Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumoniae NCTR 1.

An amidase capable of degrading acrylamide and aliphatic amides was purified to apparent homogeneity from Klebsiella pneumoniae NCTR 1. The enzyme is a monomer with an apparent molecular weight of 62,000. The pH and temperature optima of the enzyme were 7.0 and 65 degrees C, respectively. The purified amidase contained 11 5,5-dithiobis(2-nitrobenzoate) (DTNB)-titratable sulfhydryl (SH) groups. In the native enzyme 1.0 SH group readily reacted with DTNB with no detectable loss of activity. Titration of the next 3.0 SH groups with DTNB resulted in a loss of activity of more than 70%. The remaining seven inaccessible SH groups could be titrated only in the presence of 8 M guanidine hydrochloride. Titration of SH groups was strongly inhibited by carboxymethylation and KMnO4, suggesting the presence of SH groups at the active site(s). Inductively coupled plasma-atomic emission spectrometry analysis indicated that the native amidase contains 0.33 mol of cobalt and 0.33 mol of iron per mol of the native enzyme. Polyclonal antiserum against K. pneumoniae amidase was raised in rabbits, and immunochemical comparisons were made with amidases from Rhodococcus sp., Mycobacterium smegmatis, Pseudomonas chlororaphis B23, and Methylophilus methylotrophus. The antiserum immunoprecipitated and immunoreacted with the amidases of K. pneumoniae and P. chlororaphis B23. The antiserum failed to immunoreact or immunoprecipitate with other amidases.     

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.     

L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.     

Dichloromethane dehalogenase of Hyphomicrobium sp. strain DM2.

Dichloromethane dehalogenase, a highly inducible glutathione-dependent enzyme catalyzing the conversion of dichloromethane into formaldehyde and inorganic chloride, was purified fivefold with 60% yield from Hyphomicrobium sp. strain DM2. The electrophoretically homogeneous purified enzyme exhibited a specific activity of 17.3 mkat/kg of protein. Its pH optimum was 8.5. The enzyme was stable at -20 degrees C for at least 6 months. A subunit molecular weight of 33,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration of native dichloromethane dehalogenase yielded a molecular weight of 195,000. Subunit cross-linking with dimethyl suberimidate confirmed the hexameric tertiary structure of the enzyme. Dichloromethane dehalogenase was highly specific for dihalomethanes. Its apparent Km values were 30 microM for CH2Cl2, 15 microM for CH2BrCl, 13 microM for CH2Br2, 5 microM for CH2I2, and 320 microM for glutathione. Several chlorinated aliphatic compounds inhibited the dichloromethane dehalogenase activity of the pure enzyme. The Ki values of the competitive inhibitors 1,2-dichloroethane and 1-chloropropane were 3 and 56 microM, respectively.     

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.     

Presence of rhodanese in the cytosolic fraction of the fruit bat (Eidolon helvum) liver

Rhodanese was isolated and purified from the cytosolic fraction of liver tissue homogenate of the fruit bat, Eidolon helvum, by using ammonium sulphate precipitation and CM-Sephadex C-50 ion exchange chromatography. The specific activity was increased 130-fold with a 53% recovery. The K(m) values for KCN and Na(2)S(2)O(3) as substrates were 13.5 +/- 2.2mM and 19.5 +/- 0.7 mM, respectively. The apparent molecular weight was estimated by gel filtration on a Sephadex G-100 column to be 36,000 Da. The optimal activity was found at a high pH (pH 9.0) and the temperature optimum was 35 degrees C. An Arrhenius plot of the heat stability data consisted of two linear segments with a break occurring at 35 degrees C. The apparent activation energy values from these slopes were 11.5 kcal/mol and 76.6 kcal/mol. Inhibition studies on the enzyme with a number of cations showed that Mg(2+), Mn(2+), Ca(2+), and Co(2+) did not affect the activity of the enzyme, but Hg(2+) and Ba(2+) inhibited the enzyme.     

