AMP metabolism by the marine bacterium Vibrio (Beneckea) natriegens: purification and properties of adenylate kinase

1981 ◽  
Vol 27 (10) ◽  
pp. 1053-1059 ◽  
Author(s):  
Karamchand Ramotar ◽  
Michael A. Pickard
Keyword(s):  
Molecular Weight ◽  
Adenylate Kinase ◽  
Gel Electrophoresis ◽  
Marine Bacterium ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Atp Formation ◽  
Purification And Properties ◽  
Beneckea Natriegens

Adenylate kinase (EC 2.7.4.3) has been purified 484-fold from extracts of Vibrio natriegens to a specific activity of 1350 μmol ADP formed∙min−1∙mg protein−1. The preparation was 97% pure as judged by gel electrophoresis and exhibited molecular weight values of 29 000 by gel filtration and 32 000 by SDS–gel electrophoresis. The isoelectric point was at pH 4.7. Only ATP (Km 0.067 mM), ADP (Km 0.45 mM), and AMP (Km 0.12 mM) exhibited high activity as substrates, though dATP or dAMP could serve as cosubstrates with AMP or ATP, respectively, at reduced rates. The equilibrium constant in the direction of ATP formation was 1.09, and the pH optimum in both directions was broad, from pH 7.2 to pH 7.6. Enzyme activity was sensitive to the thiolalkylating agents iodacetamide and p-chloromercuriphenyl sulfonate.

scholarly journals PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

2011 ◽  
Vol 14 (3) ◽  
pp. 5-11
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Properties ◽  
Temperature Optima

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

scholarly journals The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1970 ◽  
Vol 118 (1) ◽  
pp. 15-23 ◽  
Author(s):  
K. Balasingam ◽  
W. Ferdinand
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Disc Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Major Form ◽  
Purification And Properties ◽  
Minimal Molecular Weight

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14

1993 ◽  
Vol 39 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Mohamed Blaghen ◽  
Dominique J. M. Vidon ◽  
Mohamed Said El Kebbaj
Keyword(s):  
Molecular Weight ◽  
Yersinia Enterocolitica ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mercury Resistance ◽  
Thiol Compounds ◽  
Mercuric Reductase ◽  
Purification And Properties ◽  
Layer Chromatography

A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70 000. The isolated enzyme had a molecular weight of about 200 000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140 000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105 000 composed of two identical subunits of 52 000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.Key words: Yersinia enterocolitica, mercury resistance, mercuric reductase.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

Partial purification and properties of an L-arabinose dehydrogenase from Azospirillum brasiliense

1983 ◽  
Vol 29 (2) ◽  
pp. 242-246 ◽  
Author(s):  
Norman J. Novick ◽  
Max E. Tyler
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Gel Filtration ◽  
Reducing Agent ◽  
Partial Purification ◽  
Ph Optimum ◽  
Glycine Buffer ◽  
Purification And Properties ◽  
Filtration Technique

An L-arabino-aldose dehydrogenase responsible for the oxidation of L-arabinose to L-arabino-γ-lactone has been purified 59-fold from L-arabinose grown cells of Azospirillum brasiliense. The dehydrogenase was found to be specific for substrates with the L-arabino-configuration at carbons 2, 3, and 4. Km values for L-arabinose of 75 and 140 μM were found with NADP and NAD as coenzymes, respectively. The enzyme had a pH optimum of 9.5 in glycine buffer and was stable when heated to 55 °C for 5 min. No enhancement of activity in the presence of any divalent cation or reducing agent tested was found. L-Arabinose dehydrogenase had a molecular weight of 175 000 as measured by the gel filtration technique.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals Isolation of a human hepatic 60 kDa aspartylglucosaminidase consisting of three non-identical polypeptides

1989 ◽  
Vol 262 (1) ◽  
pp. 189-194 ◽  
Author(s):  
M Baumann ◽  
L Peltonen ◽  
P Aula ◽  
N Kalkkinen
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Inherited Disease ◽  
Ph Optimum ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Molecular Masses ◽  
Polypeptide Chains

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.

scholarly journals The purification and properties of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli and Proteus mirabilis

1971 ◽  
Vol 123 (4) ◽  
pp. 493-500 ◽  
Author(s):  
J. W. Dale ◽  
J. T. Smith
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Resistance Factor ◽  
Ph Optimum ◽  
E Coli ◽  
R Factor ◽  
Purification And Properties

1. The β-lactamase specified by the R-1818 resistance factor in Escherichia coli was purified 300-fold; the resulting preparation gave a single peak on Sephadex G-100 and a single band on polyacrylamide-gel electrophoresis. 2. The β-lactamase specified by the same R-factor in Proteus mirabilis was purified over 2000-fold, but was still far from pure. The specific activity of this preparation was one-fifth that of the purified enzyme from E. coli. 3. The two enzymes were shown to be identical as regards substrate specificity, pH optimum, Km values and molecular weight. 4. It is suggested that the low β-lactamase activity of extracts of P. mirabilis (R-1818), about 5% of that from E. coli (R-1818) in crude extracts, could be due to inefficient transcription of the R-factor DNA by Proteus RNA polymerase.

Purification and properties of phosphoenolpyruvate carboxylase from green leaves of maize

1979 ◽  
Vol 44 (6) ◽  
pp. 1835-1840 ◽  
Author(s):  
Jaroslav Mareš ◽  
Jana Barthová ◽  
Sylva Leblová
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Chloride Ions ◽  
Magnesium Ions ◽  
Ph Optimum ◽  
Green Leaves ◽  
Purification And Properties ◽  
Fructose 1,6 Bisphosphate ◽  
No Activation

Phosphoenolpyruvate carboxylate was isolated from green leaves of maize (Zea mays L.) by a procedure including fractionation with ammonium sulphate, chromatography on DEAE-cellulose and preparative electrophoresis on polyacrylamide gel. The specific activity of the electrophoretically homogeneous enzyme was 23 U/mg. Its molecular weight was about 405 000, pH optimum was within the range 7.9 to 8.3, Km for phosphoenolpyruvate was 1.05 . 10-3 and the apparent Km for the magnesium ions was 8.0 . 10-4M. The enzyme was inhibited by malate, aspartate, citrate, pyruvate, ATP and ADP and chloride ions. It was strongly activated by glycine and glucose 6-phosphate and to a lesser degree by glucose 1-phosphate and fructose 1,6-bisphosphate; no activation by orthophosphate and 3-phosphoglycerate was observed.

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