Gel Chromatographic Methods Research Articles

The role of calcium for stability and activity of an extracellular proteolytic enzyme from Staphylococcus aureus.

The inactivation by EDTA of an extracellular protease, produced by Staphylococcus aureus, strain V8, was studied by the use of radioactive enzyme, gel chromatography and quantitative immunological methods. It was shown that the removal of Ca2+ from the enzyme by treatment with EDTA (5 mM) at 4° C resulted in irreversible loss of its activity on casein. This inactivation was not accompained by any measurable change in the molecular weight or immunoprecipitating properties of the protease. If treatment with EDTA were performed at 37° C, the loss of activity on casein would be followed by a subsequent loss of the immunoprecipitating properties of the enzyme, which was shown to be due to degradation of the enzyme. The degradation started after the loss of detectable enzyme activity. Thus it may be suggested that the removal of Ca2+ from the enzyme resulted in an altered tertiary structure of the protease in which the active site was hidden so that it could not reach the large substrate molecules i.e. casein and that, though inactive against casein, the calcium‐free enzyme was capable of degrading itself to peptides with molecular weights in the range 4,000–7,000.

The molecular weight of pig liver microsomal carboxylesterase

The enzyme carboxylesterase, isolated from the microsomes of pig liver, was found to have a molecular weight of 180,000 in dilute salt solutions as determined by the method of sedimentation equilibrium. In the presence of 6 m guanidine hydrochloride, 0.1 M β-mercaptoethanol, the molecular weight, uncorrected for preferential solvation, was found to be 61,000, also by the method of sedimentation equilibrium. The molecular weight determined for the enzyme (reduced and alkylated with acrylonitrile) in 6 m guanidine hydrochloride by the method of analytical gel chromatography was found to be 58,200. The method of disc gel electrophoresis in sodium dodecyl sulfate yielded a molecular weight of 62,000. The conclusion of the study is that the native carboxylesterase molecule is comprised of three subunits each with a molecular weight of approximately 60,000.

Casein interactions as studied by gel chromatography and ultracentrifugation

Gel chromatographic and ultracentrifugal methods were employed to demonstrate structural differences in the soluble casein complexes obtained by several pathways. In solutions containing the purified κ- and α s1-casein fractions or whole acid casein at pH 6.85, I 0.26, 30 °C, an equilibrium mixture is established which promotes the formation of a unit weight ratio complex with a Stokes radius of 114 Å and M r 700 000. At the low protein concentrations obtained by zonal gel chromatography, the complex dissociates to yield free κ-casein. Whole casein isolated by high speed centrifugation and freed of calcium at pH 6.85 (first cycle casein) is shown to be an associating system with the polymeric species in rapid equilibrium. First cycle casein dissociates at low protein concentrations to a unit complex with a diameter of about 100 Å and M r approx. 270 000. The unit complex is shown by gel electrophoresis to contain κ-, α s1 - and β-caseins. The mild preparative methods employed in obtaining whole casein by centrifugation at pH 6.6 probably yield complexes more representative of their native quarternary structure than the casein complexes discussed previously. Our results, therefore, support a model of the native casein micelle built up from a 100-Å-diameter complex containing all the casein fractions.

A convenient gel chromatography method for determining the molecular weight distributions and averages of synthetic polypetide collagen models

A convenient gel chromatography method for determining the molecular weight distributions and averages of synthetic polypetide collagen models

The elastase-like enzymes from Streptomyces griseus (pronase). Isolation and partial characterization.

Three elastase‐like enzymes, designated Enzyme I, II and III, were purified from a commercial preparation of Streptomyces griseus (pronase) by successive chromatography on DEAE‐cellulose at pH 9.5 and CM‐cellulose at pH 5.5. Electrophoresis on cellulose acetate at pH 6.9, on polyacrylamide gels at pH 4.5 and 9.5 and exclusion chromatography on Sephadex G‐75 indicated that all three fractions were essentially homogenous. The amino acid composition of the three enzymes was determined and their molecular weights were estimated by gel‐chromatographic methods. All three enzymes possess strong esterolytic activity when assayed on a specific elastase substrate N‐acetyl‐L‐alanyl‐L‐alanyl‐L‐alanine methyl ester (AcAla3OMe) and are active, to a smaller extent, on N‐acetyl‐L‐tyrosine ethyl ester. Their activity on α‐N‐benzoyl‐L‐arginine ethyl ester was negligible. All the enzymes have strong proteolytic activity at slight alkaline pH and Enzyme II and III possess also an elastolytic activity on congo red elastin. All three enzymes are inhibited by diisopropylfluorophosphate. Enzymes II and III were slowly inhibited by N‐tosyl‐L‐phenylalanine chloromethane. Enzyme I was inhibited by the esterolysis products of AcAla3OMe, thus indicating that the same active site controls both esterolytic activities.

Reversible alteration of the structure of enzymes in acidic solution

Enzyme kinetic studies are presented which demonstrate the activating effect of phosphate on the conversion of pyruvate to lactate by rabbit muscle lactate dehydrogenase. A simple method of active enzyme gel chromatography is used to preclude the possibility that this effect is due to redistribution of enzyme between tetrameric and dissociated states as the result of preferential binding of phosphate to the tetrameric enzymatic form. By analysis of the kinetic results in terms of an ordered two-substrate mechanism, the source of the activation is traced to enhancement of the strength of the enzyme-NADH interaction, primarily because of an increase in the rate constant for the formation of the binary enzyme-coenzyme complex. Preliminary estimates of the relevant equilibrium constants from the kinetic data indicate that the binding of phosphate to rabbit muscle lactate dehydrogenase leads to a two- to fourfold increase in the intrinsic association constant for the interaction between NADH and the enzyme under the conditions (pH 7.4, I = 0.15) used to study the activation phenomenon.