This program of twenty years aims to understand cell function in terms of organelles, membranes and membrane components. The focus on the level of the membrane is on two purified membrane components, D-Beta-hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, prepared from heart and liver mitochondria which has an absolute requirement for lecithin for enzymic activity and the calcium pump protein (CPP) from sarcoplasmic reticulum of fast-twitch skeletal muscle, an ATP-driven membrane pump which removes Ca2+ from the sarcoplasm enabling muscle to relax. The reconstitution approach is being used to prepare membranes of defined components, lipid environment and to vary the lipid/protein composition of the membrane. Studies are ongoing to elucidate the molecular architecture of the membrane and to relate structure to the exercise of function. We continue directing a variety of approaches, both biochemical and biophysical, to characterize the nature of lipid-protein interactions in membranes including: 1) attempts to obtain two- and three-dimensional crystals of the membrane proteins so that the structure of these components can be elucidated; 2) studies of the influence of the lipid environment on function; 3) deuterium, proton and phosphorus NMR to measure the motional characteristics of the phospholipid and the exchange rate of """"""""boundary"""""""" phospholipid with bilayer phospholipid. For BDH, a key question is the basis of the role of lipid; what makes this dehydrogenase distinct from many others which do not require lipid for function. Chemical derivatization studies and primary sequence analysis are in progress. Diffraction studies of oriented multilayers containing BDH are also designed to reveal orientation of BDH in the membrane. We previously found, in diabetes, increases in the saturation of select fatty acids esterified to phospholipid in the mitochondrial membrane of liver occurred within a week after insulin deprivation and the level of BDH decreased over a period of several weeks. The basis for this decrease will be studied. A second major thrust is to define, in fast-twitch skeletal muscle, the nature of the Ca2+ release process in excitation-contraction coupling. the approach is to isolate and study highly purified membrane components including plasmalemma, terminal and longitudinal cisternae, transverse tubule, and triads (the junctional association of terminal cisternae and transverse tubule). The emphasis is to define, in molecular terms, the nature of the triad junctional association and how Ca2+ release is triggered across this intracellular junction.
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