Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases

Abstract

Solutions of crystalline beta-lactamase I and beta-lactamase II, prepared by Kuwabara (1970), were examined in the ultracentrifuge and their sedimentation coefficients, diffusion coefficients, molecular weights and heterogeneity determined. Each sample was shown to consist of a major component comprising at least 97% of the material and a minor component of much higher molecular weight. The molecular weights of the major components were 27800 for beta-lactamase I and 35600 for beta-lactamase II. Emphasis is placed on a straightforward practical way of analysing the sedimentation-equilibrium results on mixtures of two macromolecular components rather than on a strict theoretical solution. Appendices describe the theory of systems at both chemical and sedimentation equilibrium and the procedure for calculating the combined distribution of two components.

Bacillus cereus 569/H β-lactamase I: Structure-function relationship

“…The molecular weight of the purified enzyme preparation, estimated from SDS-polyacrylamide electrophoresis, is 27,500, which agrees well with previously published data (25). In addition, the specific activity of the enzyme (0.35 moles benzylpenicillin hydrolyzed/mg protein/h at 30 ~ pH 7.0) is similar to that reported by PATIL and DAY (30).…”

“…In addition, the specific activity of the enzyme (0.35 moles benzylpenicillin hydrolyzed/mg protein/h at 30 ~ pH 7.0) is similar to that reported by PATIL and DAY (30). However, wide variations exist in the amino acid content of ~-lactamase I isolated in different laboratories (6,22,25); thus, in this case, amino acid composition appears to be an unsatisfactory method for determining purity.…”

Extracellular enzyme synthesis in the genus Bacillus.

“…These enzymes are coded for by different genes (unlike the situation in B. licheniformis) and no immunological affinities between them have been detected. /8-Lactamase I, an enzyme with relatively little activity on many cephalosporins, displays many of the properties typical of the penicillinases from other gram-positive sources, e.g., Staphylococcus aureus (136,192,313). ,3-Lactamase U has a number of unusual chemical and enzymological properties that set it apart from other penicillinases.…”

The molecular weight of a yeast pyruvate kinase and its subunits

“…and to be independent of protein concentration. This substantial linearity indicated that the genuine subunits (having a molecular weight M,) were of one size, but the departure from linearity near the bottom of the cell also indicated the presence of a species of larger molecular weight (Ma) which was estimated to be 80,000 by a subtraction method assuming a non-interacting mixture [ 1 l] The analysis of Lloyd and Peacocke [6] was applied when curved Lamm plots were obtained and yielded values of Ma /n/i,, which were fairly constant at 3.8 t 0.30 at different r. Both analyses therefore indicated the presence mainly of subunits of molecular weight 19,500-22,000 and also of a much smaller proportion of a species of Mw of about 80,000-90,000.…”

“…The initial concentrations of pyruvate kinase were determined by differential refractometry at 8". The white-light fringe method of Richards and Schachman [ 51 was used for the determination of the hinge points and measurements of the photographs were made by means of a microcomparator [6] . A molecular weight for the native enzyme was also determined at 16,200 rpm by the meniscus-depletion technique of Yphantis 171.…”