化のう連鎖球菌におけるペニシリン結合たんぱく Ｉ ペニシリン結合たんぱくの性状とそれらに対するβ‐ラクタム系抗生物質の親和性

Abstract

The inner membrane isolated from strain TK-4958 of Streptococcus pyogenes (Str .pyogenes) was incubated with benzyl [14C] penicillin ([14C]-PCG) at 30°C for 10 min. Then, penicillin-binding proteins (PBPs) were fractionated by dodecylsulphate-polyacrylamide gel electrophoresis, after solubilisation of the membrane by the addition of sodium lauroyl sarcosinate and non-radioactive benzylpenicillin (PCG). The properties of PBP s isolated from Str. pyogeneswere compared with those of PBPs of E. coli and the affinities of various β-lactam antibiotics were determined with their competition for the binding of [14C]-PCG to each of the PBPs. The results obtained are as follows:(1) Six PBP s, PBP-1 A (mol. wt.: 96, 000), PBP-1 Bs (mol.wt.: 80, 000, 78, 000), PBP-3 (mol.wt.: 69, 000), PBP-4 (mol.wt.: 52, 000), PBP-5 (mol.wt.: 45, 000) and PBP-6 (mo.wt.: 40, 000), were identified in Str.pyogenes and this species was not estimated to have the same PBP as the PBP-2 of E.coli.(2) These PBPs from Str. pyogenes were more heat labile than the PBPs of E. coli. E specially PBP-1 Bs was almost completely inactivated at 50°C for 10 min., and the property of [14 C]-PCG binding was abolished.(3) The [14C]-PCG bound to PBP-5 or -6 of E. coli was easily released, while almost no release of [14 C]-PCG from the PBPs of S tr. pyogenes was observed. From this fact, the PBP-5 and -6 of this species were suggested to have no penicillinase activity.(4) The binding affinities of various β-lactams for PBP-1 A, -1 Bs and -3, which are considered to be most closely related to lysis of bacteria, were compared by the 50% inhibitive concentration (molar ratio) of [14 C]-PCG binding by competition of β-lactams for [14 C]-PCG to these PBPs or by kinetics of β-lactams. Cephaloridine and PCG had the highest affinities to these PBPs followed by such cephalosporins as cefotiam, cephalothin and cefazolin, and ampicillin. However, there was difference in either Km value or Vmax/Km value. Piperacillin, cloxacillin and cefmetazole had lower affinities for the PBPs than the above mentioned another antibiotics, and mecillinam had almost no affinity.From these results, it was suggested that β-lactams, which have high affinities for PBPs of gram-negative bacilli, did not always show high affinities for those of gram-positive cocci.