Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters.

Human platelets, prelabeled with [32P]phosphate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37 degrees C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G-proteins and the catalytic moiety. Less than 0.01 mol of [32P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the alpha-subunit of the GTP binding protein Gi. TPA, however, caused the incorporation of 0.67-1.1 mol of [32P]phosphate per mol of catalyst while 0.13-0.2 mol were found in the absence of TPA. Lack of modification of the alpha-subunit of Gi was also indicated by the results of reconstitution experiments with purified Gi alpha from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added G1 alpha, than that from TPA treated cells. While beta, gamma-subunits were like-wise inhibitory no difference dependent on platelet-pretreatment could be observed.

Calcium-dependent distance changes in binary and ternary complexes of troponin.

Calcium-dependent distance changes have been determined by resonance energy transfer in binary and ternary troponin complexes in order to collect evidence for the structural rearrangements which are part of the hypothetical trigger mechanism of skeletal muscle contraction. Donor and acceptor fluorophores were either intrinsic tryptophans in subunits with a favourable sequence from different species, quasi-intrinsic Tb3+ ions bound to troponin C or extrinsic labels attached to specific cysteine or methionine residues. All chemically modified subunits proved fully active in conferring calcium sensitivity onto myosin ATPase. Nine distances were determined between five sites which allowed construction of a three-dimensional lattice representing the spatial distribution of four sites in the ternary complex of troponin C, I and T. Distances in binary complexes were nearly unaltered upon addition of the third subunit. Regulatory calcium binding caused distance changes of the order of 0.7-1.1 nm. In view of the large displacements of the hypothetical mechanism, they turned out to be smaller than anticipated. The fluorophoric sites selected may be localized in a zone of the troponin complex which happens to be relatively little affected by the mechanism. Alternatively, amplification of the moderate changes seen here would require the complete set of thin filament proteins.