Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

pH-sensitive control of arginase by Mn(II) ions at submicromolar concentrations

The manganese dependence of arginase was reinvestigated with extracts of mouse liver to see whether more physiological properties were displayed than have been reported for the purified enzyme. In a preincubation with Mn(II) ions at 37 degrees C the enzyme underwent a slow and reversible activation. At least 90-95% of the activation achieved was dependent on Mn2+. However, no Mn2+ was required for catalytic activity in the assay. The activation showed little dependence upon pH over the range 6.5-9.5, whereas the catalytic activity increased 12-fold in apparent accord with the titration curve of an ionizable group of pKa 7.9. The Mn2+ dependence of arginase activation obeyed Michaelis-Menten kinetics, with Kd varying from 0.3 microM at pH 6.8 to 0.08 microns at pH 7.7. Free Mn2+ concentrations were established in these assays with a trimethylenediaminetetraacetate-Mn buffer. Vmax increased about three-fold over this range. The calculated arginase activity at 0.05 microM Mn2+ increases about nine-fold over this physiological pH range. An enzyme model is proposed to explain these findings. The activity of arginase at "physiological" [Mn2+] and the pronounced pH dependence conferred upon it are consistent with a recently revised role for the urea cycle in the control of bicarbonate and pH in the body. It appears possible that arginase loses Mn2+ sensitivity during the usual purification.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

Purification and characterization of the sesquiterpene cyclase trichodiene synthetase from Fusarium sporotrichioides

The sesquiterpene cyclase, trichodiene synthetase, has been purified from a supernatant fraction of Fusarium sporotrichioides by hydrophobic interaction, anion exchange, and gel filtration chromatography. Purified enzyme had a specific activity 15-fold higher than that previously reported for preparations of terpene cyclases. Molecular weight determinations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography indicated the enzyme to be a dimer with a subunit of Mr 45,000. The requirement of Mg2+ (Km 0.1 mM) for activity could be partially substituted with Mn2+ at a concentration of 0.01 mM, but higher concentrations of Mn2+ were inhibitory. Maximum activity was observed between pH 6.75 and pH 7.75. The Km for farnesyl pyrophosphate was 0.065 microM.     

Wheat germ phosphoglycerate mutase: evidence for a metalloenzyme

Wheat germ phosphoglycerate mutase, exposed to 3.4 M guanidinium chloride at 22 degrees C and pH 7.8, slowly undergoes time-dependent inactivation which can be fully reversed by adding excess Co2+ or Mn2+ to a 50-fold dilution of the denaturing medium. Titration of the denatured enzyme with either Co2+ or Mn2+ shows that wheat germ mutase preferentially binds Co2+. Assuming 1:1 complexation between metal atom and protein, the apparent dissociation constants (Kd) for E Co2+ and E Mn2+ at 22 degrees C and pH 8.7 are approximately 1.06 and 1.84, respectively. Other metal atoms (e.g., Cr2+, Cu2+, Fe2+, Fe3+, Mg2+, and Ni2+) have no effect in restoring the apoenzyme's catalytic activity. At low concentrations (0.11-0.23 mM) Zn2+ partially restores activity, but promotes protein precipitation at elevated concentrations. Evidence suggests that all bisphosphoglycerate-independent phosphoglycerate mutases require either an intra- or an extramolecular metal atom in order to function. Attempts to characterize wheat germ mutase as a glycoprotein have yielded negative results.     

A galactokinase of Mycobacterium sp. 279 galactose mutant

A galactokinase and the other enzymes of a galactose catabolic pathway were found in Mycobacterium sp. 279 galactose mutant. The galactokinase was partially purified in a procedure involving ammonium sulfate precipitation, Sephadex G-100 filtration and DEAE-cellulose chromatography. The enzyme was 170-fold purified with 25% of recovery. It was most active at pH 7.8-8.0 in the presence of Mg2+, CO2+, Mn2+ or Fe2+ ions. The molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 41,700. The apparent Michaelis constants for galactose and ATP in spectrophotometric test were 1.0 mM and 0.29 mM, respectively. Mercuric compounds at concentration of 0.4 mM completely blocked the enzyme. The galactokinase was quite stable during storage at moderatory temperatures and neutral pH but underwent rapid inactivation on heating above 50 degrees C.     

