Binding of antibody to the active site of the adenosine triphosphatase of sarcoplasmic reticulum.
1. The thiol group of fragmented sarcoplasmic reticulum that is protected from reaction with N-ethylmaleimide by 1 mM ATP was labelled with N-ethyl-[2,3-14C2] maleimide. Autoradiography after electrophoresis of this material on dodecylsulphate/polyacrylamide gels showed that this group is located on the polypeptide chain of the ATPase. 2. The ATP-protected thiol group of fragmented sarcoplasmic reticulum has been labelled by treatment with either 1-(2,4,-dinitrophenylamino), 6-(N-maleimido) hexane or N, N'-bis(2,4-dinitrophenyl)-L-cystine. The total dinitrophenyl contents of the dinitrophenyl-vesicle conjugates found by spectrophotometry were in good agreement with the ATP-protected thiol content, especially in the case of the N,N'-bis(2,4-dinitrophenyl)-L-cystine-treated vesicles. Fluorescence-quenching titrations of anti-dinitrophenyl-antibody tryptophyl fluorescence with the dinitrophenyl-vesicle conjugates showed that not all the dinitrophenyl groups were available for combination with antibody. 3. Phospholipase C(EC 3.1.4.3) digestion of ATP-protected, N-ethylmaleimide-treated vesicles, labelled with dinitrophenyl groups using N,N'-bis(2,4-dinitrophenyl)-L-cystine, caused the dinitrophenyl groups to become completely inaccessible to anti-dinitrophenyl-antibody, although no dinitrophenyl groups were lost during the incubation. This indicates a possible crowding together of the ATPase molecules as the effective membrane area was reduced.
D000251:Adenosine Triphosphatases / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D008301:Maleimides; D012967:Sodium Dodecyl Sulfate; D004228:Dithionitrobenzoic Acid; D000251:Adenosine Triphosphatases; D005033:Ethylmaleimide