scholarly journals Partial purification and properties of l-asparagine synthetase from mouse pancreas

1979 ◽  
Vol 181 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Harry A. Milman ◽  
David A. Cooney
Keyword(s):  
Specific Activity ◽  
Asparagine Synthetase ◽  
Partial Purification ◽  
Synthetase Activity ◽  
Mouse Pancreas ◽  
Purification And Properties ◽  
Bean Trypsin Inhibitor ◽  
Thiol Reagent ◽  
High Concentrations ◽  
Glutaminase Activity

l-Asparagine synthetase was partially purified from mouse pancreas to a final mean specific activity of 0.10 unit/mg of protein. The enzyme exhibited an l-glutaminase activity which was not affected by l-asparate, NH4Cl, ATP–MgCl2, l-glutamate, AMP (sodium salt) or sodium pyrophosphate. The l-glutamine-dependent l-asparagine synthetase activity of the partially purified enzyme from mouse pancreas was markedly decreased by freezing for 7 days at −87°C in the presence of 1mm-dithiothreitol, but effectively protected from inactivation by high concentrations (10mm) of the thiol reagent. The l-glutaminase activity of the enzyme was inhibited by antagonists of l-glutamine (e.g. 6-diazo-5-oxo-l-norleucine, 5-chloro-4-oxo-l-norvaline, 5-diazo-4-oxo-l-norvaline and NSC-163501) and thiol-reactive compounds (e.g. 2-amino-4-arsenophenol hydrochloride, maleimide, mucochloric acid and ZnCl2), but not by aminomalonic acid, the next lower homologue of l-aspartate, nor by l-homoserine β-adenylate, an analogue of the presumed transitory covalent intermediate. The complete forward reaction catalysed by l-asparagine synthetase from mouse pancreas appears to be irreversible and essentially stoicheiometric under the conditions examined. Mouse pancreas contains a proteolytic inhibitor of l-asparagine synthetase separable from the enzyme by ion-exchange column chromatography. The inhibitor is activated by incubation at 4°C for 110h and inactivated by soya-bean trypsin inhibitor, di-isopropyl phosphorofluoridate and boiling.

The purification and properties of an amino acid arylamidase from bovine milk

1969 ◽  
Vol 47 (2) ◽  
pp. 173-178 ◽  
Author(s):  
A. Mellors
Keyword(s):  
Amino Acid ◽  
Milk Fat ◽  
Specific Activity ◽  
Bovine Milk ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Divalent Metal Ions ◽  
Purification And Properties ◽  
Milk Fat Globule ◽  
High Concentrations

An amino acid arylamidase is present in bovine milk and is associated with the "microsomes" of the milk-fat globule membrane. It has been purified by DEAE-cellulose chromatography of a 0.1 M NaCl extrast of milk microsomes. The specific activity of the purified arylamidase was increased 12 700-fold over that of the milk. Three peaks of arylamidase activity could be recognized after the chromatography. One form was apparently bound to casein. The major peak of arylamidase activity hydrolyzes lysyl-, alanyl-, valyl-, and arginyl-β-naphthylamides at similar rates, with little activity against glycyl- and histidyl-β-naphthylamides. The arylamidase requires the restoration of sulfhydryl groups by dithiothreitol for maximum activity. It is inhibited by EDTA and some divalent metal ions, and only calcium ions restore the EDTA-inactivated enzyme. The optimum pH for the hydrolysis of lysyl-β-naphthylamide is pH 7.7, and high concentrations of this substrate are inhibitory.

scholarly journals Partial purification and characterization of a novel neutral proteinase from human uterine cervix

1980 ◽  
Vol 185 (2) ◽  
pp. 443-450 ◽  
Author(s):  
Akira Ito ◽  
Hiroko Ihara ◽  
Yo Mori
Keyword(s):  
Uterine Cervix ◽  
Gel Filtration ◽  
Reaction System ◽  
Partial Purification ◽  
High Concentration ◽  
Neutral Proteinase ◽  
Bean Trypsin Inhibitor ◽  
Cervical Stroma ◽  
High Concentrations ◽  
Hydrolysed Casein

1. Human uterine cervical stroma was found to contain a Ca2+-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×105 by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.

scholarly journals Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

1977 ◽  
Vol 161 (2) ◽  
pp. 357-370 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Transport Chain ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.

scholarly journals The biosynthesis of granulose by Clostridium pasteurianum

1974 ◽  
Vol 144 (3) ◽  
pp. 503-511 ◽  
Author(s):  
R L Robson ◽  
R M Robson ◽  
J G Morris
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Partial Purification ◽  
Clostridium Pasteurianum ◽  
Saturation Curve ◽  
Wild Type ◽  
Competitive Inhibitors ◽  
Fine Control ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like α-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose–α-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5′-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (Ki=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5′-phosphate were competitive inhibitors, ADP being most effective (Ki about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.

