Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa

We report here the purification and characterization of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme (PMI-GMP) which catalyzes both the phosphomannose isomerase (PMI) and guanosine 5'-diphospho-D-mannose pyrophosphorylase (GMP) reactions of the Pseudomonas aeruginosa alginate biosynthetic pathway. The PMI and GMP activities co-eluted in the same protein peak through successive fractionation on hydrophobic interaction, ion exchange, and gel filtration chromatography. The purified enzyme migrated as a 56,000 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native protein migrated as a monomer of 54,000 molecular weight upon gel filtration chromatography. The apparent Km for D-mannose 6-phosphate was 3.03 mM, and the Vmax was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent Km values of 20.5 microM and 29.5 microM for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction Vmax (5,680 nmol/min/mg of enzyme) was comparable to the reverse reaction Vmax (5,170 nmol/min/mg of enzyme), and the apparent Km for GDP-D-mannose was determined to be 14.2 microM. Both reactions required Mg2+ activation, but the PMI reaction rate was 4-fold higher with Co2+ as the activator. PMI (but not GMP) activity was sensitive to dithiothreitol, indicating the involvement of disulfide bonds to form a protein structure capable of PMI activity. DNA sequencing of a cloned mutant algA gene from P. aeruginosa revealed that a point mutation at nucleotide 961 greatly decreased the levels of both PMI and GMP in a crude extract.     

Purification and characterization of a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.     

Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg-1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS-PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated Km is 6.3 microM and Vmax 9.8 nKat.     

Ornithine decarboxylase from calf liver. Purification and properties

Ornithine decarboxylase (ODC) was purified 25000-fold from calf liver to apparent homogeneity by methods developed to circumvent the lability of the enzyme. Appropriate ratios of sample protein applied to column size and/or gradient size were derived for each purification procedure (ion-exchange, gel filtration ahd hydroxylapatite chromatography, electrophoresis, and thiol affinity chromatography) to maintain enzymatic activity. The enzyme was labile to dilution at all steps of the purification; the inclusion of poly(ethylene glycol) or additional protein decreased but did not eliminate the activity loss. The purified enzyme had a Stokes radius of 3.14 and a molecular weight of 54000. The Km for ornithine was 0.12 mM, and pyridoxal phosphate was 2.0 microM; the pH optimum for the decarboxylation reaction was 7.0. Analysis by sievorptive ion-exchange chromatography indicated the presence of three ionic forms. In the presence of Tris-barbital buffer containing thioglycolic acid, the ODC preparation assumed an apparent molecular weight of 100000 and a Stokes radius of 4.5 and retained full catalytic activity.     

Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Bacteriolytic enzyme induced from pyocinogenic Pseudomonas aeruginosa. Purification and characterization of PR1-lysozyme.

A bacteriolytic enzyme, PR1-lysozyme, has been purified from the lysate of mitomycin C-induced pyocinogenic Pseudomonas aeruginosa, by acrinol treatment, Amberlite CG-50 chromatography, ammonium sulfate fractionation, Sephadex G-100 gel filtration and two cycles of SP-Sephadex C-50 chromatography. Homogeneity of the preparation was demonstrated by three electrophoretic techniques. PR1-lysozyme is a basic protein (pI, 9.4) and consists of a single polypeptide chain having a molecular weight of 24,000. The amino acid composition of the protein was analyzed, and no cystein residue was found among more than 210 amino acid residues. The optimum pH for enzymatic activity was 6.4 and the enzyme exhibited about 50 to 70 times greater specific activity than hen egg-white lysozyme when assayed with chloroform-killed P. aeruginosa as a substrate. By analyzing the products of enzymatic action on purified peptidoglycan of P. aeruginosa, the enzyme was identified as an N-acetylmuramidase, i.e., the same classification as hen-egg-white lysozyme. PR1-lysozyme did not show any activity towards intact cells of gram-positive and gram-negative bacteria tested. However, the enzyme was able to lyse chloroform-killed gram-negative and gram-positive bacteria.     

Purification and characterization of benzoyl-L-arginine p-nitroanilide hydrolase from etiolated leaves of Zea mays

Benzoyl-L-arginine p-nitroanilide hydrolase in the etiolated leaves of Zea mays L. has been purified 1,266-fold by a combination of gel filtration, ion exchange, and hydrophobic chromatography with a recovery of 13%. The specific activity of the purified enzyme is 5.7 units/mg protein. The enzyme is an acidic protein with a pI value of 4.6 and optimum pH of 8.2. The molecular weight of the enzyme was estimated to be 59,000. Substrate inhibition was observed at a concentration higher than 30 microM BAPA and the apparent Km for BAPA was 29 microM at pH 8.0. The enzyme activity was inhibited by sulfhydryl reagents, leupeptin, antipain, and N-tosyl-L-lysine chloromethyl ketone. The inhibitor study suggests that the enzyme belongs to the class of the sulfhydryl proteases.     

