Separation and characterization of endopolygalacturonase and exopolygalacturonase from peaches

Two polygalacturonases (PG I and PG II) have been separated from extracts of ripe peaches (Prunus persica) by chromatography on Sephadex G-100. PG I hydrolyzes polygalacturonic acid from the nonreducing ends of the molecules, releasing galacturonic acid as the product. It functions optimally at pH 5.5, requires Ca²⁺ for activity, and hydrolyzes low molecular weight substrates most rapidly. In contrast, PG II cleaves the molecular chain of the substrate randomly with a pH optimum at about 4. This enzyme is most reactive with substrates of intermediate molecular weight. It catalyzes the release of water-soluble, but 70% ethanol-insoluble, pectin from washed peach cell walls.     

Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.1.Ga was separated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carboxylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electrophoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P2 carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme inhibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I enzyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensitive to 6-phosphogluconate.     

Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of the human placenta.

Three activity peaks hydrolysing L-cystine-di-beta-naphthylamide (CysNA) and two activities hydrolysing L-leucine-beta-naphthylamide (LeuNA) were separated by gel filtration on Sepharose 6B from human placental tissue. The enzyme activities in the void volume and the solubilized enzyme activities with both substrates apparently are bound and free forms of the same enzymes (I) since detergent treatment caused a total disappearance of the activities in the void volume. The second distinct enzyme (II) was highly soluble and detected only with CysNA. The particle-bound enzyme(s) had a pH optimum at 6.5 with CysNA and at about 7.5 with LeuNA. They were highly sensitive to EDTA, could be reactivated by Co2+ and Zn2+ and were more sensitive to Ni2+ and L-methionine than the soluble enzyme II. The former enzyme(s) tolerated thermal treatment better than the soluble enzyme II. The solubilized free enzyme(s) I had a molecular weight of about 309,000. The soluble enzyme II was resistant to EDTA. Its optimum was at pH 6.0 and an estimate of 76,000 for the molecular weight was obtained.     

Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide, but not to isonicotinic acid: differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis

Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (Mr, 137) to isonicotinamide (Mr, 122), not to isonicotinic acid (Mr, 123), in the presence or absence of H2O2. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis.

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation, purification and properties of β-lactamase I and β-lactamase II from Bacillus cereus 569/H/9

1. A procedure was devised which is suitable for the isolation of beta-lactamase I and beta-lactamase II from Bacillus cereus 569/H/9 on a large scale. After adsorption on to Celite both enzymes were eluted in good yield and separated by chromatography on Sephadex CM-50. 2. beta-Lactamase I was separated into three main components by isoelectric focusing and into two components by chromatography. 3. The Zn(2+)-requiring beta-lactamase II obtained by this procedure had a lower molecular weight (22000) than beta-lactamase I (28000) and also differed from the latter in containing one cysteine residue. 4. The beta-lactamase II contained no carbohydrate, but showed the thermostability of the enzyme isolated earlier as a protein-carbohydrate complex. 5. Amino acid analyses and tryptic-digest ;maps' indicate that some degree of homology between beta-lactamase I and beta-lactamase II is possible, but that beta-lactamase I is not composed of the entire sequence of beta-lactamase II together with an additional peptide fragment. 6. A 6-methylpenicillin and a 7-methylcephalosporin showed much lower affinities for both enzymes than did penicillins and cephalosporins themselves.     

Studies on the oligosaccharide heterogeneity of the isoelectric forms of the lower molecular weight acid phosphatase of frog liver

1. The lower molecular weight, heterogeneous acid phosphatase (AcPase) from the frog liver (Rana esculenta) containing AcPase I, II, III and IV was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient. 2. The blotted enzyme bands were characterized by their different binding patterns obtained with the lectins concanavalin A, wheat germ agglutinin (WGA), Lens culinaris hemagglutinin (LcH) and peanut agglutinin (PNA). 3. In situ neuraminidase treatment reduced the staining intensity of some WGA-bands and increased that of PNA-bands. 4. The finding that AcPases I, II, III and IV differ in their carbohydrate chain composition, together with previous results showing different bioactivities of AcPases III and IV, indicates a correlation between the glycosylation state of enzyme forms and their physiological action.     

Properties of two molecular forms of beta-glucuronidase from the mollusc Littorina littorea L.

The occurrence of the two molecular forms, I and II, in the beta-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and II of beta-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4--4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p-nitrophenyl beta-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein beta-D-glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This beta-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with beta 1 leads to 3 linkages; it had practially no activity on certain glycosaminoglycans. Hg2+ and glucaro-1,4-lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.     

Protein expression in Escherichia coli minicells containing recombinant plasmids specifying trimethoprim-resistant dihydrofolate reductases.

