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 贝克的酵母 长期目标是阐明蛋白质磷酸化和 涉及酵母信号转导过程的有限蛋白水解 与哺乳动物细胞的比较。 这个具体目标是：（i） 证明免疫沉淀的酵母菌本构氮的底物性质 氧化物合酶，M-钙蛋白酶，MU-钙蛋白酶，钙蛋白酶，髓磷脂样蛋白， 通过对免疫沉淀的酵母进行测试，整联蛋白（alpha-6亚基） 蛋白激酶C（PKC）Alpha，beta，伽玛：缺陷蛋白酶 酿酒酵母（贝克的酵母）用于制备大多数酶 和蛋白质。 磷酸化的底物经过SDS-PAGE和 通过放射自显影分析。 （ii）区分免疫沉淀 酵母PKC（Alpha，Beta，Gamma，Delta，Epsilon，Theta，Iota，Lambda） 辅因子（Ca2+，磷脂酰丝氨酸，二酰基甘油）的需求， 亚细胞分布，底物特异性，等电pH和 分子质量。 （iii）研究酵母活性的改变 一氧化氮合酶被酵母PKCα磷酸化后； 这 通过生产Cistrulline监测活动的变化。 （iv）到 评估酵母菌钙的活动的改变 用酵母pkcalpha磷酸化（使用前磷酸化营地 依赖性蛋白激酶以32p标记为蛋白水解底物）。 （v） 研究PKCALPHA的酵母热稳定调节蛋白； 这 调节蛋白是通过对预沸的原油酵母菌制备的 提取到用线性NAC1梯度洗脱的DE-52柱。 这 抑制机制，分子质量和等电pH将为 决定。 （vi）确定酵母S-100-的调节作用（a） 像蛋白质上清液的蛋白质磷酸化一样 抗= s = 100和抗钙调蛋白的免疫沉淀后。 这些研究可能会洞悉信号转导过程， 从而使某些病理疾病的治疗受益 酵母或真菌感染。 它也可能有效 处理贝克酵母中代谢过程的生产 有价值的（基因工程）产品，以及其他工业 申请。 此外，三个少数本科生将 接受与酶学相关的生物医学研究的培训。