The overall goals of this project are to gain insight into the mechanisms which regulate 6-phosphofructo-1-kinase (PFK) isozymes during development and also to better understand the role of fructose-2, 6-bisphosphate (fructose-2, 6-P2) during development.
Our specific aims i nclude measurement of the PFK activity and examination of the properties and subunit composition of the PFK isozymes in the following tissues: (1) fetal heart and muscle during the week prior to partition, (2) different types of muscle fibers during the first month of neonatal maturation, and (3) diaphragm, brain, liver, kidney, and testes during fetal and neonatal development. The native PFK isozymes pools will be studied by measuring the effect of subunit-specific antibodies on total activity, by resolution of extracted activity by QAE-Sephadex column chromatography, and by determining the types and amounts of the PFK subunits which are present in the native PFK isozyme pools employing SDS-polyacrylamide gel electrophoresis, immunoblotting or silver staining and quantitative gel scanning. In order to appreciate the physiological significance of the various PFK isozyme pools, we will measure their kinetic/regulatory properties. Also, since the native PFK isozymes appear to be assembled by random association of the subunit(s) which are expressed, it is important to initiate studies which are designed to uncover the mechanisms which determine subunit levels. We will initiate these studies by measuring the rates of subunit synthesis during development. To this end, we have successfully developed a method for measuring rates of incorporation of radioactive phenylalanine into the different types of subunits. In addition, the extent of labeling of the phe-tRNA pool and the amount of phenylalanine residues in each subunit will be measured and used to normalize the incorporation data. Since fructose-2, 6-P2 is an important regulator of PFK activity and appears to be metabolized in the liver by one type of enzyme and in nonhepatic tissue by another type of enzyme, we propose to characterize the type(s) which are present during heart, muscle and liver development. Completion of the work proposed in this grant application should significantly increase our knowledge concerning many aspects of the regulation of PFK and consequently, glycolysis in developing tissues at a time when glucose is a primary source of the ATP which is required for tissue growth proper development and maintenance of normal cellular processes.
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