The kinetics of inhibition of Vigna catjang cotyledon and buffalo liver arginase by L-proline and branched-chain amino acids

The effect of proline, isoleucine, leucine, valine, lysine and ornithine under standard physiological conditions, on purified Vigna catjang cotyledon and buffalo liver arginases was studied. The results showed that V. catjang cotyledon arginase is inhibited by proline at a lower concentration than buffalo liver arginase and the inhibition was found to be linear competitive for both enzymes. Buffalo liver arginase was more sensitive to inhibition by branched-chain amino acids than V. catjang cotyledon. Leucine, lysine, ornithine and valine are competitive inhibitors while isoleucine is a mixed type of inhibitor of liver arginase. We have also studied the effect of manganese concentration which acts as a cofactor and leads to activation of arginase. The optimum Mn2+ concentration for Vigna catjang cotyledon arginase is 0.6 mM and liver arginase is 2.0 mM. The preincubation period required for liver arginase is 20 min at 55 degrees C, the preincubation period and temperature required for activation of cotyledon arginase was found to be 8 min at 35 degrees C. The function of cotyledon arginase in polyamine biosynthesis and a possible role of branched chain amino acids in hydrolysis of arginine in liver are discussed.     

The kinetics of inhibition of Vigna catjang cotyledon and buffalo liver arginase by L-proline and branched-chain amino acids

The effect of proline, isoleucine, leucine, valine, lysine and ornithine under standard physiological conditions, on purified Vigna catjang cotyledon and buffalo liver arginases was studied. The results showed that V. catjang cotyledon arginase is inhibited by proline at a lower concentration than buffalo liver arginase and the inhibition was found to be linear competitive for both enzymes. Buffalo liver arginase was more sensitive to inhibition by branched-chain amino acids than V. catjang cotyledon. Leucine, lysine, ornithine and valine are competitive inhibitors while isoleucine is a mixed type of inhibitor of liver arginase. We have also studied the effect of manganese concentration which acts as a cofactor and leads to activation of arginase. The optimum Mn^{2 +} concentration for Vigna catjang cotyledon arginase is 0.6 mM and liver arginase is 2.0 mM. The preincubation period required for liver arginase is 20 min at 55°C, the preincubation period and temperature required for activation of cotyledon arginase was found to be 8 min at 35°C. The function of cotyledon arginase in polyamine biosynthesis and a possible role of branched chain amino acids in hydrolysis of arginine in liver are discussed.     

Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver.

The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.     

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na+/H+ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na+-resistance on an antiporter-deficient E. coli strain, nor did they confer Na+/H+ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.     

Purification and properties of liver arginase from teleostean fish Clarias batrachus (L.).

Liver arginase of Clarias batrachus has been purified to 56.3-fold employing ammonium sulphate fraction, DEAE-cellulose and CM-cellulose chromatography. Bidirectional polyacrylamide gel electrophoresis shows the presence of two isoenzymes of arginase. The enzyme has a molecular weight of about 87,000 and Km 15.38 mM for L-arginine, optimum pH 9.5 and temperature 37 degrees C. Ornithine and leucine as competitive whereas valine and isoleucine act as non-competitive inhibitors with respect to L-arginine as substrate.     

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na⁺/H⁺ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na⁺-resistance on an antiporter-deficient E. coli strain, nor did they confer Na⁺/H⁺ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.     

Kinetic properties and substrate specificities of two recombinant human N-acetylglucosaminyltransferase-IV isozymes

N-acetylglucosaminyltransferase (GnT)-IV catalyzes the formation of the GlcNAcβ1-4 branch on the GlcNAcβ1-2Manα1-3 arm of the core structure of N-glycans. Two human GnT-IV isozymes (GnT-IVa and GnT-IVb) had been identified, which exhibit different expression profiles among human tissues and cancer cell lines. To clarify the enzymatic properties of the respective enzymes, their kinetic parameters were determined using recombinant full-length enzymes expressed in COS7 cells. The K _m of human GnT-IVb for UDP-GlcNAc was estimated to be 0.24 mM, which is 2-fold higher than that of human GnT-IVa. The K _m values of GnT-IVb for pyridylaminated (PA) acceptor sugar chains with different branch numbers were 3- to 6-fold higher than those of GnT-IVa. To compare substrate specificities more precisely, we generated recombinant soluble enzymes of human GnT-IVa and GnT-IVb with N-terminal flag tags. Both enzymes showed similar substrate specificities as determined using fourteen PA-sugar chains. They preferred complex-type N-glycans over hybrid-types. Among the complex-type N-glycans tested, the relative activities of both enzymes were increased in proportion to the number of GlcNAc branches on the Man α1-6 arm. The Man α1-6 arm of the acceptors was not essential for their activities because a linear pentasaccharide lacking this arm, GlcNAcβ1-2Manα1-3Manβ1-4GlcNAcβ1-4 GlcNAc-PA, was a substrate for both enzymes. These results indicate that human GnT-IVb exhibits the same acceptor substrate specificities as human GnT-IVa, although GnT-IVb has lower affinities for donors or acceptors than GnT-IVa. This suggests that GnT-IVa is more active than GnT-IVb under physiological conditions and that it primarily contributes to the biosynthesis of N-glycans.     

Purification, characterization and substrate specificities of xylanase isoenzymes from Melanocarpus albomyces IIS 68

The ascomycetous fungus Melanocarpus albomyces when grown on wheat straw produced seven extracellular xylanase isoenzymes, designated as la, Ib, Ic, IIa, lib, llc, and lId. All seven xylanases were purified to homogeneity by gel filtration and ion-exchange chromatography. The molecular mass (kDa) of la, lb, Ic, Ila, lIb, lIc, and lId were estimated to be 22.9, 20.7, 18.6, 31.3, 25.4, 38.5, and 34.3, respectively by SDS-PAGE, and 23.7, 20.5, 17.1, 31.7, 25.1, 39.8, and 32.2, respectively by gel filtration. The isoelectric points of Ia, lb, Ic, Ila and IIb were found to be 6.2, 6.3, 6.4, 3.7, and 4.4, respectively. The activity of the isoenzymes was dependent on the type of the xylan substrates; Xylanases Ia, lb, and Ic showed highest specific activity toward larchwood xylan (an arabinoglucuronoxylan), IIa and Ilc toward birchwood xylan (a glucuronoxylan), and llb and IId toward beechwood xylan (a glucuronoxylan). Four isoenzymes la, lb, Ic, and Ila had an arabinose-releasing property on larchwood xylan. Application of specific isoenzymes as prebleaching agents in paper manufacture is proposed.     

Properties and substrate specificities of two neuraminidases from Trichomonas fetus

Trichomonas fetus, a protozoon belonging to the class of flagellates causes vaginal infections in cows, leading to sterility or abortion in early stage of pregnancy. Two neuraminidases were isolated from the culture medium and purified by various procedures of gel chromatography, ion exchange chromatography, and by affinity chromatography on N-(4-nitrophenyl)-oxamic acid-Sepharose 4B. The molecular weights of the two neuraminidases were determined as 320 000 (enzyme I) and 38 000 (enzyme II) respectively. However, enzyme I seems to consist of two isoenzymes containing four subunits of almost equal molecular weight. The pH optima of both enzymes depend on the substrates and range from pH 4.7 to 5.5. Due to the type of substrate, the Michaelis constants (Km) vary between 5.0 x 10(-2)M and 6.6 x 10(-3)M for enzyme I and between 1.4 x 10(-2)M and 4.9 x 10(-3)M for enzyme II. Among the different groups of NeuAc-containing substrates, i.e. glycoproteins, glycolipids, oligosaccharides and synthetic ketosides, enzyme I preferably cleaves high molecular weight glycoprotein type substrates whereas enzyme II shows higher affinities to low-molecular weight oligosaccharides. The ganglioside II3NeuAcGgOse4Cer is susceptible to both enzymes only after removal of the lipophilic ceramide residue. Both enzymes show differences in the specificity towards alpha 2 leads 3 to 3, alpha 2 leads to 6, and alpha 2 leads to 8 glycosidic linkages of NeuAc. Taking the rate of cleavage of the alpha 2 leads to linkage in II3NeuAc-Lac as 100, enzyme I reveals 65 for the alpha 2 leads to 6 linkage in II6NeuAc-Lac, and 15 for the alpha 2 leads to 8 linkage in II3(comes from 2 alpha NeuAc8)2-Lac, whereas enzyme II exhibits values around 50 for both the alpha 2 leads to 6- and the alpha 2 leads to 8-linked substrates. The activity of neuraminidase I and II is not influenced by Ca2 but is inhibited by Cu2, Hg2, ann 4-hydroxymercurisulfonic acid. The inhibition by Hg2 and by the latter is reversible with enzyme I by addition of dithioerythritol.     

Purification and properties of the arginase from Jerusalem artichoke tubers

Arginase [l-arginine amidinohydrolase] in Jerusalem artichoke tubers occurs in a particulate fraction from which it was released in active form by detergent treatment. The particulate enzyme was purified 450-fold with ca 3% yield. The enzyme has a MW of ca 140 000 and pI of 5.3. The enzyme required Mn²⁺ for activity and was unstable when Mn²⁺ was removed. In tissue extracts the K_m for arginine was ca 1OmM, but when purified the K_m (arginine) was 145 mM. The artichoke arginase was shown to be more substrate specific than other plant and animal arginases which have been described, and to be very sensitive to competitive inhibition by indospicine, ornithine and citrulline.     

