Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.     

Purification and characterization of an alpha-glucosidase from Rhizobium sp. (Robinia pseudoacacia L.) strain USDA 4280.

A novel alpha-glucosidase with an apparent subunit mass of 59 +/- 0. 5 kDa was purified from protein extracts of Rhizobium sp. strain USDA 4280, a nodulating strain of black locust (Robinia pseudoacacia L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 +/- 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35 degrees C. The activity increased in the presence of NH4+ and K+ ions but was inhibited by Cu2+, Ag+, Hg+, and Fe2+ ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl alpha-glucoside was the fluorogenic substrate. The enzyme was more active with alpha-glucosides substituted with aromatic aglycones than with oligosaccharides. This alpha-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl alpha-D-glucopyranoside (Km, 0.141 microM; Vmax, 6.79 micromol min-1 mg-1) and with p-nitrophenyl alpha-D-glucopyranoside (Km, 0.037 microM; Vmax, 2.92 micromol min-1 mg-1). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.     

Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea.

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of L-tryptophan by dimethylallyl diphosphate to form 4-(gamma,gamma-dimethylallyl)-L-tryptophan. The enzyme from mycelia of Claviceps purpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an alpha 2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for KM and Vmax were determined in the metal-free EDTA buffer (KMDMAPP, 14 microM; KML-tryptophan, 40 microM; Vmax, 215 nmol min-1 mg-1), 4 mM CaCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 17 microM; Vmax, 504 nmol min-1 mg-1), and 4 mM MgCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 12 microM; Vmax, 455 nmol min-1 mg-1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.     

Purification and characterization of phosphoenolpyruvate phosphomutase from Pseudomonas gladioli B-1.

Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B-1 isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by cleaving the C-P bond of PnPy, but the C-P bond-forming reaction is physiologically significant. The C-P bond-forming activity of PEPPM was confirmed with a purified protein. The molecular mass of the native enzyme was estimated to be 263 and 220 kDa by gel filtration and polyacrylamide gel electrophoresis, respectively. A subunit molecular mass of 61 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the native protein was a tetramer. The optimum pH and temperature were 7.5 to 8.0 and 40 degrees C, respectively. The Km value for PnPy was 19 +/- 3.5 microM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 microM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the Km values were 3.5 +/- 1.4 microM for Mg2+, 16 +/- 5 nM for Mn2+, 3.0 +/- 1.5 microM for Zn2+, and 1.2 +/- 0.2 microM for Co2+.     

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.     

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, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646.

An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/- 3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/- 75, 29.3 +/- 3.1, and 57.3 +/- 12.5 microM, respectively. The Vmax was determined to be 0.902 +/- 0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/- 2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/- 30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined.     

Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.     

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.     

Malic enzyme in human liver. Intracellular distribution, purification and properties of cytosolic isozyme

In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction. Extramitochondrial malic enzyme has been isolated from the post-mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, ADP-Sepharose-4B and Sephacryl S-300 to apparent homogeneity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 mumol.min-1.mg protein-1, which corresponds to about 10,000-fold purification. The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is a tetramer composed of identical-molecular-mass subunits. The isoelectric point of the isolated enzyme was 5.65. The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction. The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP-linked decarboxylation reaction varied with malate concentration. The Km values determined at pH 7.2 for malate and NADP were 120 microM and 9.2 microM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 microM and 27.9 mM, respectively. The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. The Km values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD-dependent decarboxylation reaction was not reversible. The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetate and NADPH-linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP-linked oxidative decarboxylation of malate, respectively. The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.     

Rat kidney histamine N-methyltransferase: purification and partial characterization

Histamine N-methyltransferase (HMT, EC 2.1.1.8) was purified 8,420-fold in 44% yield from rat kidney. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous as determined by gel electrophoresis and was stable for at least five months at -80 degrees C. The apparent molecular weight of the enzyme was found to be 31,500 as determined by gel filtration through Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was determined to be 5.4. The Km's for histamine and S-adenosyl-L-methionine were 12.4 +/- 1.3 microM and 10.2 +/- 0.5 microM, respectively. When S-adenosyl-L-methionine was the variable substrate, the Ki's for S-adenosyl-L-homocysteine and S-adenosyl-D-homocysteine were 31.9 +/- 3.4 microM and 32.0 +/- 3.5 microM, respectively. When histamine was the variable substrate, the Ki for S-adenosyl-L-homocysteine was 11.8 +/- 0.6 microM. Comparison of physico-chemical and catalytic properties of the rat kidney and the guinea pig enzymes suggest that these proteins have similar structural and catalytic characteristics.     

