The long-term goals of this project are to biochemically characterize and define the physiologic functions of transglutaminases in vascular cells. A sensitive transglutaminases assay utilizing 3H-putrescine incorporation into dimethyl casein and endogenous cellular proteins will be used to biochemically characterize the transglutaminase activity in cultured bovine aortic endothelial cells (ABAE) and vascular smooth muscle cells (VSM). The subcellular localization of the transglutaminase in vascular cells will also be determined, with assays of cytoplasm, crude membrane and nuclear fractions. The effects of Mg-ATP, calcium, calmodulin, ionic and osmotic strength on the transglutaminase activity in sonicates of cultured vascular cells will be studied. The effectfs of calcium, the electrophoretic mobility and fibrinogen cross-linking pattern of the ABAE cell sonicates suggest that the endothelial transglutaminase has properties unlike other previously describes tissue treanglutaminases. A procedure is outlined to isolate the vascular transglutaminase from either bovine aortic tissue or from large-scale tissue cultures of ABAE and VSM cells. The purified enzyme will be characterized by SDS-PAGE, agarose gel electrophoresis, and enzyme kinetics using various substrances. The ability of polyamines to serve as transglutaminase substrates in vascular cells will also be studied. The ability of 3H-putrescine or its metabolic products to be incorporated into transglutaminase-mediated linkages with protein-bound glutamine groups will be analyzed in confluent and non-confluent ABAE and VSM cells. The specific proteins which have transglutaminase-coupled radioactive polyamines will be analyzed by SDS-PAGE and autoradiography. The effect of lipoproteins on both ABAE and VSM transglutaminase activity will also be studied and correlated with their effects on cell growth. These studies are designed to provide further information on both the biochemistry of vascular transglutaminases and how they regulate cell proliferation. These data may further explain the role of transglutaminases in normal vascular proliferation involved in wound healing and in the pathologic vascular proliferation of atherosclerosis.
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