Studies on nitrate reductase of Clostridium perfringens. Purification, some properties, and effect of tungstate on its formation.

Nitrate reductase (NaR) linked to reduced methyl viologen from Clostridium perfringens was purified by ammonium sulfate precipitation. DEAE-cellulose chromatography, disc electrophoresis on polyacrylamide gel, and triple DEAE-Sephadex chromatography. The specific activity was increased 1,200-fold with a yield of 9%. The purified preparation was nearly homogeneous in disc electrophoresis. It was brown, and its spectrum showed a slight shoulder near 420 nm as well as a peak at 280 nm. The molecular weight was found to be 90,000 based on s020,w (5.8S) and 80,000 by Sephadex G-100 gel filtration. In SDS-polyacrylamide electrophoresis, it showed only a single band with a molecular weight of 90,000; it had no subunit structure. The isoelectric point was pH 5.5, and the optimum pH was 9. Mn2+, Fe2+, Mg2+, and Ca2+ stimulated the activity. Km for nitrate was 0.10 mM, and nitrate was stoichiometrically reduced to nitrite in the presence of 2 mM Mn2+. Ferredoxin fraction obtained from extracts of the bacterium was utilizable as an electron donor at pH 8. Cyanide and azide inhibited the enzyme. The formation of NaR was induced by nitrate and inhibited by 0.5 mM tungstate, but recovered in the presence of 0.1 mM molybdate; NaR of C. perfringens appears to be a molybdo-iron-sulfur protein.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

The devapamil-binding site of the purified skeletal muscle receptor for organic-calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+.

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3- methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of (3-[2-(3,4-dimethoxyphenyl)ethyl]- methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitr ile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4 degrees C and 30 degrees C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha 1 subunit, with an apparent Kd of 13 +/- 2.6 nM at 4 degrees C in the presence of 0.4 microM Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4 microM to 100 mM. The same results were obtained at 30 degrees C, although the ligand-receptor complex was not stable at Ca2+ concentrations below 10 microM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (KD) values than the equilibrium experiments performed using 1 nM and 0.4 microM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (KD) by decreasing 10-fold the association rate at 4 degrees C. The dissociation rate was increased about 10-fold by 5 microM devapamil or 100 microM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptors in the absence of Ca2+ and devapamil at 30 degrees C, but not at 4 degrees C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 microM. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4 microM Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.     

2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate, a new nucleotide analogue that acts as an affinity label of pyruvate kinase

A new reactive adenine nucleotide has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 5'-monophosphate (2-BDB-TAMP). Adenosine 5'-monophosphate 1-oxide was synthesized by reaction of AMP with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 5'-monophosphate (TAMP). The final product was generated by reaction of TAMP with 1,4-dibromobutanedione. The structure of 2-BDB-TAMP was determined by UV, 1H NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Rabbit muscle pyruvate kinase is inactivated by 2-BDB-TAMP at pH 7.0 and 25 degrees C. The inactivation rate exhibits a nonlinear dependence on the reagent concentration with KI = 0.57 mM. Protection against inactivation is provided by ADP and ATP, in the presence of Mn2+, as well as by phosphoenolpyruvate, in the presence of K+; in addition, partial protection is provided by AMP plus Mn2+. Incubation of pyruvate kinase with 0.075 mM 2-BDB-TAMP for 70 min in the absence of protective ligands leads to incorporation of 1.55 mol of reagent/mol of enzyme subunit when the enzyme is 53% inactive. In the presence of ADP and Mn2+, only 0.96 mol of reagent/mol of subunit is incorporated at 70 min, while the enzyme retains 100% activity. Similar results were obtained in the presence of ATP plus Mn2+. Assuming that the groups modified in the absence of ligands include those modified in the presence of the nucleotides, the 53% inactivation can be attributed to the modification of 0.59 (1.55-0.96) group per enzyme subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.     

