Studies using mitochondria from spf ash mice, which are severely deficient in ornithine transcarbamylase, will clarify the mechanism of the specific requirement for ornithine of carbamyl phosphate synthetase (ammonia) in situ. Experiments using liver mitochondria from normal, spf, and spf ash mice will establish the effects of interactions of carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase with the inner membrane on their catalytic properties, and the effect that the mutations have on the channelling extramitochondrial ornithine to ornithine transcarbamylase. The relative contribution of this enzyme and of carbamyl phosphate synthetase (ammonia) to the control of citrulline synthesis will be analyzed. Experiments using rat and mouse liver mitochondria will be done to obtain quantitative information on the role of matrix carbonic anhydrase in the supply of HCO3- to carbamyl phosphate synthetase (ammonia) and to pyruvate carboxylase, and to determine the Km values of these enzymes in situ for HCO3-. The significance of this work lies in the information it will yield on the properties of proteins under the conditions which prevail in cells, and on how enzymes and metabolic pathways function in situ. In their normal environment, many enzymes appear to have kinetic properties different from those they exhibit in solution. Examples are several of the enzymes of the pathway of urea synthesis, two of which (carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase) are in the mitochondrial matrix, and the remaining three in the cytoplasm. Work to date shows that, although these enzymes are soluble, several of their substrates are channelled between cell compartments or enzymes, suggesting that the enzymes are organized within the cell. In addition, these studies show that some kinetic characteristics of the two mitochondrial enzymes in situ differ from those they exhibit in solution.***//

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
8819440
Program Officer
Robert Uffen
Project Start
Project End
Budget Start
1989-03-01
Budget End
1994-02-28
Support Year
Fiscal Year
1988
Total Cost
$437,800
Indirect Cost
Name
University of Southern California
Department
Type
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90089