DIFFERENT ISOZYMIC FORM OF CGMP-DEPENDENT PROTEIN KINASE

CGMP 依赖性蛋白激酶的不同同工形式

• 批准号: 3240108
• 负责人: JACKIE David CORBIN
• 金额: $ 20.45万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3240108
• 关键词: atrial natriuretic peptide; calcium; chemical binding; complementary DNA; cyclic AMP; cyclic GMP; cyclic nucleoside monophosphate; enzyme mechanism; enzyme structure; enzyme substrate; guinea pigs; high performance liquid chromatography; ion exchange chromatography; isozymes; laboratory rabbit; laboratory rat; molecular cloning; nitroglycerin; nucleic acid probes; protein kinase; protein purification; protein sequence; vascular smooth muscle

Recent studies, including our own, have pointed to cGMP-dependent protein kinase as the mediator of cGMP-induced relaxation of vascular smooth muscle in response to agonists such as atrial natriuretic factor, nitrovasodilators (e.g., nitroglycerin), methylxanthines (e.g., caffeine) and metabolites (e.g., EDRF). This proposal is based primarily on two features of cGMP-dependent protein kinase discovered in this laboratory over the past year: (1) Soluble fractions of vascular smooth muscle tissues contain a novel isozymic form named type I beta, in addition to the well-characterized type I alpha found in other tissues; (2) The cGMP-dependent protein kinase exists as chromatographically separable cGMP-deficient and cGMP-bound forms. The tissue and species distribution of types I alpha and I beta will be examined. The newly discovered type I beta will be purified to homogeneity and determined if it is a different gene product from type I alpha. Other physical and kinetic characteristics of the two enzymes will be compared, including protein and peptide substrate specificity. If it is established that type I beta, like type I alpha, has two different cGMP binding sites on each of two subunits, then a thorough study of cGMP binding kinetics and cGMP analog specificity for each site will be undertaken. Where possible, both isozymes will be utilized to study the function of each cGMP binding site by isolating cGMP-deficient and cGMP-bound forms of the enzymes. For some of these experiments, enzyme containing cGMP bound at particular sites will be characterized following isolation by the cGMP-induced DEAE "charge shift" procedure recently established. The "charge shift" will also be used as a novel approach to determine if cGMP or cAMP is bound to each site of type I alpha or I beta in the basal state of vascular smooth muscle, which might act as a "priming" device for activation; and to determine if the various agents mentioned above which modulate cGMP levels,.as well as agents which modulate cAMP and calcium, will change the amount of cyclic nucleotide bound to each site. The role of intra-versus interchain interactions in the cGMP activation mechanism will also be investigated by studies of the monomeric kinase (the native enzyme is dimeric), found recently to be produced by proteolysis of the native enzyme. Also complementary for sequencing and for future studies of the cGMP binding sites will be the attempted cloning of the cDNA and genomic DNA for type I alpha and I beta.

最近的研究，包括我们自己的研究，都指出了cgmp依赖蛋白