PROTEIN KINASE C SUBSTRATES, REGULATORY PROTEINS, AND YEAST PROTEINS

蛋白激酶 C 底物、调节蛋白和酵母蛋白

• 批准号: 6107181
• 负责人: WU-NAN KUO
• 金额: $ 15.1万
• 依托单位: BETHUNE-COOKMAN COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107181
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; autoradiography; biological signal transduction; calpain; cyclic GMP; developmental genetics; enzyme activity; enzyme substrate; fungal proteins; immunoprecipitation; integrins; microorganism growth; nitric oxide synthase; phosphorylation; protease inhibitor; protein kinase C; proteolysis; transcription factor

Protein Kinase C Substrates and Regulatory Proteins, and S-100-Like Protein of Baker's Yeast The long-term objectives are to elucidate the protein phosphorylation and limited proteolysis involved in yeast signal transduction process in comparison with that of mammalian cells. This specific aims are: (I) To prove the substrate nature of immunoprecipitated yeast constitutive nitric oxide synthase, m-calpain, mu-calpain, calpastatin, myelin-like protein, and integrin (alpha-6 subunit) by testing against immunoprecipitated yeast protein kinase C (PKC) alpha, beta, gamma: Protease-deficient Saccharomyces cerevisiae (baker's yeast) is used to prepare most enzymes and proteins. Phosphorylated substrates are subjected to SDS-PAGE and analyzed by autoradiography. (II) To differentiate immunoprecipitated yeast PKC (alpha, beta, gamma, delta, epsilon, theta, iota, lambda) with cofactor (Ca2+, phosphatidylserine, diacylglycerol) requirement, subcellular distribution, substrate specificity, isoelectric pH and molecular mass. (iii) To investigate the alteration of activity of yeast nitric oxide synthase after being phosphorylated by yeast PKC alpha; The change in activity is monitored by the production of cistrulline. (IV) To evaluate the alteration of activity of yeast calpains after being phosphorylated by yeast PKCalpha (using preautophosphorylated cAMP- dependent protein kinase labeled with 32p as a proteolytic substrate). (V) To investigate yeast heat-stable regulatory protein(s) of PKCalpha; The regulatory protein(s) are prepared by subjecting preboiled crude yeast extract to the DE-52 column eluted with linear NaC1 gradient. The inhibitory mechanism, molecular mass, and isoelectric pH will be determined. (VI) To identify the regulatory effect (a) of yeast S-100- like protein on the protein phosphorylation of yeast supernatant obtained after immunoprecipitations with anti=S=100 and anti-calmodulin. These studies may provide insight into the signal transduction process, thereby benefitting the treatments of certain pathological disorders, and yeast or fungal infections. It could also facilitate effective manipulation of the metabolic processes in baker's yeast for the production of valuable (genetic engineering) products, and for other industrial applications. Furthermore, three minority undergraduate students will receive training in enzymology-related biomedical research.

蛋白激酶C底物和调节蛋白以及S-100样蛋白 面包酵母 长期目标是阐明蛋白质磷酸化和 参与酵母信号转导过程的有限蛋白水解 与哺乳动物细胞相比。 具体目标是：（一） 证明免疫沉淀酵母组成型硝酸盐的底物性质 氧化物合酶，m-钙蛋白酶，μ-钙蛋白酶，钙蛋白酶抑制剂，髓磷脂样蛋白， 和整合素（α-6亚基）通过测试对免疫沉淀酵母 蛋白激酶C（PKC）α、β、γ：蛋白酶缺陷型 酿酒酵母（面包酵母）用于制备大多数酶 和蛋白质。 将磷酸化底物进行SDS-PAGE， 通过放射自显影术分析。 (II)为了区分免疫沉淀 酵母PKC（α，β，γ，δ，β，θ，iota，λ）， 辅因子（Ca 2+、磷脂酰丝氨酸、甘油二酯）需求， 亚细胞分布、底物特异性、等电pH和 分子量 (iii)研究酵母菌活性的变化 一氧化氮合酶被酵母PKC α磷酸化后， 活性的变化通过顺曲林的产生来监测。 (IV)到 酵母钙蛋白酶活性的变化 磷酸化的酵母PKCalpha（使用preautophosphorylated cAMP- 用32 p标记的依赖性蛋白激酶作为蛋白水解底物）。 （五） 研究PKCalpha的酵母热稳定调节蛋白; 通过使预煮的粗酵母 提取物上DE-52柱，用线性NaCl梯度洗脱。 的 抑制机制、分子量和等电pH将是 测定 (VI)为了鉴定酵母S-100的调节作用（a）， 样蛋白对蛋白磷酸化酵母上清液获得 在用抗S=100和抗钙调蛋白进行免疫沉淀后。 这些研究可能会提供深入了解信号转导过程， 从而有利于某些病理性疾病的治疗， 酵母菌或真菌感染。 它还可以促进有效的 控制面包酵母的代谢过程， 有价值的（基因工程）产品，以及其他工业 应用. 此外，三名少数民族本科生将 接受酶学相关生物医学研究方面的培训。