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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK014632-25
Application #
3225248
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1975-04-01
Project End
1990-03-30
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
25
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Arts and Sciences
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37203
Adami, P; Duncan, T M; McIntyre, J O et al. (1993) Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme. Biochem J 292 ( Pt 3):863-72
Marks, A R; McIntyre, J O; Duncan, T M et al. (1992) Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart. J Biol Chem 267:15459-63
Radermacher, M; Wagenknecht, T; Grassucci, R et al. (1992) Cryo-EM of the native structure of the calcium release channel/ryanodine receptor from sarcoplasmic reticulum. Biophys J 61:936-40
Hazarika, P; Kaetzel, M A; Sheldon, A et al. (1991) Annexin VI is associated with calcium-sequestering organelles. J Cell Biochem 46:78-85
Marks, A R; Taubman, M B; Saito, A et al. (1991) The ryanodine receptor/junctional channel complex is regulated by growth factors in a myogenic cell line. J Cell Biol 114:303-12
Marks, A R; Fleischer, S; Tempst, P (1990) Surface topography analysis of the ryanodine receptor/junctional channel complex based on proteolysis sensitivity mapping. J Biol Chem 265:13143-9
Rudy, B; Dubois, H; Mink, R et al. (1989) Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme. Biochemistry 28:5354-66
Nagasaki, K; Fleischer, S (1989) Modulation of the calcium release channel of sarcoplasmic reticulum by adriamycin and other drugs. Cell Calcium 10:63-70
Marks, A R; Tempst, P; Hwang, K S et al. (1989) Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc Natl Acad Sci U S A 86:8683-7
McGrew, S G; Inui, M; Chadwick, C C et al. (1989) Comparison of the calcium release channel of cardiac and skeletal muscle sarcoplasmic reticulum by target inactivation analysis. Biochemistry 28:1319-23

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