Subcellular localization of cyclic GMP-dependent protein kinase and its substrates in vascular smooth muscle.

作者：Harlan E. Ives, JOHN E. CASNELLIE, Paul Greengard, James D. Jamieson · 发表于：Journal of Biological Chemistry · 年份：1980 · DOI：10.1016/s0021-9258(19)85772-1 · 被引用次数：60 · 研究领域：Calpain Protease Function and Regulation、Bioactive Compounds and Antitumor Agents、Signaling Pathways in Disease

The subcellular location of cGMP-dependent protein kinase and its substrates in rabbit aortic medial smooth muscle was studied by subjecting homogenates to a single 4 X lo' g-min centrifugation on an 8.5 to 60% sucrose density gradient at low ionic strength.Substrate proteins GO to GI co-sedimented with a median density of 1.10, while the enzyme (measured by binding of the photoaffinity ligand 8-N3-C3'P]cIMP) was found both at the top of the gradient (with soluble proteins) and in a second peak with median density of 1.12.Gradient fractions were characterized by morphology, assay of the marker enzymes galactosyltransferase, 5'- AMPase, phosphodiesterase I, NADPH-cytochrome c (P-450) reductase, acid phosphatase, succinate cytochrome c reductase, and determination of RNA, DNA, and protein.Fractions rich in cGMP-dependent protein kinase and its substrates corresponded closely with markers for the plasma membrane, Golgi complex, and endoplasmic (sarcoplasmic) reticulum; electron microscopy indicated that these fractions contained membrane sheets and smooth closed vesicles of various sizes.To improve the resolution of the smooth membranous organelles, homogenates were pretreated with digi- tonin prior to centrifugation.The cGMP-dependent protein kinase substrates exhibited a large shift in density parallel with markers for the plasma membrane, whereas markers for the other organelles exhibited smaller shifts in density.In conjunction with the findings of the accompanying paper (Ca...