Radiolabeling of natural adenosine triphosphatase inhibitor with phenyl (14C)isothiocyanate and study of its interaction with mitochondrial adenosine triphosphatase. Localization of inhibitor binding sites and stoichiometry of binding.
The natural ATPase inhibitor (IF1) from beef heart mitochondria was labeled with phenyl (14C)isothiocyanate [(14C)PITC]. Chemical labeling by (14C)PITC does not modify the inhibitory properties of IF1, provided the number of residues of (14C)PITC bound per molecule of IF1 is lower than five to six, which corresponds to the average labeling of roughly half of the available lysine residues in IF1. This partially labeled, fully active, IF1 was used to determine the binding stoichiometry of IF1 with respect to F1 and to localize the inhibitor binding sites in F1-ATPase. The pattern of loss of ATPase activity of F1 with increasing amounts ot (14C)PITC-IF1 indicated that the ATPase activity is fully inhibited when 1 mol of IF1 is bound to 1 mol of F1. As F1 contains at least 2 beta subunits, this points to a half-site reactivity of F1 with respect to IF1. Sites of interaction between (14C)PITC-IF1 and F1 subunits were investigated by the use of two cross-linking reagents which act as "zero length" cross-linkers, 1-ethyl-3-[(dimethylamino)propyl]carbodiimide (EDAC) and N-(ethoxycarbonyl)-2-ethoxydihydroquinoline (EEDQ); the products of cross-linking were analyzed by NaDodSO4-polyacrylamide gel electrophoresis. IF1 was found to bind preferentially to the beta subunit of F1. Among the cross-linked products formed by reaction of EDAC or EEDQ with subunits of F1, one of them, the beta gamma dimer, did not accumulate when IF1 was added to F1 prior to cross-linking.
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors*; D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D017879:Isothiocyanates; D007553:Isotope Labeling; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011506:Proteins / Q000502:physiology*; D011804:Quinolines; D013861:Thiocyanates; D014357:Trypsin; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D017879:Isothiocyanates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011506:Proteins; D011804:Quinolines; D013861:Thiocyanates; D014357:Trypsin; D000097795:ATPase Inhibitory Protein; C005441:phenylisothiocyanate; C000470:EEDQ