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.     

Unique Hepatic Cytosolic Arginase Evolved Independently in Ureogenic Freshwater Air-Breathing Teleost,Heteropneustes fossilis

Hepatic cytosolic arginase (ARG I), an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N^ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150–200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.     

Purification and some properties of five endo-1,4-beta-D-xylanases and a beta-D-xylosidase produced by a strain of Aspergillus niger.

Five different xylanases and a beta-D-xylosidase in the culture medium of Aspergillus niger have been purified to homogeneity from 13- to 52-fold by a procedure of gel and hydroxylapatite chromatography. The strain was isolated from soil of the African equatorial forest. Gel chromatography of the purified enzymes indicated that three of the xylanases have molecular weights of 31,000 and the other two xylanases have molecular weights of 50,000. beta-D-Xylosidase has a molecular weight of 78,000. The pH curves of the xylanases were quite diverse and showed pH optima ranging from 4.0 to 6.5. Characteristic action patterns were obtained for each of the purified xylanases by gel chromatography of the xylan digests on Bio-Gel P-2. The enzymes degraded arabinoxylan by an endomechanism, producing L-arabinose, D-xylose, xylobiose, and a mixture of branched arabinose-xylose and D-xylose oligosaccharides. All xylanases seemed to be capable of liberating L-arabinose from either arabinoxylan or the arabinose-xylose oligosaccharides. Branched arabinose-containing D-xylose oligosaccharides were slowly hydrolyzed, so that these sugars accumulate in the digest. Two xylanases showed relatively broad substrate specificity and were able to degrade also crystalline cellulose. beta-D-Xylosidase showed optimal activity at pH 6.7 to 7.0 and at 42 degrees C. The Km for o-nitrophenyl-beta-D-xylopyranoside was 0.22 mM and xylotriose was hydrolyzed more rapidly than xylobiose.     

Purification of the beta-glucosidase from Sclerotinia sclerotiorum

A beta-glucosidase (EC 3.2.1.21) has been isolated from culture filtrates of the fungus Sclerotinia sclerotiorum. The protein was purified by gel filtration on a column of Bio-Gel P-300 and by ion exchange chromatography on DEAE-Bio-Gel A. The molecular weight, determined by gel filtration, was 240,000. Km values for the enzyme towards p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 0.10 mM and 1.23 mM. The beta-glucosidase activity was found to be strongly associated with a beta-xylosidase (EC 3.2.1.37) activity, suggesting that both activities could be represented in a single protein complex.     

High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.     

alpha-Amylase inhibitor from fungus Cladosporium herbarum F-828

A strain of fungus Cladosporium herbarum extracellularly produced an inhibitor specific for mammalian alpha-amylase. The inhibitor was purified 81-fold by freeze-thawing, heat treatment, and column chromatography on DEAE-cellulose, Sephadex G-75, DEAE-Sephacel, and Bio-Gel P-100. An apparent molecular weight of approximately 18,000 was estimated for the inhibitor using Bio-Gel P-100 filtration. The purified inhibitor preparation was a glycoprotein containing about 10% carbohydrate. The amino acid analysis of the inhibitor showed abundances of Gly, Asp, Glu, Ser, Ala, and Thr residues. The inhibitor was stable between pH 5 and 12 at 4 degrees C, and below 80 degrees C at pH 7.0. A binary complex formation out of equimolar amounts of the inhibitor and alpha-amylase, was demonstrated by polyacrylamide gel electrophoresis, and Bio-Gel P-100 chromatography. Kinetic studies exhibited that the inhibitor noncompetitively inhibited the enzyme reaction with a Ki value of 2.3 approximately 4.8 x 10(-10) M, by combining with the enzyme molecule at a different site from the substrate binding site.     

A simple and rapid high recovery protocol for the purification of arginase

Summary A protocol of purification is devised that gave high recovery of homogeneous arginase from ox erythrocytes. The protocol involved haemolysis, heat treatment, CM- and DEAE-Sepharose chromatography, arginine AH-Sepharose chromatography and molecular sieving through Biogel P-150, all in the presence of 2-mercaptoethanol. It afforded (a) elution of arginase as a single peak, (b) almost 2900 fold purification, (c) 30% recovery and (d) electrophoretically homogeneous arginase. The purified arginase exhibited (a) M.W. of 115,000, (b) dissociation upon SDS treatment into homologous monomers of 30 kDa, (c) optimum pH and temperature 11.5 and 55° C, respectively and (d) Km value for L-arginine-HCl 2.2–2.3 mM, characteristic of the ureotelic type.     

β-Glucosidase Catalyzing Specific Hydrolysis of an Iridoid β-Glucoside from Plumeria obtusa

An iridoid β-glucoside, namely plumieride coumarate glucoside, was isolated from the Plumeria obtusa （white frangipani） flower. A β-glucosidase, purified to homogeneity from P. obtusa, could hydrolyze plumieride coumarate glucoside to its corresponding β-O-coumarylplumieride. Plumeria β-glucosidase is a monomeric glycoprotein with a molecular weight of 60.6 kDa and an isoelectric point of 4.90. The purified β-glucosidase had an optimum pH of 5.5 for p-nitrophenol （pNP）-β-D-glucoside and for its natural substrate. The Km values for pNP-β-D-glucoside and Plumeria β-glucoside were 5.04±0.36 mM and 1.02±0.06 mM, respectively. The enzyme had higher hydrolytic activity towards pNP-β-D-fucoside than pNP-β-D-glucoside. No activity was found for other pNP-glycosides. Interestingly, the enzyme showed a high specificity for the glucosyl group attached to the C-7＂ position of the coumaryl moiety of plumieride coumarate glucoside. The enzyme showed poor hydrolysis of 4-methylumbelliferyl-β-glucoside and esculin, and did not hydrolyze alkyl-β-glucosides, glucobioses, cyanogenic-β-glucosides, steroid β-glucosides, nor other iridoid β-glucosides. In conclusion, the Plumeria β-glucosidase shows high specificity for its natural substrate, plumieride coumarate glucoside.     

Purification and properties of an extracellular beta-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.     

Purification and properties of bovine liver lysosomal adenosine 5'-phosphosulphate sulphohydrolase. A non-specific enzyme with pyrophosphatase and phosphodiesterase activities.

Bovine liver lysosomal adenosine 5'-phosphosulphate sulphohydrolase (EC 3.6.2.1) was purified to apparent homogeneity. The molecular weight of the enzyme was 53 000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis and 56 000 by BioGel P-150 gel filtration. The substrate specificity of the enzyme was studied. The several substrates towards which the enzyme preparation showed high activity were used to establish that a single enzyme was responsible for the different activities. This multiple specificity provides a possible explanation of the physiological role of lysosomal adenosine 5'-phosphosulphate sulphohydrolase.     

Major isoforms of starch branching enzymes in premature seeds of kidney bean (Phaseolus vulgaris L.).

Developing seeds of the kidney bean (Phaseolus vulgaris L.) contain several isoforms of starch branching enzymes. Two of them, KBE1 and KBE2, which are the major forms in the premature seeds, were purified as a single band of protein on SDS-PAGE and native PAGE by chromatographies on DEAE-Sepharose, Bio-Gel P-200, and amylose-binding Sepharose 6B. The enzymes had similar pH optimum (7.0), pH stability (7.0-9.5), temperature optimum (25-30 degrees C), and temperature stability (up to 40 degrees C). Additionally, both were inhibited by various divalent metal ions and activated by citrate. Finally, though their N-terminal amino acid sequences were identical, their molecular masses and affinities for amylose differed; 80 kDa and 1.27 mM for KBE1 and 77 kDa and 0.74 mM for KBE2.     

Arginase from rat fibrosarcoma. Purification and properties

Arginase from rat fibrosarcoma was purified about 1900-fold and its properties were compared with those of the enzyme from liver and kidney. Arginase from fibrosarcoma was a neutral protein of molecular weight 120,000 with a K_m value of 11 mM for arginine. The activation energy was 7.2 kcal/mol and the pH optimum was 10. The fibrosarcoma enzyme was immunologically different from that of the liver. The arginase from fibrosarcoma closely resembled the arginase from the kidney in its electrophoretic, kinetic and immunological properties.     

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.     

Expression, purification, and biochemical properties of arginase from Bacillus subtilis 168

The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The K_m and V_max values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.     

Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

Purification and characterization of arginase from Neurospora crassa

We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.     

Purification and characterization of bacterial aryl alcohol oxidase from <Emphasis Type="Italic">Sphingobacterium</Emphasis> sp. ATM and its uses in textile dye decolorization

Aryl alcohol oxidase (AAO) produced by dye decolorizing bacteria Sphingobacterium sp. ATM, was purified 22.63 fold to a specific activity of 21.75 μmol/min/mg protein using anion exchange and size exclusion chromatography. The molecular weight of the purified AAO was found to be 71 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and confirmed by zymography of AAO using L-dopa. The enzyme showed substrate specificity towards veratryl alcohol, followed by n-propanol. The optimum pH and temperature of purified AAO were found to be 3.0 and 40°C, respectively. The K _m and V _max of AAO was 1.1615 mM and 3.13 mM/min when veratryl alcohol was used as substrate. Sodium azide showed maximum inhibition while ethylenediamine tetra acetic acid (EDTA), L-cysteine and dithiothreitol showed slight inhibition. Metal ions also showed slight inhibition. HPLC analysis confirmed the degradation of Direct Red 5B. The metabolite obtained after decolorization of Direct Red 5B was characterized as 3 diazenyl 7 [-(phenyl carbonyl) amino] naphthalene-2-sulfonic acid using GC-MS analysis.     

Purification and Some Properties of Cell Wall-bound Invertases from Sugar Beet Seedlings and Aged Slices of Mature Roots

Cell wall-bound invertases (EC 3.2.1.26) from both sugar beet seedlings and aged slices of mature roots were purified to homogeneity separately with CM-cellulose chromatography and Bio-Gel P-150 gel filtrations. The enzymes behaved similarly throughout the purification procedures. The purified enzymes are identical as characterized by specific activity, gel electrophoretic mobility, K_m for sucrose and raffinose (1.33 and 4.0 millimolar, respectively), mobility on Bio-Gel P-150 (molecular weight 28,000), optimum pH (4.6 to 5.0), optimum temperature, and dependence on NaCl concentration for insolubilization by DNA. The results suggest that the enzymes may be encoded for by the same structural gene.     

Purification and characterization of a phytase from Pseudomonas syringae MOK1

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40 degrees C. The Michaelis constant (Km) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu2+, Cd2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.