Domain structure of rat liver carbamoyl phosphate synthetase I.

S G Powers-Lee,K Corina

doi:10.1016/s0021-9258(18)66714-6

Abstract

Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions. The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the fragments were determined by automated Edman degradation. Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments. The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase. The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides. The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides. The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected. In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.

Highlights

Folded structural domains of rat liver Elastase and trypsin were supplied by Sigma and protein molecular carbamoyl phosphate synthetase I have been identified weight standards by Bethesda Research Laboratories
The hydrolysis of carbamoyl phosphate synthetase I (M, 160,000) by either trypsin or elastase proceeded in two stages, with twoalternaing the carbamoyl phosphate synthetase I solution over an agarosehexane-guanosine 5”triphosphate column under conditions where only glutamate dehydrogenase was bound (6)
The enzymatic activity of carbamoyl phosphate synthetase I was assayed by the coupledpyruvate kinase-lactate dehydrogenase system as previously described (5)

Summary

Domain Structure of Rat Liver Carbamoyl Phosphate Synthetase I*

MgATP or N-acetylglutamate alters the rate of elastase inactivation, but the studydid not involve any structural analysis (4). We have utilized limited proteolysis by elastase and trypsin to demonstrate that rat livercarbamoyl phosphate synthetase I is organized into independentlyfolded domains. Folded structural domains of rat liver Elastase and trypsin were supplied by Sigma and protein molecular carbamoyl phosphate synthetase I have been identified weight standards by Bethesda Research Laboratories. Study of other large proteins Proteolytic Digestion of Carbamoyl Phosphate Synthetase has shown that they are organized into several independent I-Partial digestion of carbamoyl phosphate synthetaseI with structural domains(3).A previousstudy of rat liver carbamoyl trypsin yielded distinct polypeptides which could be detected phosphate synthetase I has shown that theenyzme is subject by SDS-polyacrylamide gel electrophoresis. This hydrolysis proceeded by two alternative routes, each of which had two stages (Fig. 3). In the firsrtoute, which was followed primarily when there was no addition, a 140kDa fragment was again formed initially, and again the remaining 20-kDa portion of the protein was cleaved to small

NO AODITION

OF CPS