Purification and characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

Extracts of bovine neurointermediate pituitary secretory granules and frozen bovine neurointermediate pituitary contain multiple forms of peptidylglycine alpha-amidating monooxygenase (PAM) activity differing in apparent molecular weight and in charge. Metal chelate affinity chromatography, substrate affinity chromatography, and gel filtration resulted in the purification of two forms of amidation activity from frozen bovine neurointermediate pituitary: PAM-A, apparent molecular weight 54,000, was purified 7,000-fold and PAM-B, apparent molecular weight 38,000, was purified 21,000-fold. Enzyme activity of similar molecular weights was observed in the starting material. Purified PAM-A and PAM-B correspond to two of the three charge forms present in crude extracts, and both exhibited optimal activity at alkaline pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PAM-B revealed the presence of two bands with apparent molecular weights of 42,000 and 37,000; autoradiography of 125I-labeled PAM-B revealed only the same two bands, and 125I-labeled PAM-B co-eluted with enzyme activity during gel filtration. PAM-A was still heterogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties of purified PAM-A and PAM-B were very similar to those of amidation activity in crude extracts: activity was reduced upon removal of molecular oxygen; activity was stimulated by the addition of CuSO4 and eliminated by the addition of diethyldithiocarbamate; activity was stimulated by the addition of ascorbate, with optimal levels of ascorbate increasing as the concentration of peptide substrate was increased. In the presence of 1.25 mM ascorbate, PAM-B exhibited a Km of 7.0 microM for D-Tyr-Val-Gly and a Vmax of 84 nmol/micrograms/h.     

Sea urchin sperm guanylate cyclase. Purification and loss of cooperativity.

The Lubrol-dispersed guanylate cyclase from sea urchin sperm was purified and isolated essentially free of detergent by GTP affinity chromatography, DEAE-Sephadex chromatography, and gel filtration. After removal of the detergent, the enzyme remained in solution in the presence of 20% glycerol. The specific activity of the purified enzyme was about 12 mumol of guanosine 3':5'-monophosphate (cyclic GMP) formed - min-1 - mg of protein-1 at 30 degrees, an activity about 4600 times that of a soluble guanylate cyclase purified recently from Escherichia coli (Macchia V., Varrone, S., Weissbach, H., Miller, D.L., and Pastan, I. (1975) J. Biol. Chem. 250, 6214-6217). The cyclic GMP phosphodiesterase activity was negligible and adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase was not detectable in the purified preparation. Cyclic AMP formation from ATP occurred at a rate of 0.002% of that of guanylate cyclase. In the absence of phosphodiesterase or guanosine triphosphatase inhibitors, 100% of the added GTP was converted to cyclic GMP. The purified enzyme required Mn2+ for maximum activity, the relative rates in the presence of Mg2+ or Ca2+ being less than 0.6% of the rates with Mn2+. The purified enzyme displayed classical Michaelis-Menten kinetics with respect to MnGTP (apparent Km is approximately equal to 170 muM) in contrast to the positively cooperative kinetic behavior displayed by the unpurified, detergent-dispersed, or particulate guanylate cyclase. The molecular weight of the purified enzyme was approximately 182,000 as estimated on Bio-Gel A-0.5m columns equilibrated in the presence or absence of 0.1 M NaCl. The unpurified, detergent-dispersed enzyme also migrated with an apparent molecular weight of 182,000 on columns equilibrated with 0.5% Lubrol WX and 0.1 M NaCl, but it migrated as a large aggregate (molecular weight is greater than 5 X 10(5)) on columns equilibrated in the absence of either the detergent of NaCl. After gel filtration, the unpurified, dispersed enzyme still yielded positive cooperative kinetic patterns as a function of MnGTP. Na dodecyl-SO4 gel electrophoresis of the enzyme after the DEAE-Sephadex or the gel filtration steps resulted in two major protein bands with estimated molecular weights of 118,000 and 75,000. Whether or not these protein bands represent the subunit molecular weights of guanylate cyclase is unknown at present.     

NADP-dependent isocitrate dehydrogenase from bass (Dicentrarchus labrax L.) liver

The isocitrate dehydrogenase from bass liver was purified to homogeneity by gel filtration, affinity and ion exchange chromatographies. The molecular weight was estimated by gel filtration chromatography to about 120,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed it to be a dimeric protein. The enzyme showed maximum activity in the pH range between 7.0 and 8.0 while its maximum activity was at pH 7.5. DL-Isocitrate and Mn2+ stabilized the enzyme, while NADP had the opposite effect. The Km for isocitrate was 0.31 mM and the Km for NADP was 36 microM.     

Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila).

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723.

The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized. In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723. The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps. Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da. The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect. The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture. The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH. The enzyme required Fe2+ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40 degrees C. The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.     

蓖麻β-半乳糖苷酶的纯化及其基本性质

蓖麻籽黄化苗中存在高活性β-半乳糖苷酶。经硫酸铵分级分离、DEAE-纤维素离子交換层析、Sephadex G-100、CM-Sephadex和DEAE-Sephadex层析纯化。活性收率为6.4％,纯化倍数达107倍。纯化了的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示两条蛋白带,其相应分子量分别为3.25×10~4和2.94×10~4。用Sephadex G-200分子筛层析法测得分子量为6.7×10~4。综合上述结果推测该酶是由两个不同的亚基构成。以邻硝基苯酚-β-半乳糖苷为底物测得该酶的表观Km为5.9×10~(-3)mol/L。最适pH和最适温度分别为4.5和50℃。酸碱稳定区域在pH4.6—7.5之间。不同浓度缓冲液以及不同种类缓冲液、不同金属离子对酶活性影响均进行了讨论。     

Purification and characterization of a soluble phospholipase A2 from guinea pig lung

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Purification and characterization of thymidine kinase from regenerating rat liver

Thymidine kinase (EC 2.7.1.21) from regenerating rat liver has been purified 70,000-fold to apparent homogeneity by affinity chromatography. Molecular weight of the native enzyme was found to be about 54,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band with a molecular weight of 26,000, suggesting that thymidine kinase is a dimer of very similar or identical subunits. The Michaelis constant for thymidine is 2.2 microM. ATP acts as a sigmoidal substrate with a 'Km' of 0.2 mM. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be sequential.     

Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and an apparent Km for leukotriene A4 between 2 X 10(-5) and 3 X 10(-5) M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases.