Author(s): Lei Yu and Li Jia-tai
Pages: 321-325+320
Year: 1988 Issue:  5
Journal: Chinese Journal of Antibiotics
Keyword:  Penicillin-Binding Proteins; Mode of Action; Beta-lactams;
Abstract: A method of analytical research on the detection of penicillin-binding proteins (PBPs) with electrophoresis on the dodecylsulphate-polyacrylamide slab gel and then auto-radiography -was described in detail in this study.The main procedures were as follows: 1) preparation of bacterial membranes by super-centrifugation and sonication; 2) obtaining the target proteins with their competitive binding to C-14 benzylpenicillin or to the "test" antibiotics; 3) separation of PBPs by sodium docecylsulphate-polyacrylamide slab gel in a discontinuous buffer system; 4) mixed with PPO, X-ray explosure of the dried gel under ultr-cold and microdensitometer scanning for PBPs demonstrated by dark bends within the gel; and 5) calculation of IC 50 applied with cubic-spline interpolation. Some trick steps were stressed, for instance, standardisation of the method, concentration of membrane protein, isotope-labelled antibiotics, and auto-radiography, etc.This study proved that the method, used by most of the laboratories in many countries in the field of molecular pharmacology,is a simple, sensitive and accurate one which should be introduced into our country for the research on penicillin-binding proteins on the basis of molecular pharmacology in the aspect of evaluation of new betalactams.