Purification and Properties of Thymidylate Synthetase from Pig Thymus

1972 ◽  
Vol 50 (4) ◽  
pp. 352-362 ◽  
Author(s):  
V. S. Gupta ◽  
J. B. Meldrum
Keyword(s):  
Catalytic Activity ◽  
Sulfonic Acid ◽  
Specific Activity ◽  
Gel Filtration ◽  
Thymidylate Synthetase ◽  
Heat Inactivation ◽  
Synthetase Activity ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Sulfhydryl Compounds

Thymidylate synthetase of pig thymus has been separated into two principal forms (designated I and II, based on their order of elution) by chromatography on CM-Sephadex. By the use of (NH4)2SO4 the synthetase activity was separated into two fractions, and these were further purified by gel filtration using Sephadex G-100 and chromatography on CM-Sephadex. The highest specific activity obtained for I and II was 10.4 and 16.3 μmol of thymidine-5′-phosphate per hour per milligram of protein at 25° and pH 7.3 which represents a purification of 1680- and 2630-fold, respectively. Electrophoretically, I and II appear to be 70–80% pure. The Michaelis constants of 7.4 × 10−6 M, 1.7 × 10−5 M, and 1.8 × 10−4 M for II with respect to deoxyuridine-5′-phosphate, 5,10-methlenetetrahydrofolate, and uridine-5′-phosphate, respectively, have been determined. A double pH optima in the range of 6.6–6.8 and 7.2–7.4 in 2-N-morpholinoethane sulfonic acid buffer was exhibited by both forms. Forms I and II showed maximal catalytic activity only in the presence of sulfhydryl compounds (60 mM) and also had the ability to methylate uridine-5′-phosphate, although at a slower rate (ca. 28% and 13%, respectively) compared with the rate of methylation of deoxyuridine-5′-phosphate. Both deoxyuridine-5′-phosphate and tetrahydrofolate (to a lesser extent) afforded protection to II against heat inactivation.

scholarly journals Partial purification and properties of Halobacterium cutirubruml-alanine dehydrogenase

1977 ◽  
Vol 161 (2) ◽  
pp. 313-320 ◽  
Author(s):  
E K Kim ◽  
P S Fitt
Keyword(s):  
Reductive Amination ◽  
Partial Purification ◽  
Oxidative Deamination ◽  
Alanine Dehydrogenase ◽  
Amination Reaction ◽  
Absolute Requirement ◽  
Purification And Properties ◽  
High Concentrations

1. Halobacterium cutirubrum L-alanine dehydrogenase was purified approx. 100-fold. 2. It has a mol. wt. of 72 500, about one-third that of two well-studied alanine dehydrogenases from non-halophiles. 3. The activity of the enzyme increases with temperature up to 70 degrees C, but the protein itself is not thermostable. 4. In the reductive amination reaction, the enzyme is fully active in the presence of high concentrations of K+, Na+ or NH4+ and partially active with Cs+ or Li+, but for oxidative deamination it has an absolute requirement for K+.

Role of pancreatic L-asparagine synthetase in homeostasis of L-asparagine.

1979 ◽  
Vol 236 (6) ◽  
pp. E746 ◽  
Author(s):  
H A Milman ◽  
D A Cooney ◽  
D M Young
Keyword(s):  
Amino Acid ◽  
Exocrine Pancreas ◽  
Specific Activity ◽  
Pancreatic Enzyme ◽  
High Specific Activity ◽  
Asparagine Synthetase ◽  
Mouse Pancreas ◽  
Normal Limits ◽  
Intravenous Treatment

L-Asparagine synthetase from mouse pancreas was found to be associated principally with the exocrine pancreas and to be dependent on the age of the animal, but not on gender, diet, or the presence of tumor under the conditions examined. The function of the pancreatic enzyme appears to be to supply L-asparagine for the synthesis of pancreatic proteins. This function is suggested by the high specific activity of L-asparagine in pancreatic proteins after intravenous treatment of BDF1 mice with L-[U-14C]asparatate. The pancreas is also able to function as a storage depot for L-asparagine under conditions in which the concentration of the amino acid in the blood is in excess. Unlike the liver, the pancreas is unable to add L-asparagine to the circulation when the concentration of the amide is below normal limits.

scholarly journals Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions

1980 ◽  
Vol 189 (3) ◽  
pp. 553-560 ◽  
Author(s):  
K S Srivenugopal ◽  
P R Adiga
Keyword(s):  
Specific Activity ◽  
Hydrolytic Activity ◽  
Lathyrus Sativus ◽  
Partial Purification ◽  
Natural Occurrence ◽  
Thiol Compounds ◽  
Reaction Conversion ◽  
Purification And Properties ◽  
Plant Embryo

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine–Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis–Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine–hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.

scholarly journals Partial purification and properties of frog liver tyrosine aminotransferase

1977 ◽  
Vol 163 (3) ◽  
pp. 411-417 ◽  
Author(s):  
J J Ohisalo ◽  
S M Andersson ◽  
J P Pispa
Keyword(s):  
Pyridoxal Phosphate ◽  
Specific Activity ◽  
Rana Temporaria ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Complex Form ◽  
Tyrosine Aminotransferase ◽  
Partial Purification ◽  
Purification And Properties ◽  
Frog Rana Temporaria

Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5′-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.

Partial purification and properties of an endo-xylanase from cucumber seeds

1991 ◽  
Vol 81 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Cesar V. Mujer ◽  
Dale W. Kretchman ◽  
A. Raymond Miller
Keyword(s):  
Partial Purification ◽  
Purification And Properties
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