Purification and characterization of an esterase hydrolyzing monoalkyl phthalates from Micrococcus sp. YGJ1

An esterase that specifically hydrolyzes medium-chain (C(3)-C(5)) monoalkyl phthalates was purified from phthalate-grown Micrococcus sp. YGJ1. The enzyme activity was split into two fractions by hydrophobic chromatography on Phenyl Sepharose, and the enzymes were purified to homogeneity from each fraction. The purified enzymes showed similar properties with respect to molecular mass (60 kDa), subunit molecular mass (27 kDa), N-terminal amino acid sequence, optimal pH (about 7.5), temperature-dependence, substrate specificity, and inhibitor susceptibility. The enzymes showed no activity toward various dialkyl phthalates or aliphatic carboxyl esters. 2-Mercaptoethanol effectively protected the enzymes from spontaneous inactivation. Diethylpyrocarbonate, p-chloromercuribenzoate, Hg(2+), and Cu(2+) strongly inhibited the enzymes, while phenylmethylsulfonyl fluoride produced weak inhibition, and various metal chelating reagents were ineffective. These findings show that the enzymes bear a close resemblance to the putative phthalate ester hydrolase (PehA) of Arthrobacter keyseri 12B.     

酸性α-淀粉酶的分离纯化与酶学性质研究

纯化了枯草芽胞杆菌xm-1菌株酸性α-淀粉酶,并对其酶学性质进行了研究。通过硫酸铵沉淀和Sephadex G-75凝胶层析将酸性α-淀粉酶粗酶液纯化了32.5倍,活力回收率为10.0%。酶性质测定结果表明,该酸性α-淀粉酶分子量约为60kD,最适反应温度为45℃、最适作用pH5.0,该酶在pH3.4-6.0下稳定,高温耐受性差。Cu2+、Zn2+、EDTA对酶有不同程度的抑制作用,Ca2+和Mn2+对酶具有较强的激活作用。     

Arylsulphatases in human brain: purification and characterization of an isoluble arylsulphatase

After solubilization with 0.5% (w/v) lysolecithin an arylsulphatase was purified 30-fold from human brain. By this procedure, 82% of the activity was recovered in the 100,000 g supernatant fluid. Solubilization of the enyzme was dependent on lysolecithin concentration but not on the time of incubation. The enzyme was purified using ethanol and ammonium sulphate fractionations. The purified protein showed a single band on acrylamide gel electrophoresis in two different buffer systems. On ultracentrifugation, a sharp symmetrical peak was obtained with a s_20,w value of 5.4 and an apparent molecular weight of 103,000 daltons was calculated. A molecular weight of 105,000 daltons was obtained by sucrose density gradient. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed the presence of two subunit species with molecular weights of 47,000 and 25,000 daltons. The enzyme was unstable at 04°C but could be stored in a frozen state without much loss of activity. 4-Methylumbelliferone-sulphate was used as substrate in these studies and the product, methylumbelliferone, was quantified fluorometrically. The enzyme had an optimum pH of 6.8. A higher activity was exhibited in imidazole buffer than in acetate buffer. Enzyme activity was linear up to 30 min of incubation. The enzyme showed a K_m of 37.7 μm for 4-methylumbelliferone-sulphate. Ammonium sulphate at 5 mm produced a slight activation of the enzyme. Borate, silver and sulphite ions inhibited enzyme activity, whereas p-chloromercuribenzoate, and cyanide, arsenite, fluoride and phosphate ions caused very little inhibition. The chemical enzymatic hydrolysis of the native enzyme revealed the presence of 2 mol of sialic acid per mole of the enzyme. Enzymatic removal of sialic acid did not affect the activity of the enzyme; therefore, the sialic acid moiety was not required for enzyme activity.     

Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific D-carbamoylase guarantee the total conversion from D,L-5-monosubstituted hydantoins with a low velocity of racemization to optically pure D-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of Agrobacterium tumefaciens, C58 and LBA4404, in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000 Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55 degrees C with L-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni(2+) and Co(2+) and strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with Mn(2+), EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).     

