scholarly journals The active site of penicillinase from Staphylococcus aureus PC1. Isolation of a specific covalent complex with the substrate quinacillin

1975 ◽  
Vol 149 (2) ◽  
pp. 397-401 ◽  
Author(s):  
R Virden ◽  
A F Bristow ◽  
R H Pain
Keyword(s):  
Staphylococcus Aureus ◽  
Sodium Dodecyl Sulphate ◽  
Active Site ◽  
Sodium Dodecyl ◽  
Carboxyl Group ◽  
Guanidinium Chloride ◽  
Group 3 ◽  
Ph Range ◽  
Hydrolysis Of ◽  
Covalent Complex

1. The penicillinase-catalysed hydrolysis of quinacillin was quenched by addition of 5 m-guanidinium chloride or 1% (w/v) sodium dodecyl sulphate, and the quenched reaction mixture was dialysed exhaustively against solutions of the denaturant. 2. Irreversibly bound quinacillin was shown by titration with HgCl2 to be covalently attached to the protein by the β-lactam carboxyl group. 3. The derivative was found to be stable over the pH range 3.5-8.5. 4. Chymotryptic hydrolysis of the product and subsequent fractionation showed that quinacillin was bound to one or possibly two peptides.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals p-NN′-phenylenebismaleimide, a specific cross-linking agent for F-actin

1978 ◽  
Vol 175 (3) ◽  
pp. 1023-1032 ◽  
Author(s):  
P Knight ◽  
G Offer
Keyword(s):  
Quantitative Analysis ◽  
Sodium Dodecyl Sulphate ◽  
Actin Filaments ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Cross Linking ◽  
Maximum Value ◽  
Cross Links ◽  
The Cross ◽  
Hydrolysis Of

Covalent cross-links can be inserted between the subunits of F-actin by using p-NN′-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN′-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.

scholarly journals Cytochrome P450 102A2 Catalyzes Efficient Oxidation of Sodium Dodecyl Sulphate: A Molecular Tool for Remediation

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Irene Axarli ◽  
Ariadne Prigipaki ◽  
Nikolaos E. Labrou
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Reaction Rate ◽  
Sodium Dodecyl ◽  
Cytochrome P450s ◽  
Ph Values ◽  
Maximum Value ◽  
Endogenous Compounds ◽  
Ph Range ◽  
Bell Shaped Curve

Bacterial cytochrome P450s (CYPs) constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. In the present work we report the characterization of CYP102A2 from B. subtilis with a focus on its substrate specificity. CYP102A2 is more active in oxidation of sodium dodecyl sulphate (SDS) than any other characterized CYP. The effect of SDS and NADPH concentration on reaction rate showed nonhyperbolic and hyperbolic dependence, respectively. The enzyme was found to exhibit a bell-shaped curve for plots of activity versus pH, over pH values 5.9–8.5. The rate of SDS oxidation reached the maximum value approximately at pH 7.2 and the pH transition observed controlled by two ps in the acidic and basic pH range. The results are discussed in relation to the future biotechnology applications of CYPs.

scholarly journals An active-site-directed adenosine triphosphate analogue binds to the β-subunits of factor F1 mitochondrial adenosine triphosphatase with its triphosphate moiety

1979 ◽  
Vol 182 (2) ◽  
pp. 617-619 ◽  
Author(s):  
V L Drutsa ◽  
I A Kozlov ◽  
Y M Milgrom ◽  
Z A Shabarova ◽  
N I Sokolova
Keyword(s):  
Adenosine Triphosphate ◽  
Sodium Dodecyl Sulphate ◽  
Active Site ◽  
Adenosine Triphosphatase ◽  
Covalent Binding ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Beta Subunit ◽  
Mixed Anhydride ◽  
Adenosine Triphosphatase Activity

