PEPTIDE PROCESSING ENZYMES IN FAT/FAT MICE

说明(改编自申请人的摘要)：在建议的 在工作中，首席调查员将研究以下内容：1)他将 羧肽酶F(CPF)的酶学性质研究及比较 用从牛中提纯的酶转化为羧基肽酶E(CPE) 脑下垂体和大鼠脑。他将扩大对底物的研究 对自然发生的卵裂的专一性和包容性研究 多肽。在这种情况下，将会做出强有力的努力来确定 用高效液相分析和一种改进的方法分析特定的切割产物 称为基质辅助激光解吸电离的质谱学 飞行时间质谱仪(MALDITOF-MS)。第二个具体目标 就是确定CPF的分布。这将在开始时完成 根据酶的活性，接下来将使用 抗血清升高至CPF，最后通过一次mRNA表达研究 克隆的cdna是可用的。具体目标3是实际上 克隆CPF全长基因。这主要是基于聚合酶链式反应。 利用已知N-末端序列和已知N-末端序列的策略 其他CP酶的同源区。最后，校长 研究人员将评估CPF在肥胖/肥胖小鼠中是否受到调节 在酶、蛋白质和RNA水平上都是如此。这将包括 对不同年龄的小鼠的研究，因为胖/胖小鼠显示 不同年龄有不同程度的高血糖。

DESCRIPTION (Adapted from the applicant's abstract): In the proposed work, the principal investigator will study the following: 1) he will study the enzymatic properties of carboxypeptidase F (CPF) and compare those to Carboxypeptidase E (CPE) using enzymes purified from bovine pituitary and rat brain. He will expand the studies of substrate specificity and including the study of cleavage of natural occurring peptides. In this case, there will be a strong effort to identify the specific cleavage products using both HPLC analysis and a modification of mass spectrometry called matrix assisted laser desorption ionization time of flight mass spectrometry (MALDITOF-MS). The second specific aim is to determine the distribution of CPF. This will be done initially based on enzymatic activity, this will be followed by studies using antisera raised to CPF and finally by studies of mRNA expression once the cDNA clone is available. Specific aim 3 is the proposal for actually cloning the full length CPF cDNA. This is based primarily on a PCR strategy taking advantage of the known N-terminal sequence and known homologous regions in other CP enzymes. Finally, the principal investigator will evaluate whether CPF is regulated in the fat/fat mouse both at the enzymatic, protein and RNA levels. This will include studies of mice of different ages, since the fat/fat mouse shows different degrees of hyperglycemia at different ages.