Active site histidine in pig liver aminolevulic acid dehydratase modified by diethylpyrocarbonate and protected by Zn2+ ions

1. The effect of diethylpyrocarbonate (DEP) (0.1-0.35 mM) on the purified pig liver amino-levulic acid dehydratase (ALA-D) containing 0.3 g-atoms Zn/subunit, under different pHs (6.0-7.5), temperature (0-18 degrees C) and time (0-60 min) was studied. 2. Three histidyl residues/subunit were modified by DEP (0.2 mM, pH 6.8), but activity was completely lost after the first one had reacted, indicating the presence of one histidine residue essential for ALA-D catalysis. Reactivation by treatment with hydroxylamine (0.7 mM, pH 7.0) confirmed that only histidine and no other nucleophile amino acids were directly involved in DEP inhibition. 3. Zn ions (0.5 mM) and the substrate ALA (5-10 mM) protected against DEP inactivation, protection was dependent on pH. 4. Sn, Se, Hg, Cd, Mn, Co and Pb (0.01-0.1 mM) did not significantly protect ALA-D against inactivation. 5. It is concluded that the substrate and Zn binding sites and the essential histidyl residues are in close proximity in the active center. It is proposed that in the catalytic synthesis of porphobilinogen from ALA, histidine groups have the specific role of transporting protons from the aqueous media to a hydrophobic active site.     

Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli.

Partial unfolding of dodecameric glutamine synthetase (GS) from Escherichia coli has been studied by differential scanning calorimetry (DSC). A single endotherm (tm = 51.6 +/- 0.1 degrees C and delta Hcal = 211 +/- 4 kcal/mol of enzyme) was observed in DSC experiments with Mn.GS in the presence of 1.0 mM free Mn2+ and 100 mM KCl at pH 7. The dodecameric structure of Mn.GS was retained throughout heating cycles, and thermal transitions were reversible as shown by rescans [with 6-18 mg of GS (Mr 622,000) from 15 to 68 degrees C at 20-60 degrees C/h] and by greater than 93% recovery of activity. A cooperative ratio delta Hcal/delta HvH of 1.6 +/- 0.1 and deconvolution analysis show two cooperative units (two-state transitions): t1 = 50.4 and t2 = 51.7 degrees C; the ratio of the relative sizes of thermally labile domains is approximately 1:2 as judged by delta H2/delta H1 approximately equal to 2. However, the thermally induced overall enthalpy change (0.34 cal/g) for GS dodecamer is only 5-10% of that for thermal unfolding of small globular proteins at 50 degrees C. The t1 and t2 values from deconvolutions of DSC data agree with t0.5 values previously calculated from spectral measurements of temperature-induced exposures of approximately 0.7 of 2 Trp and approximately 2 of 17 Tyr per subunit, respectively [Shrake et al. (1989) Biochemistry 28, 6281-6294], over a 14 degrees C temperature range using both stabilizing and destabilizing conditions for Mn.GS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 2-ketoaldehydes in mold: purification and characterization of glyoxalase I from Aspergillus niger

Glyoxalase I catalyzing the conversion of methylglyoxal into S-lactoylglutathione in the presence of glutathione was purified approximately 1,400-fold with 2.9% activity yield from mold, Aspergillus niger. The enzyme consisted of a single polypeptide chain with a relative molecular weight of 36,000 on both SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The enzyme was most active at pH 7.0, 35-37 degrees C. Among the various aldehydes tested, the enzyme was active on methylglyoxal and 4,5-dioxovalerate with Km values of 1.25 and 0.87 mM, respectively. The activity of the enzyme was completely inhibited by Zn2+ at 0.5 mM. An equimolar amount of EDTA (0.5 mM) protected the enzyme from inactivation by Zn2+. EDTA competitively (K1 = 1.3 mM) inhibited the activity of the enzyme. Fe2+ was a potent activator for the enzyme, the activation being approximately 2.4-fold at 0.5 mM.     