The reaction of the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid and soluble mitochondrial adenosine triphosphatase is accompanied by the covalent binding of one molecule of the inhibitor to a molecule of the enzyme and results in the inhibition of adenosine triphosphatase activity by more than 90%. The electrophoresis of adenosine triphosphatase modified by reaction with the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that the inhibitor is bound to the beta-subunit of the enzyme. The results suggest that ATP may also bind to the beta-subunit of the adenosine triphosphatase with its triphosphate moiety.

scholarly journals The action of progesterone and diethylstilboestrol on the dehydrogenase and esterase activities of a purified aldehyde dehydrogenase from rabbit liver

1977 ◽  
Vol 161 (1) ◽  
pp. 123-130 ◽  
Author(s):  
R J S Julian
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Rabbit Liver ◽  
Polyacrylamide Gels ◽  
Catalytically Active ◽  
Steroid Sensitive ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of ◽  
Esterase Activities

A steroid-sensitive aldehyde dehydrogenase (EC 1.2.1.3) was purified from rabbit liver and is homogeneous by the criterion of electrophoresis in polyacrylamide gels with or without sodium dodecyl sulphate. The enzyme is tetrameric, of subunit mo.wt. 48 300, and contains no tightly bound zinc. The fluorescence of the protein is decreased in the presence of progesterone, which is inhibitory to the reactions catalysed by the enzyme. When NADH is bound to the enzyme, the fluorescence of the coenzyme is augmented to an extent independent of the presence of steroids or acetaldehyde. The purified enzyme catalyses the oxidation of acetaldehyde and glucuronolactone, and the hydrolysis of 4-nitrophenyl acetate. Each of these reactions is inhibited by progesterone in such a manner as to suggest the formation of a catalytically active enzyme-hormone complex. Diethylstilboestrol inhibits the hydrolysis of esters by this enzyme, but stimulates the oxidation of aldehydes, except at low aldehyde concentrations; the ligand is then inhibitory. NADH inhibits the hydrolysis of 4-nitrophenyl acetate by the enzyme in a partially competitive fashion.

scholarly journals The active site of β-glucosidase from Botryodiplodia theobromae. Effects of pH and dioxan on enzyme-catalysed reactions

1977 ◽  
Vol 167 (3) ◽  
pp. 831-833 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Maximum Velocity ◽  
Carboxyl Group ◽  
Michaelis Constant ◽  
Botryodiplodia Theobromae ◽  
Effects Of Ph ◽  
Group 2 ◽  
Beta Glucosidase ◽  
Hydrolysis Of

1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) of Botryodiplodia theobromae Pat in the absence or presence of added dioxan was found to be dependent on the ionization of two groups, which appeared to be a carboxyl group and an imidazole group. 2. Dioxan increased the Michaelis constant, Km, but decreased the maximum velocity, V.

Irreversible heat denaturation of bovine α-lactalbumin

1986 ◽  
Vol 53 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Lesley C. Chaplin ◽  
Richard L. J. Lyster
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heat Denaturation ◽  
Two Dimensional ◽  
Native Protein ◽  
Denatured Protein ◽  
Hydrolysis Of

SUMMARYThe irreversible heat denaturation of α-lactalbumin (α-la) in 0·1 M-phosphate, pH 7·0, at 100 °C was studied using polyacrylamide-gel electrophoresis (PAGE). PAGE revealed two groups of bands, one moving faster than native α-la and one slower, in addition to some denatured protein which remained at the origin and some residual native α-la. The faster group had unchanged molecular weight, but an increase in charge, partly due to hydrolysis of glutamine and asparagine residues. The slower group was shown by two-dimensional sodium dodecyl sulphate-PAGE to be oligomers of denatured α-la; formation of the smaller oligomers preceded the larger ones. The oligomers reverted to monomers in the presence of dithiothreitol, showing that they were disulphide-linked aggregates of denatured α-la. Immuno-blots of the gels showed that both fast and slow groups of bands had irreversibly lost most of the antigenicity of the native protein.

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