Purification and some properties of arginase from human lung.

Arginase from human lung has been isolated and purified about 100-fold. During the purification procedure the enzyme was stabilized by Mn2+. The molecular weight determined by Sephadex G-150 gel filtration was found to be 120 000. The enzyme is highly specific towards L-arginine. Incubation of the enzyme with EDTA for 60 min at pH 7.5 and 37 degrees C results in dissociation into inactive subunits of mol. wt. 30 000. On addition of Mn2+ ion to the inactivated enzyme, the subunits reassociate into the native form of the enzyme of mol. wt. 120 000, and the activity is restored.     

Purification and properties of soluble and bound gamma-glutamyltransferases from radish cotyledon

Soluble and cell wall bound gamma-glutamyltransferases (GGTs) were purified from radish (Raphanus sativus L.) cotyledons. Soluble GGTs (GGT I and II) had the same M(r) of 63,000, and were composed of a heavy subunit (M(r), 42,000) and a light one (M(r), 21,000). The properties of GGT I and II were similar. Bound GGTs (GGT A and B) were purified to homogeneity from the pellet after the extraction of soluble GGTs. GGT A and B were monomeric proteins with an M(r) of 61,000. The properties of GGT A and B were similar. Thus, bound GGTs were distinguished from soluble GGTs. The optimal pHs of soluble and bound GGTs were about 7.5. Both soluble and bound GGTs utilized glutathione, gamma-L-glutamyl-p-nitroanilide, oxidized glutathione and the conjugate of glutathione with monobromobimane as substrates, and were inhibited by acivicin, but soluble GGTs were also distinguished from bound GGTs with regard to these properties.     

Histidine ammonia-lyase from rat liver. Purification, properties, and inhibition by substrate analogues.

Histidine ammonia-lyase (EC 4.3.1.3) from rat liver was purified more than 250-fold to near homogeneity. Electrophoretic determinations indicated a native molecular weight of approximately 200,000. The enzyme has a pH optimum of approximately pH 8.5. The minimum Km for L-histidine was 0.5 mM at pH 9.0. The Michaelis constant in the physiological pH range was, however, more than 2.0 mM. D-alpha-hydrazinoimidazolylpropionic acid was found to be a potent competitive inhibitor of liver histidine ammonia-lyase (Kis=75 muM); the L enantiomer of this compound was less effective in this regard. The enzyme was also inhibited competitively by L-histidine hydroxamate (Kis=0.4 mM), and to a lesser extent by L-histidinol, D-histidine, and glycine. Failure of a wide variety of other histidine analogues to inhibit the enzyme substantially indicates high specificity of the active site for L-histidine. No alternate substrates were identified for the enzyme. DL-alpha-Hydrazinophenylpropionic acid, the alpha-hydrzino analogue of phenylalanine, was similarly shown to be a very potent competitive inhibitor of a mechanistically similar L-phenylalanine ammonia-lyase purified from Rhodotorula glutinis. The properties of histidine ammonia-lyase from rat liver differ significantly from those of the enzyme from Pseudomonas fluorescens which has been studied most extensively to date.     

Structural basis for different substrate specificities of two ADP-ribose pyrophosphatases from Thermus thermophilus HB8

ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with Mg2+, with Mg2+ and AMP, and with Mg2+ and a nonhydrolyzable ADPR analogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg2+ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.     

Purification and properties of arginase of rat kidney

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.     

Purification and properties of buffalo serum albumin

Based on a detailed study of the solubility of serum albumin, a procedure for its purification by selective ammonium sulphate precipitation has been developed. Using buffalo serum, first extraneous proteins were precipitated by making the serum 2.26 M saturated with ammonium sulphate at pH 7.0 and then albumin was precipitated from the supernatant at 1.9 M ammonium sulphate concentration at pH 4.2. The overall yield of serum albumin thus isolated was about 55% with a purity of 97%. The protein preparation gave a single nearly symmetrical peak on Sephadex G-100 column and virtually a single band on polyacrylamide gel electrophoresis in the presence and absence of SDS. Buffalo serum albumin has a molecular weight of 69,000 Da. The hydrodynamic properties such as Stoke's radius (3.70 nm), diffusion coefficient (6.03 X 10(-7) cm2/s) and frictional ratio (1.36) obtained by analytical gel chromatography, bilirubin binding characteristics and its interaction with anti-bovine serum albumin antiserum suggest that buffalo serum albumin is very similar to BSA in its molecular properties.