Determination of some kinetic and characteristic properties of glutathione S-transferase from bovine erythrocytes

Glutathione S-transferase was purified from bovine erythrocytes and some kinetic and characteristic properties of the enzyme were investigated. The purification procedure was composed of preparation of homogenate and Glutathione-Agarose affinity chromatography. Thanks to the procedure, the enzyme was purified 6,800 fold with 97% yield and a specific activity of 136 EU/mg proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), one band with a mass of 27 kDa was found. The native molecular weight of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimum pH, stable pH, optimum temperature, and optimum ionic strength were determined as 7.0, 6.5 in K-phosphate buffer, 20 degrees C, 0.1 M K-phosphate, respectively. The best activity was obtained with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed with different substrates. Vmax, Km, and kcat values were calculated as 402.63 +/- 4.99 EU/mg proteins, 0.7447 +/- 0.0007 mM, and 11436 min(-1) for CDNB, and 88.00 +/- 2.30 EU/mg proteins, 0.3257 +/- 0.0012 mM, and 477 min(-1) for GSH, respectively, by using Lineweaver-Burk graphs obtained from 1/V versus 1/[CDNB] and 1/[GSH].     

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.     

Dephosphorylation of phytate by using the Aspergillus niger phytase with a high affinity for phytate.

A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.     

Purification and properties of a glutathione peroxidase from Southern bluefin tuna (Thunnus maccoyii) liver

A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 micromol NADPH oxidised min(-1) mg(-1) protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 microM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX.     

Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).     

Purification and properties of mitochondrial monoamine oxidase type A from human placenta

A high yield purification scheme for monoamine oxidase A from human placental mitochondria is described. The enzyme is solubilized by a combination of treatment with phospholipase A and C and extraction with Triton X-100 and further purified by partitioning between dextran and polyethylene glycol polymers. The enzyme was obtained in 35% yield and high purity on DEAE-Sepharose CL-6B chromatography. This product, 90% catalytically active, showed a single major and several minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further purification could be achieved by additional chromatography using Bio-Gel HTP, but concomitant loss of catalytic activity occurred (enzyme remained about 60% active). The difference extinction coefficient for flavinox--flavinred at 456 nm was 10,800 +/- 350 m-1 cm-1. A sulfhydryl to flavin ratio of 7.5 was obtained when enzyme was denatured with sodium dodecyl sulfate, reduced with 2-mercaptoethanol, and titrated with 2,2'-dipyridyl disulfide. Anaerobic titration with 0.5 eq of sodium dithionite gave rise to the red anionic flavin radical, and full reduction was observed on further addition of reagent. The Km value for kynuramine was essentially the same for mitochondria (0.12 mM) and enzyme after DEAE-Sepharose CL-6B chromatography (0.17 mM). The concentration of clorgyline and deprenyl required for 50% inactivation also remained essentially unchanged. Incubation of the enzyme with 2,2'-dipyridyl disulfide caused inactivation in a biphasic manner with apparent second-order rate constants of 1230 M-1 min-1 and 235 M-1 min-1 for the rapid and slow phase, respectively. This inactivation was largely abolished by the inclusion of the competitive inhibitor amphetamine (Ki = 20 microM) in the incubation mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit molecular mass of 60-64 kDa, about 1.5-2.5 kDa higher than human liver monoamine oxidase B.     

Isolation and properties of oxaloacetate keto-enol-tautomerases from bovine heart mitochondria

Two highly purified proteins with quite different properties capable of oxaloacetate keto-enol-tautomerase activity (oxaloacetate keto-enol-isomerase, EC 5.3.2.2) were isolated from the bovine heart mitochondrial matrix. The first protein has an apparent molecular mass of 37 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-200 gel filtration. It is quite stable upon storage at 40 degrees C and reaches the maximal catalytic activity at pH 8.5 with a half-maximal activity at pH 7.0. The enzyme is specifically inhibited by oxalate and diethyloxaloacetate. When assayed in the enol----ketone direction at 25 degrees C (pH 9.0), the enzyme obeys a simple substrate saturation kinetics with Km and Vmax values of 45 microM and 74 units per mg of protein, respectively; the latter value corresponds to the turnover number of 2700 min-1. The second protein has an apparent molecular mass of 80 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-300 gel filtration. The enzyme is rapidly inactivated at 40 degrees C and shows a sharp pH optimum of activity at pH 9.0. The enzyme can be completely protected from thermal inactivation by oxaloacetate and dithiothreitol. The kinetic parameters of the enzyme as assayed in the enol----ketone direction at 25 degrees C (pH 9.0) are: Km = 220 microM and Vmax = 20 units per mg of protein; the latter corresponds to the turnover number of 1600 min-1. The enzyme activity is specifically inhibited by maleate and pyrophosphate. About 30% of the total oxaloacetate tautomerase activity in crude mitochondrial matrix is represented by the 37 kDa enzyme and about 70% by the 80 kDa protein.