Purification and Characterization of an l-Amino Amidase from Mycobacterium neoaurum ATCC 25795

An l-amino amidase from Mycobacterium neoaurum ATCC 25795 responsible for the enantioselective resolution of dl-alpha-methyl valine amide was purified and characterized. The purification procedure included ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography, which resulted in a homogeneous preparation of the enzyme with a native molecular mass of 136 kDa and a subunit molecular mass of 40 kDa. The purified enzyme displayed the highest activity at 50 degrees C and at pH 8.0 and 9.5. The enzyme was strongly inhibited by the metal-chelating agent 1,10-phenanthroline, the disulfide-reducing agent dithiothreitol, and the cysteine proteinase inhibitor iodoacetamide. The purified amino amidase showed a unique l-enantioselective activity towards a broad range of both alpha-H- and alpha-alkyl-substituted amino acid amides, with the highest activity towards the cyclic amino acid amide dl-proline amide. No activity was measured with dl-mandelic acid amide nor with the dipeptide l-phenylalanine-l-leucine. The highest catalytic efficiency (k(cat)/K(m) ratio) was measured with dl-alpha-allyl alanine amide, dl-alpha-methyl phenylalanine amide, and dl-alpha-methyl leucine amide.     

Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.     

Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1

A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+). The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).     

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.     

Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26

An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. Methylene-blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino-acid analysis indicated that this photooxidative loss of activity corresponds to the modification of one histidine residue per monomer of protein.     

Purification and properties of bovine liver plasma membrane 5' nucleotidase

5'-Nucleotidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin-A-Ultrogel and 5' AMP-Sepharose 4B followed by AcA-54-Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5'-Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane-bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane-bound enzyme showed a wide range of pH optimum (7.5-8.3). An Arrhenius plot of the membrane-bound enzyme shows a break at 28 degrees C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+.     

Purification and Characterization of an Enantioselective Amidase from Pseudomonas chlororaphis B23

An amidase produced by Pseudomonas chlororaphis B23 was purified and characterized. The purification procedure used included ammonium sulfate precipitation and hydrophobic, anion-exchange, gel filtration, and ceramic hydroxyapatite chromatography steps. This amidase has a native molecular mass of about 105 kDa and is a homodimer whose subunits have a molecular mass of 54 kDa. The enzyme exhibited maximal activity at 50(deg)C and at pH values ranging from 7.0 to 8.6. We found no evidence that metal ions were required, and the enzyme was inhibited by several thiol reagents. This amidase exhibited activity against a broad range of aliphatic and aromatic amides and exhibited enantioselectivity for several aromatic amides, including 2-phenylpropionamide (enantiomeric excess [ee] = 100%), phenylalaninamide (ee = 55%), and 2-(4-chlorophenyl)-3-methylbutyramide (ee = 96%), but not 2-(6-methoxy-2-naphthyl)propionamide (the amide form of naproxen) (ee = 0%). The characteristics of the P. chlororaphis B23 amidase are the same as the characteristics of enantioselective amidases described by Mayaux et al. (J. F. Mayaux, E. Cerbelaud, F. Soubrier, D. Faucher, and D. Petre, J. Bacteriol. 172:6764-6773, 1990; J. F. Mayaux, E. Cerbelaud, F. Soubrier, P. Yeh, F. Blanche, and D. Petre, J. Bacteriol. 173:6694-6704, 1991) and Kobayashi et al. (M. Kobayashi, H. Komeda, T. Nagasawa, M. Nishiyama, S. Horinouchi, T. Beppu, H. Yamada, and S. Shimizu, Eur. J. Biochem. 217:327-336, 1993).     

Purification and characterization of an oxygen-stable carbon monoxide dehydrogenase of Methanothrix soehngenii

Carbon monoxide dehydrogenase was purified to apparent homogeneity from Methanothrix soehngenii. In contrast with the carbon monoxide dehydrogenases from most other anaerobic bacteria, the purified enzyme of Methanothrix soehngenii was remarkably stable towards oxygen and it was only slightly inhibited by cyanide. The native molecular mass of the carbon monoxide dehydrogenase of Methanothrix soehngenii determined by gel filtration was 190 kDa. The enzyme is composed of subunits with molecular mass of 79.4 kDa and 19.4 kDa in an alpha 2 beta 2 oligomeric structure. The enzyme contains 1.9 +/- 0.2 (n = 3) mol Ni/mol and 19 +/- 3 (n = 3) mol Fe/mol and it constitutes 4% of the soluble cell protein. Analysis of enzyme kinetic properties revealed a Km of 0.7 mM for CO and of 65 microM for methyl viologen. At the optimum pH of 9.0 the Vmax was 140 mumol of CO oxidized min-1 mg protein-1. The enzyme showed a high degree of thermostability.     

Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells

An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.