Studies on the sarcoplasmic reticulum (Ca'2'+ + Mg'2'+) - ATPase.
Tryptic digestion of SRV labelled with N-cyclohexyl-N'-(4-dimethylamino--naphthyl) carbodiimide (NCD-4), was shown to expose Ca^2+-protectable NCD-4-labelled amino acid residues, manifested by a time dependent quench (38%) of the initial fluorescence of the preparation. This finding is discussed in terms of a 3D model of the (Ca2+ + Mg2+)-ATPase. The inhibition of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by the diphenolic antioxidants: 4,4'-isopropylidene diphenol, 4-hydroxy, 4'-methoxy isopropylidene diphenol, 4,4'dimethoxy isopropylidene diphenol and bis (2-hydroxy-3,5-methyl phenyl) methane, was studied, using the fluorescence response to bound NCD-4. They were shown to modulate conformational transitions of the ATPase subsequent to Ca2+ binding. It is suggested that the diphenolic antioxidants may have a target/binding site on the ATPase polypeptide via which they compete with Ca2+ ion binding and translocation. The stoichiometry of incorporation into the ATPase of tritated NCD-4 was studied. The ATPase was incubated with [3H]-NCD-4 for 4h. in the presence and in the absence of Ca2+ ions and then treated with trypsin for 35 mins. The four fragments: A(Mr= 50KD), B(Mr= 45KD), A1(Mr= 30KD) and A2(Mr= 20KD) were purified by preparative SDS-PAGE. The stoichiometry of incorporation of the radioactive label into fragments A1, A2 and the A2 subfragments SI (Mr= 13KD) and SII(Mr= 8.5KD) from CNBr cleavage, was measured by liquid scintillation counting of the gel eluates. In the absence of Ca2+, fragment A2 was found to incorporate 2.3 nmol [^3H]-NCD-4 per nmol of protein more than the equivalent preparation in the presence of Ca^2+. Subfragment S_I was shown to incorporate roughly equal amounts of label in both cases. S_II, however, in the absence of Ca^2+ was found to incorporate 1.15-2.3 nmol [^3H]-NCD-4 more than the equivalent preparation in the presence of Ca^2+. These differences were attributed to inhibitor binding at the Ca^2+-binding sites of the enzyme. However, discrepancies in the absolute incorporation values when experiments were compared, were related to loss of bound label due to nucleophilic attack. Four isomers of NCD-4 were prepared and evaluated in terms of inhibitory capacity towards the (Ca^2+ + Mg^2+)-ATPase. Preliminary evidence suggested that even related (isomeric) carbodimides can label different sites on the ATPase polypeptide. The synthesis and the evaluation of (N-cyclohexyl, N'-dimethylamino phenylazophenyl) carbodiimide (ACD) as a chromophoric probe for the Ca^2+ sites of the ATPase, is also described: this compound emerged as a relatively poor inhibitor of the enzyme, despite showing favourable chromophoric properties.