CHARACTERIZATION OF THE ESSENTIAL GLUTAMIC ACID RESIDUE OF THE MITOCHONDRIAL F(1)-ATPASE THAT REACTS WITH NITROGEN-ETHOXYCARBONYL - 2-ETHOXY-1,2-DIHYDROQUINOLINE (EEDQ, AMPHIPATHIC CATIONS).

摘要

The inactivation of the bovine heart mitochondrial F(,1)-ATPase with the carboxyl activating reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), in the presence of {lcub}('3)H{rcub}aniline at pH 7.0 leads to the covalent incorporation of ('3)H into the enzyme. The amount of ('3)H incorporated per mol of F(,1)-ATPase increased with increasing protein concentration and increased with decreasing pH. On 94% inactivation of the enzyme at 21 mg per ml by 0.9 mM EEDQ in the presence of {lcub}('3)H{rcub}aniline, 4.14 mols of {lcub}('3)H{rcub}anilide were formed per mol enzyme. Isolation of the subunits after this large scale inactivation showed the following incorporation of {lcub}('3)H{rcub}anilide per mol subunit: (alpha), .35; (beta), 1.0; (gamma), 0.01; and (delta) plus (epsilon), 0.06. Examination of the tryptic digests of the isolated subunit showed the presence of a single, major tryptic peptide, which when purified was shown by automatic Edman degradation to have the following sequence for 16 residues: E*-V-A-A-F-A-Q-F-G-S-D-L-D-A-A-T, where E* represents the {lcub}('3)H{rcub}anilide of glutamic acid. The labeled residue corresponds to (alpha)-Glu-402 of the E. coli F(,1)-ATPase. Examination of the tryptic digests of the isolated (beta) subunit by HPLC showed that about 24% of the ('3)H incorporated was non-specifically associated with several peptides, none of which were labeled to an appreciable extent. Another 28% of the ('3)H incorporated into the (beta) subunit was isolated in a tryptic peptide, which, as shown by automatic Edman degradation contained the (gamma)-{lcub}('3)H{rcub}anilide of (beta)-Glu-341. About 48% of the ('3)H incorporated into the subunit was found to be present as the (gamma)-{lcub}('3)H{rcub}anilide of (beta)-Glu-199, the same residue which is labeled when the enzyme is inactivated by dicyclohexyl{lcub}('14)C{rcub}carbodiimide. After carrying out inactivations with EEDQ in the presence of {lcub}('3)H{rcub}aniline under a variety of conditions, only the labeling of (beta)-Glu-199 appeared to be associated with the loss of enzyme activity.; The pharmacologically active amphipathic cations, chlorpromazine, dibucaine, tetracaine are reversible inhibitors of MF(,1)-ATPase. The inactivation of MF(,1) by DCCD was protected by these amphipathic cations. Chlorpromazine also protects the enzyme against inactivation by EEDQ. The relative effectiveness of the reagents as protectors against inactivation by DCCD correlates well with their effectiveness as inhibitors. The amphipathic cation quinacrine and its alkylating derivative quinacrine mustard are structural analogs of chlorpromazine. Evidence presented here suggests that (beta)-Glu-199 is modified in MF(,1) during inactivation of MF(,1) by quinacrine mustard.