PROTEIN KINASE C SUBSTRATES, REGULATORY PROTEINS, AND YEAST PROTEINS

蛋白激酶C底物和调节蛋白，以及S-100样蛋白 贝克酵母菌 长期的目标是阐明蛋白质的磷酸化和 酵母菌信号转导过程中的有限蛋白分解 与哺乳动物细胞进行比较。这一具体目标是：(I) 免疫沉淀酵母组分硝酸盐底物性质的证明 氧化合成酶，m-钙蛋白，m-钙蛋白，钙蛋白，类髓鞘蛋白， 和整合素(α-6亚基)通过免疫沉淀酵母试验 蛋白激酶C(PKC)α、β、伽马：缺乏蛋白酶 酿酒酵母(面包酵母)是用来制备大多数酶的 和蛋白质。对磷酸化底物进行十二烷基硫酸钠-PAGE和 通过放射自显影进行分析。(Ii)鉴别免疫沉淀 酵母PKC(阿尔法、贝塔、伽马、德尔塔、埃西隆、西塔、IOTA、波长) 辅因子(钙离子、磷脂酰丝氨酸、二酰甘油)需要量， 亚细胞分布、底物专一性、等电pH和 分子质量。(三)调查酵母菌活性变化 一氧化氮合酶被酵母PKCα磷酸化后； 活性的变化通过产生胞二磷胆碱来监测。(Iv)至 酵母钙解酶活性变化的评价 酵母PKCalpha的磷酸化(使用前自动磷酸化的cAMP- 用32P标记的依赖蛋白激酶作为蛋白降解底物)。(五) 研究PKCalpha酵母热稳定调节蛋白(S) 调节蛋白(S)是通过将预煮的粗酵母 萃取至DE-52色谱柱，用线性NaCl梯度洗脱。这个 缓蚀机理、分子质量和等电点pH 下定决心。(Vi)确定酵母S-100的调节作用- 酵母上清液中类蛋白质的蛋白磷酸化 免疫沉淀后，抗S=100，抗钙调素。 这些研究可能提供对信号转导过程的洞察， 从而有益于某些病理性疾病的治疗，以及 酵母或真菌感染。它还可以促进有效的 面包酵母生产中代谢过程的调控 有价值的(基因工程)产品，以及其他工业产品 申请。此外，三名少数民族本科生将 接受与酶学相关的生物医学研究培训。

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.