溜曲霉—β—N—乙酰氨基己糖苷酶的纯化与性质

A beta-N-acetylhexosaminidase from mycelium-free culture filtrate of Asp tamarii S215 was purified to PAGE homogenous by ammonium sulfate and polyethylene glycol fractionation precipitation followed by Sephadex G-50 desalt, DEAE-Sephadex A-25 chromatography, hydroxyapatite chromatography and DEAE-Sephadex A-25 rechromatography with 170-fold purification and 24.7% recovery. The ratio of the beta-GlcNAcase and beta-GalNAcase was 2.5 and remained constant throughout the purification. The Mr estimated with concentration gradient PAGE was 140,000 and subunit Mr determined with SDS-PAGE was 72,000, the number of subunit were 2. The pI was 4.2 determined by PAGIEF. The optimum pH was 5.5-6.5 and 5.0-6.0 for beta-GlcNAcase and beta-GalNAcase respectively with stable pH range 5.5-8.3 for both. The optimum temperature was 60 degrees C for beta-GlcNAcase and beta-GalNAcase. The residual activity of beta-GlcNAcase was 52.7% after treated at 50 degrees C for 8 h and it was 44.9% for beta-GalNAcase. The residual activities of both were down to 1% after treated at 62 degrees C for 10 min. The activity was slightly activated by Mn2+ or Fe2+, while strongly inhibited by Hg2+ and slightly by Ag+, Cu2+, Pb2+, Cd2+ or Zn2+. Analyses of amino acids composition showed that the beta-HexNAcase contained about 24.2% acidic amino acids and 14.9% basic amino acids and only 0.6% methionine.     

Ureidoglycolate amidohydrolase from developing French bean fruits (Phaseolus vulgaris [L.].)

Ureidoglycolate is an intermediate of allantoin catabolism in ureide-transporting legumes. This report describes the first purification of ureidoglycolate degrading activity (UGDA) from plant tissue in which the enzyme has been separated from urease. The enzyme from developing fruits of Phaseolus vulgaris has been purified 48-fold to give a preparation free of allantoinase and urease activity. UGDA was inhibited by EDTA while the Vmax was increased in the presence of Mn2+. The Km values for ureidoglycolate in the presence and the absence of Mn2+ were 2.0 and 5.4 mM, respectively. In the absence of Mn2+ UGDA was heat labile at 40 degrees C, but in the presence of Mn2+ the activity was stable up to temperatures of 60 degrees C. The Mr of UGDA was determined to be 300,000 by gel filtration chromatography and the pH optimum ranged from pH 7.0 to 8.5. Ammonia was determined to be the nitrogen-containing product of UGDA by a microdiffusion assay. This enzyme should therefore be described as ureidoglycolate amidohydrolase. The activity was shown to be associated with peroxisomes by fractionation of a crude extract on a sucrose density gradient. The products of ureidoglycolate degradation are glyoxylate, ammonia, and presumably carbon dioxide, which can be readily utilized by pathways of metabolism that are known to be present in this organelle.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Hepatic tyrosine aminotransferase from the rainbow lizard Agama agama: purification and some properties

Tyrosine aminotransferase, induced by dexamethasone in the liver of the rainbow lizard, Agama agama, was extracted under optimal conditions which yield the native undegraded enzyme; purified by heat treatment at 65 degrees C, ammonium sulfate precipitation, chromatography on DEAE-Sephacel and Sephadex G-150-120 and then characterized. The enzyme was purified over 2000-fold to a specific activity of 2653 units/mg of protein. It had an optimum pH of 7.6 in potassium phosphate buffer, KmTyr: 1.0 mM; K alpha-KGm: 0.32 mM; Vmax: 1.33 nmol/min and a molecular weight of about 130,000. It was inhibited by L-glutamate (competitively, Ki, 2.5 mM), and by metal ions Ca2+, Mn2+, Zn2+, Hg2+ and Ag2+, but was unaffected by chelating agents and other divalent cations. Lizard hepatic cytosolic tyrosine aminotransferase was specific for L-tyrosine and alpha-ketoglutarate as substrates sensitive to sulfhydryl inactivation and to protection from thermal lability by alpha-ketoglutarate and pyridoxal phosphate.