The Characteristics of Arylamine N-Acetyltransferase in Pseudomonas aeruginosa

N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K _m and V _max for 2-AF increased. The K _m and V _max were 0.504 ± 0.056 mM and 31.92 ± 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5°C to 55°C, with maximum activity at 37°C. The enzyme activity was inhibited by divalent metal ions including Cu⁺⁺, Fe⁺⁺, Zn⁺⁺, Ca⁺⁺, Co⁺⁺, Mn⁺⁺, and Mg⁺⁺, suggesting that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site. Our data demonstrate that P. aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species. Received: 29 August 1997 / Accepted: 23 December 1997     

Reaction of yeast carboxypeptidase C1 with group-specific reagents.

The reactions between yeast carboxypeptidase C and the group-specific reagents, phenylglyoxal and iodoacetamide, have been studied in detail and the reactions of residue at the active site with N-tosyl-L-phenylalanine chloromethyl ketone and diisopropyl phosphorofluoridate have been confirmed. Modification of the enzyme by either phenylglyoxal or iodoacetamide results in the loss of peptidase activity, while esterase activity remains unchanged. Inactivation by phenylglyoxal appears to be the result of the modification of a single arginine residue, whereas inhibition by iodoacetamide can be correlated with the modification of a single methionine residue. Inactivation of the enzyme by either N-tosyl-L-phenylalanine chloromethyl ketone or diisopropyl phosphorofluoridate is the result of the modification of a single histidine and a single serine residue, respectively. The pattern of inhibition indicates certain analogies in the mechanism of yeast carboxypeptidase C to pancreatic chymotrypsin, on the one hand, and to carboxypeptidase A, on the other.     

Purification and characterization of a thermostable glucoamylase from Chaetomium thermophilum

Thermostable amylolytic enzymes are currently being investigated to improve industrial processes of starch degradation. A thermostable extracellular glucoamylase (exo-1, 4-alpha-D-glucanohydrolase, E.C.3.2.1.3) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by using ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Phenyl-Sepharose Fast Flow chromatography. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 64 kDa. The glucoamylase exhibited optimum catalytic activity at pH 4.0 and 65 degrees C. It was thermostable at 50 degrees C and 60 degrees C, and retained 50% activity after 60 min at 65 degrees C. The half-life of the enzyme at 70 degrees C was 20 min. N-terminal amino acid sequencing (15 residues) was AVDSYIERETPIAWN. Different metal ions showed different effects on the glucoamylase activity. Ca2+, Mg2+, Na+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, and Hg2+ cause obvious inhibition. These properties make it applicable to other biotechnological purposes.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

N-hydroxyarylamine O-acetyltransferase in hamster liver: identity with arylhydroxamic acid N,O-acetyltransferase and arylamine N-acetyltransferase

N-Hydroxyarylamine O-acetyltransferase, arylhydroxamic acid N,O-acetyltransferase, and arylamine N-acetyltransferase in hamster liver cytosol were co-purified almost to electrophoretical homogeneity by ion exchange chromatography on DEAE-cellulose, gel filtration on Cellulofine GCL-2000-sf and high-performance KB-hydroxyapatite chromatography. The molecular weight of the acetyltransferase was estimated to be 33,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The three acetyltransferase activities were inhibited by iodoacetamide, pentachlorophenol, and 1-nitro-2-naphthol. Furthermore, 2-aminofluorene, a substrate for arylamine N-acetyltransferase, inhibited the reactions of N-hydroxyarylamine O-acetyl transfer and arylhydroxamic acid N,O-acetyl transfer. These results suggest that the same enzyme catalyzes the three types of acetyl transfer reactions. The acetyltransferase could activate N-hydroxyarylamines, such as 2-hydroxyamino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, and N-hydroxy-2-aminofluorene, to the corresponding N-acetoxyarylamines, which are capable of binding to nucleic acid. Polyguanylic acid was most efficiently modified by the N-acetoxyarylamines formed by the acetyltransferase.     

嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性

探讨了液体发酵嗜热毛壳菌（Chaetomium thermophile)产生的内切β－葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀,DEAE－Seplharose Fast Flow阴离子层析,Pheny1-Sepha-rose疏水层析,Sephacry1 S-100分子筛层析等步骤便可获得凝胶电泳均一的内切β－葡聚糖酶,经12．5％SDS－PAGE和凝胶过滤层析法分离纯化酶蛋白的分子量约为67．8kD的69．8kD。该酶反应的最适温度和pH分别为60℃和4．0－4．5在pH5.0条件下,该酶在60℃下稳定：70℃保温1h后,仍保留30％的活性;在80摄氏度的半衰期为25min,金属离子内切β－葡聚糖酶的活性影响较大,其中Na^ 对酶有激活作用;Fe^2 ,Ag^ ,Cu^2 ,Ba^2 ,Zn^2 等对酶有抑制作用。该酶对结晶纤维素有没水解能力。     

The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 A resolution with a bisubstrate analog.

