Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei.
Interaction of N,N'-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30-65% inactivation over a concentration range of 5-50 microM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5 X 10(5) M-1 X min(-1). The correlation between the initial binding of [14C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F0F1-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [14C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K+-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F0F1-ATPase complex.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D009161:Mycobacterium / Q000201:enzymology*; D009168:Mycobacterium phlei / Q000201:enzymology*; D011485:Protein Binding; D011510:Proteolipids / Q000302:isolation & purification*; D006180:Proton-Translocating ATPases
D002250:Carbon Radioisotopes; D011510:Proteolipids; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases