Purification and properties of a carboxylesterase from the liver of tiger shark (Galeocerdo cuvier)

1976 ◽  
Vol 54 (5) ◽  
pp. 453-461 ◽  
Author(s):  
Keith Scott ◽  
Susan E. Hamilton ◽  
Burt Zerner
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Sedimentation Equilibrium ◽  
Acetone Powder ◽  
Tiger Shark ◽  
Equivalent Weight ◽  
Alkyl Esters ◽  
Galeocerdo Cuvier ◽  
Purification And Properties ◽  
Marked Preference

A procedure is described for the purification of a carboxylesterase from shark liver, using a chloroform-acetone powder prepared from the liver as the starting material. The yield of purified enzyme is ~50 mg from 530 g of chloroform–acetone powder. The preparation is electrophoretically homogeneous. Active-site titrations with paraoxon gave an equivalent weight of ~83 000. The molecular weight, found from sedimentation equilibrium experiments, is ~80 000. There is no evidence of any association or dissociation of this species. The enzyme shows a marked preference for aryl esters over alkyl esters, in contrast to other carboxylesterases so far studied. The amino acid composition of the purified enzyme is reported.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from sheep liver

1969 ◽  
Vol 115 (4) ◽  
pp. 639-643 ◽  
Author(s):  
R. H. Villet ◽  
K. Dalziel
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Equilibrium Studies ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Equilibrium Sedimentation ◽  
Purification And Properties

A method is described for the isolation of 6-phosphogluconate dehydrogenase from sheep liver. The product appears to be homogeneous in polyacrylamide-gel electrophoresis and in sedimentation-velocity and sedimentation-equilibrium studies in the ultracentrifuge. The molecular weight is estimated as 129000 from equilibrium sedimentation.

Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors

1980 ◽  
Vol 58 (12) ◽  
pp. 1323-1334 ◽  
Author(s):  
Nicholas E. Dixon ◽  
John A. Hinds ◽  
Ann K. Fihelly ◽  
Carlo Gazzola ◽  
Donald J. Winzor ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Binding Sites ◽  
Spectrophotometric Titration ◽  
Hydroxamic Acids ◽  
Jack Bean Urease ◽  
Equivalent Weight ◽  
Guanidinium Chloride ◽  
Jack Bean ◽  
Equilibrium Ultracentrifugation

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 ± 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of β-mercaptoethanol is ~95 000. Essentially the same subunit molecular weight (~93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride – β-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein–inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

scholarly journals Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1

1974 ◽  
Vol 139 (2) ◽  
pp. 399-405 ◽  
Author(s):  
A. J. Hacking ◽  
J. R. Quayle
Keyword(s):  
Molecular Weight ◽  
Thiol Group ◽  
Sedimentation Equilibrium ◽  
Cleavage Reaction ◽  
Acetyl Coa ◽  
Bivalent Cation ◽  
Optimal Activity ◽  
Purification And Properties ◽  
Equilibrium Data ◽  
Michaelis Constants

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg2+ or Co2+ being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7×10−4m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6×10−5m; acetyl-CoA, 1.5×10−5m; glyoxylate, 1.7×10−3m; Mg2+, 1.2×10−3m.

scholarly journals The number of catalytic sites in acetylcholinesterase

1971 ◽  
Vol 123 (2) ◽  
pp. 139-141 ◽  
Author(s):  
W. Leuzinger
Keyword(s):  
Molecular Weight ◽  
Direct Measurement ◽  
Active Site ◽  
Active Sites ◽  
Native Enzyme ◽  
Equivalent Weight ◽  
Irreversible Inhibitor ◽  
Catalytic Sites

By using two methods of titration, the number of active sites in acetylcholinesterase was determined. Either stepwise inhibition of the enzyme by an irreversible inhibitor, namely di-isopropyl phosphorofluoridate, or direct measurement of the concentration of active sites by titration with o-nitrophenyl dimethylcarbamate yielded an equivalent weight of approx. 130000 for an active site in acetylcholinesterase. This indicates two sites per molecule, since the native enzyme has a molecular weight of 260000.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Lipophilic arsenic compound(s) in the liver of a tiger shark(Galeocerdo cuvier)

1988 ◽  
Vol 2 (4) ◽  
pp. 377-384 ◽  
Author(s):  
Mariko Miyajima ◽  
Noriaki Hamada ◽  
Etsuro Yoshimura ◽  
Akira Okubo ◽  
Sunao Yamazaki ◽  
...  
Keyword(s):  
Tiger Shark ◽  
Arsenic Compound ◽  
Galeocerdo Cuvier

Biosynthesis of the Diguanosine Nucleotides. I. Purification and Properties of an Enzyme from Yolk Platelets of Brine Shrimp Embryos

1974 ◽  
Vol 52 (3) ◽  
pp. 231-240 ◽  
Author(s):  
A. H. Warner ◽  
P. C. Beers ◽  
F. L. Huang
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Artemia Salina ◽  
Temperature Optimum ◽  
Small Molecular Weight ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Purification And Properties

An enzyme that catalyzes the synthesis of P1P4-diguanosine 5′-tetraphosphate (Gp4G) has been isolated and purified from yolk platelets of encysted embryos of the brine shrimp, Artemia salina. The enzyme GTP:GTP guanylyltransferase (Gp4G synthetase) utilizes GTP as substrate, has a pH optimum of 5.9–6.0, a temperature optimum of 40–42 °C, and requires Mg2+ and dithiothreitol for optimal activity. The synthesis of Gp4G is inhibited markedly by pyrophosphate, whereas orthophosphate has no effect on the reaction. In the presence of GDP the enzyme also catalyzes the synthesis of P1,P3-diguanosine 5′-triphosphate (Gp3G), but the rate of synthesis is low compared with Gp4G synthesis and dependent upon other small molecular weight components of yolk platelets.

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