Purification and characterization of the membrane adenosine triphosphatase complex from the wild-type and N,N'-dicyclohexylcarbodiimide-resistant strains of Streptococcus faecalis.
We have purified the F1-F0 adenosine triphosphatase complex from wild-type Streptococcus faecalis ATCC 9790 and an N,N'-dicyclohexylcarbodiimide (DCCD)-resistant mutant strain, SF-dcc-8. For preliminary purification of the complex, reconstituted F1-F0, prepared from isolated F1 adenosine triphosphatase and depleted membranes, was extracted with sodium deoxycholate and fractionated by salt precipitation. By means of two-dimensional gel electrophoresis, the F1-F0 complex was purified as a single, catalytically active band in the first dimension and then resolved into constituent subunits under denaturing conditions in the second dimension. The electrophoretic purification of F1-F0 removed a delta-less form of F1 as well as other impurities, including lipoteichoic acid. Both the DCCD-sensitive and the DCCD-resistant F1-F0 adenosine triphosphatase appeared to consist of eight proteins, five of which corresponded to the F1 subunits alpha, beta,, gamma, delta, and epsilon. The F0 sector proteins, designated M27, M15, and M6, had Mr values of 27,000, 15,000, and 6,000, respectively. There appear to be multiple copies of M6 in the complex. [14C]DCCD reacted specifically and covalently with M6 in the wild-type F1-F0 but failed to label the M6 protein in the complex from the DCCD-resistant strain. It is suggested that DCCD resistance in the SF-dcc-8 mutant may be due to a modification of the M6 protein which hinders access of DCCD to the reactive site.
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000302:isolation & purification*; D002234:Carbodiimides / Q000494:pharmacology*; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight
D002234:Carbodiimides; D002241:Carbohydrates; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases