GENETICS OF PHOSPHOFRUCTOKINASE STRUCTURE AND FUNCTION

磷酸果糖激酶结构和功能的遗传学

基本信息

批准号：
3152322
负责人：
Simon H Chang
金额：
$ 9.11万
依托单位：
LOUISIANA STATE UNIV A&M COL BATON ROUGE
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-07-01 至 1987-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3152322
关键词：
6 phosphofructokinase Escherichia coli complementary DNA deoxyribonucleotides enzyme structure gene expression gene mutation genes genetic manipulation genetic regulation isozymes molecular cloning muscle nucleic acid chemical synthesis nucleic acid sequence radiotracer

项目摘要

The long range objective of this project is to gain insight into the structure and expression of the phosphofructokinase (PFK) gene from rabbit muscle in the normal and diabetic states. The information gained is expected to contribute to our understanding of the structure/function/regulation relationship of this key regulatory enzyme of carbohydrate metabolism. Our approach combines the recent techniques of recombinant DNA with those of affinity labeling and conventional methods of sequence studies. The specific aims of the project can be summarized as follows: (1) Cloning of the PFK gene and elucidation of its base sequence by the method of Maxam and Gilbert. This part constitutes the major thrust of this proposal. Through the determination of the base sequence of the PFK cDNA, the primary structure of the enzyme itself will be deduced. (2) Structure/function/regulation relationship: This part of the project involves efforts to identify the specific amino acid residues involved in the catalytic and regulatory sites of PFK. In this area, predetermined site specific mutagenesis of the cloned cDNA of PFK will be brought about and the effects of such mutations on the kinetic and allosteric behavior of the enzyme will be determined. (3) Mode of expression of the gene(s) of PFK isozymes. The genetic mechanism of expression of the three established isozymes of PFK, namely, M for muscle, L for liver and P for platelet PFK, will be studied by using 32p labeled cDNA of PFK as a hybridization probe. (4) The mechanism of diminished phosphofructokinase activity in diabetic animals. We have reported that PFK activity in muscle from streptozotocin diabetic animals was lower than that in muscle from normal controls. The possible impairment of PFK synthesis at the translation or transcription level in the diabetic state will be studied.

该项目的远距离目标是洞悉来自兔子的磷酸果糖激酶（PFK）基因的结构和表达正常和糖尿病状态的肌肉。获得的信息是有望有助于我们对该关键调节酶的结构/功能/调节关系碳水化合物代谢。我们的方法结合了最近的技术重组DNA具有亲和力标签和常规方法的DNA 序列研究。该项目的具体目的可以总结为以下内容：（1）PFK基因的克隆及其基本序列的阐明通过Maxam和Gilbert的方法。这部分构成了主要推力该提议。通过确定的基础序列 PFK cDNA，将推导酶本身的主要结构。（2）结构/功能/调节关系：项目的这一部分涉及确定涉及的特定氨基酸残基的努力 PFK的催化和调节位点。在这个领域，预定 PFK的克隆cDNA的位点特异性诱变将被带到以及这种突变对动力学和变构行为的影响将确定酶。（3）基因表达方式 PFK同工酶。三个建立的表达的遗传机制 PFK的同工酶，即肌肉的M，肝l，P for Platelet PFK，P。将使用32p标记为PFK的cDNA作为杂交探针进行研究。（4）糖尿病患者磷酸果糖激酶活性降低的机制动物。我们报道了PFK活性来自链霉菌素的肌肉糖尿病动物低于正常对照的肌肉。这在翻译或转录时可能会损害PFK合成将研究糖尿病状态的水平。