Deoxyribonucleic acid fragments containing the structural genes for several trimethoprim-resistant dihydrofolate reductases from naturally occurring plasmids were inserted into small cloning vehicles. The genetic expression of these hybrid plasmids was studied in purified Escherichia coli minicells. The type I dihydrofolate reductase, encoded by plasmid R483 and residing within transposon 7 (Tn7), had a subunit molecular weight of 18,000. The type II dihydrofolate reductase, specified by plasmid R67, had a subunit molecular weight of 9,000. These two enzymes were antigenically distinct in that anti-type II dihydrofolate reductase (R67) antibody did not cross-react with the type I (R483) protein. The trimethoprim-resistant reductase specified by plasmid R388 had a subunit molecular weight of about 10,500 and was immunologically related to the type II (R67) enzyme. A 9,000 subunit of the dihydrofolate encoded by the transposition element Tn402 was also antigenically related to the R67 reductase.     

Regulation of the intracellular calcium level in human blood platelets: cyclic adenosine 3',5'-monophosphate dependent phosphorylation of a 22,000 dalton component in isolated Ca2+-accumulating vesicles.

Two protein kinase activities have been separated from the supernatants of homogenized human blood platelets by DEAE cellulose chromatography. One of them (peak I enzyme) is an efficient stimulator of the uptake of Ca2+ into isolated membrane vesicles in the presence of cyclic AMP and ATP. The second (peak II enzyme), although equally active towards histone, exerts only about one third of the activity of the peak I enzyme. The stimulation of Ca2+ uptake is accompanied by the phosphorylation of a membrane protein with an apparent molecular weight of 22 000, which appears to play an essential role in the regulation of the intracellular Ca2+ level and hence of platelet activity.     

Purification and further characterization of enteropeptidase from porcine duodenum

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Isolation and characterization of N-long chain acyl aminoacylase from Pseudomonas diminuta

N-Long chain acyl aminoacylase II (Enzyme II) catalyzing the hydrolysis of N-long chain acyl amino acids was purified about 2,000-fold from the cell extracts of Pseudomonas diminuta with 1.8% of activity yield. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and the molecular weight was 220,000. Enzyme II differed from N-long chain acyl aminoacylase I (Enzyme I) in molecular weight, in substrate specificity, and in behavior toward temperature and pH. Enzyme II showed broader substrate specificity than Enzyme I and catalyzed the hydrolysis of lipoamino acids containing various amino acid residues, although Enzyme I was almost specific to the lipoamino acids containing L-glutamate. The extent of hydrolysis by Enzyme II reaction varied depending on the kinds of lipoamino acids and were: 100% for palmitoyl-L-glutamate, 91% for myristoyl-L-glutamate, 85% for lauroyl-L-glutamate, 54% for lauroyl-L-aspartate, 28% for stearoyl-L-glutamate and 17.5% for lauroyl-glycine.     

Purification and characterization of acid beta-D-galactosidase from rabbit spleen

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.     

Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs. I. Isolation and properties of the low-molecular-weight apoproteins.

In a continuing study of protein-lipid interactions in egg yolk, the total apoprotein mixture (i.e. the 'apovitellenins') from the high-lipid, low-density lipoprotein (density 0.97 g/ml) of the yolk from hen's eggs has been isolated in a soluble form. By gel-filtration chromatography in 6M urea the mixture has been separated into several fractions from which three new low-molecular-weight proteins (I, Ia, and II), making up about 30% of the total, have been isolated. The most plentiful of these (I) consists of stable aggregates with several identical subunits each of molecular weight about 10 000. This protein is analogous to the principal protein from the corresponding lipoprotein of emu's egg yolk, i.e. emu's apovitellenin I. Hen's apovitellenin I has a slightly different amino acid composition from that of the emu; notably it contains a sulphydryl group. The hen's protein also forms more stable aggregates that are dissociated by detergent and by guanidine hydrochloride but are stable in urea. The molecular weight of Ia is similar to that of I and the amino acid composition is the same, with the exception that Ia has a higher proportion of amide groups. It aggregates less readily than I under the same conditions. The third new protein (II, 'hens's apovitellenin II') has a molecular weight of about 20 000. It has no tyrosine or methionine residues, but contains glucosamine and has several disulphide groups. It has been isolated in very small amount only.     

Fractionation of the rat liver enzymes that hydrolyze benzoyl-arginine-2-naphthylamide.

1. The enzyme activity in the particulate fraction from rat liver that hydrolyzes alpha-N-benzoyl-DL-arginine-2-naphthylamide (Bz-Arg-NNap) has been separated into two approximately equal components by chromatography on DEAE-cellulose. One component (peak II) is completely retained by the column at low ionic strength while the other component (peak I) passes through. 2. In contrast to the enzyme in peak I, the enzyme in peak II is extremely sensitive to inhibition by leupeptin, it will hydrolyze carbobenzoxy-alanylarginylarginyl-4-methoxy-2-naphthylamine, and it will inactivate aldolase. 3. There appears to be also a minor high molecular weight component of the alpha-N-benzoyl-DL-arginyl-2-naphthylamine-hydrolyzing activity that is retained by the DEAE-cellulose but which has properties similar to those of the peak I enzyme.     

Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide,but not to isonicotinic acid: Differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis

Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (M_r, 137) to isonicotinamide (M_r, 122), not to isonicotinic acid (M_r, 123), in the presence or absence of H₂O₂. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.