Serotonin N-acetyltransferase, a member of the GNAT acetyltransferase superfamily, is the penultimate enzyme in the conversion of serotonin to melatonin, the circadian neurohormone. Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog, coenzyme A-S-acetyltryptamine, demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site. The structure of the complex also provides insight into how the enzyme may facilitate acetyl transfer. A water-filled channel leading from the active site to the surface provides a pathway for proton removal following amine deprotonation. Furthermore, structural and mutagenesis results indicate an important role for Tyr-168 in catalysis.     

Arginine residues involved in binding of tetrahydrofolate to sheep liver serine hydroxymethyltransferase.

The arginine residue(s) necessary for tetrahydrofolate binding to sheep liver serine hydroxymethyltransferase were located by phenylglyoxal modification. The incorporation of [7-14C]phenylglyoxal indicated that 2 arginine residues were modified per subunit of the enzyme and the modification of these residues was prevented by tetrahydrofolate. In order to locate the sites of phenylglyoxal modification, the enzyme was reacted in the presence and absence of tetrahydrofolate using unlabeled and radioactive phenylglyoxal, respectively. The labeled phenylglyoxal-treated enzyme was digested with trypsin, and the radiolabeled peptides were purified by high-performance liquid chromatography on reversed-phase columns. Sequencing the tryptic peptides indicated that Arg-269 and Arg-462 were the sites of phenylglyoxal modification. Neither a spectrally discernible 495-nm intermediate (characteristic of the native enzyme when substrates are added) nor its enhancement by the addition of tetrahydrofolate, was observed with the phenylglyoxal-modified enzyme. There was no enhancement of the rate of the exchange of the alpha-proton of glycine upon addition of tetrahydrofolate to the modified enzyme as was observed with the native enzyme. These results demonstrate the requirement of specific arginine residues for the interaction of tetrahydrofolate with sheep liver serine hydroxymethyltransferase.     

Arginine biosynthesis in Thermotoga maritima: characterization of the arginine-sensitive N-acetyl-L-glutamate kinase

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of approximately 4, and the 50% inhibitory arginine concentration (I0.5) was shown to increase with temperature, approaching above 65 degrees C the I0.50 observed at 37 degrees C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75 degrees C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the Km for acetylglutamate triggered by the inhibitor, but at 37 degrees C arginine also substantially decreased the Vmax of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.     

Purification and characterization of phospholipase A2 released from rat platelets

It was found that phospholipase A2 and lysophospholipase, both of which were released from thrombin-stimulated rat platelets, had high affinity to insolubilized heparin. Phospholipase A2 released from rat platelets was purified by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW (high-performance liquid chromatography, HPLC). The enzyme was near homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, and its Mr was estimated to be 13,500. The purified enzyme was labile and lost its activity within 1 h when incubated at 37 degrees C. Phospholipids or detergent in the solution protected the enzyme against inactivation. Phospholipase activity was inhibited by p-bromophenacylbromide, but not by diisopropylfluorophosphate or iodoacetamide. Lysophospholipase, which was also released from rat platelets, was separated from phospholipase A2 by chromatography on heparin-Sepharose.     

Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.     

Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa

Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.     

Purification and properties of an L-asparaginase from Cylindrocarpon obtusisporum MB-10

An L-asparaginase producing mesophilic fungus Cylindrocarpon obtusisporum MB-10 was isolated from soil. The constitutive intracellular L-asparaginase from the organism was purified. The enzyme after 65-fold purification with an overall yield of 11% and specific activity of 100 unit.mg-1 seemed to be homogeneous in native, SDS-PAGE and thin layer isoelectric focusing gel. The apparent Mr of the enzyme was 216,000, and it constituted four identical subunits. The pI of the enzyme was 5.5. It was a conjugate protein with 37.3% (w/w) carbohydrate. The enzyme was stable to storage at -20 degrees C and to repeated freezing and thawing. The L-asparaginase from the organism was very much specific for L-asparagine and did not hydrolyze D-asparagine and L-glutamine. The pH and temperature optima for the enzyme activity were 7.4 and 37 degrees C, respectively. The Km of the L-asparaginase was found to be 1 x 10(-3)M. Metal ions, such as Zn2+, Fe2+, Cu2+, Hg2+ and Ni2+ potentially inhibited the enzyme activity, while metal chelators like EDTA, CN-, cysteine, etc., enhanced the activity indicating that the enzyme was not a metalloprotein. Its activity was also enhanced in the presence of reduced glutathione but not with dithiothreitol and 2-mercaptoethanol. Differential inhibition of the enzyme activity was observed with iodoacetamide and p-chloromercuribenzoate, thus indicating possible involvement of free-SH group in the enzyme catalysis.     

