretic, ultracentrifugal, and immunologic criteria. The entertoxin has an apparent molecular weight of 102,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and its isoelectric point is 6.90. The isolated product is highly active in inducing experimental diarrhea in adult rabbits and piglets. It also elicits, in small dosage, a marked increase in adenylate cyclase activity in broken cell preparations of cat heart tissue. The enterotoxin activity is acid-labile and is destroyed by heating at 65 degrees for 30 min. It is suggested that the heat-stable enterotoxin material is derived from heat-labile enterotoxin by forming a complex with endotoxin or capsular material present in the culture supernatant.     

Purification and characterization of [acyl-carrier-protein] acetyltransferase from Escherichia coli.

A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.