Isolation, purification, and characterization of glucosamine-6-phosphate-N-acetylase from pig liver

The procedure of isolation, purification, and characterization of glucosamine-6-phosphate acetylase from the pig liver is described. The steps of purification were as follows: adsorption on hydroxylapatite, fractionation with ammonium sulfate, chromatography on cellulose phosphate, electrofocusing, and preparative gel electrophoresis. A highly purified (about 3000-fold) preparation of GlcN-6-P acetylase, with a yield of 23%, was obtained. It was found that GlcN-6-P acetylase from pig liver is heterogeneous and exists in two active forms. The characteristic features of the preparation were established: Mr, about 24 kDa; temperature optimum at 37 degrees; pH optimum at 7.45; and Km (GlcN-6-P) 3.7 x 10(-3) M and Km (AcCoA) 1.4 x 10(-3) M. The ions K+, Na+, NH4+, Mg2+, Mn2+, and CH3COO- do not stimulate the acetylase activity. The product of acetylase reaction (GlcNAc-6-P) inhibits this reaction according to the feedback process. The highly purified preparation of GlcN-6-P acetylase is unstable during storage and it is protected by ampholine or glycine from enzyme inactivation, but it is not protected by 2-mercaptoethanol.     

Evidence for an essential arginine in the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase from the fungus Fusarium oxysporum catalyzes the oxidative denitrification of primary or secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. The enzyme is inactivated in a time-dependent fashion upon treatment with the arginine-directed reagents phenylglyoxal, 2,3-butanedione, and cyclohexanedione. The inactivation shows first order kinetics with all reagents. Valerate, a competitive inhibitor of the enzyme, fully protects the enzyme from inactivation, indicating that modification is active site directed. The most rapid inactivation is seen with phenylglyoxal, with a k(inact) of 14.3 +/- 1.1 M(-1) min(-1) in phosphate buffer at pH 7.3 and 30 degrees C. The lack of increase in the enzymatic activity of the phenylglyoxal-inactivated enzyme after removing the unreacted reagent by gel filtration is consistent with inactivation being due to covalent modification of the enzyme. A possible role for an active site arginine in substrate binding is discussed.     

An activated by cobalt alkaline aminopeptidase from Bacillus mycoides

An intracellular arginine--specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of -50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-beta-naphthylamide as a substrate was at 37 degrees C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co2+ ions activated the enzyme, while Zn2+, Cu2+, Hg2+ and Mn2+ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to AmpS of Bacillus cereus and AP II of B. thuringensis.     

Purification, characterization, and amino acid composition of rabbit pulmonary bleomycin hydrolase

Bleomycin (BLM) hydrolase, a protective enzyme that inactivates the antitumor antibiotic BLM, was purified (6000-fold) to homogeneity from rabbit lungs by DEAE-Sephacel, phenyl-Sepharose chromatography, BLM-Sepharose affinity chromatography, and Mono Q fast protein liquid chromatography. The enzyme had a molecular mass of 250,000 daltons as demonstrated by Superose gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) under native conditions. Sodium dodecyl sulfate-PAGE revealed a single band of 50,000 daltons, suggesting a pentameric structure. The Km and Vmax for BLM A2 were 1.3 mM and 5.9 mumol mg-1 h-1, respectively. BLM hydrolase activity was labile, had a half-life of 25 min at 56 degrees C, 10 h at 37 degrees C, and 5 days at 4 degrees C, and was stabilized by 2 mM dithiothreitol. The enzyme had a pH optimum of 7.0-7.5 and was inhibited by N-ethylmaleimide, leupeptin, puromycin, and divalent cations such as Cu2+, Cd2+, Zn2+, and Co2+ but was unaffected by chelating agents. On the basis of Mono P chromatofocusing chromatography, three isoforms of BLM hydrolase (apparent pI's of 5.3, 4.5, and 4.3) were present in rabbit pulmonary cytosol. The elution profiles of BLM hydrolase from phenyl-Sepharose and Mono P chromatofocusing indicated that this enzyme is hydrophobic and acidic. This was confirmed by amino acid composition analysis, which demonstrated that 48% of the total amino acids of bleomycin hydrolase were hydrophobic and 37% were acidic.     

Purification and Characterization of an Arylamine N-Acetyltransferase from the Bacteria Aeromonas hydrophilia

N-acetyltransferase from Aeromonas hydrophilia was purified by ultrafiltration, DEAE-Sephacel, gel filtration chromatography on Sephadex G-100, and DEAE-5pw on high performance liquid chromatography, as judged by sodium dodecyl sulfate-polyacrylamine gel electrophoresis (SDS-PAGE) on a 12.% (wt/vol) slab gel. The enzyme had a molecular mass 44.9 kDa. The purified enzyme was thermostable at 37°C for 1 h with a half-life 28 min at 37°C, and displayed optimum activity at 37°C and pH 7.0. The K _m and V _max values for 2-aminofluorene were determined to be 0.896 mM and 2.456 nmol/min/mg protein, respectively. Among a series of divalent cations and salts, Zn²⁺, Ca²⁺, and Fe²⁺ were demonstrated to be the most potent inhibitors. Received: 10 November 1997 / Accepted: 17 February 1998