Alteration in Structure of Multifunctional Protein from Chinese Hamster Ovary Cells Defective in Pyrimidine Biosynthesis

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1979 Apr 1

PMID 36610

Citations 14

Authors

J N Davidson

D Patterson

Affiliations

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Abstract

A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd(-)A, and selected Urd(-)A revertants. The enzymes of Urd(-)A and the Urd(-)A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (ii) the aspartate transcarbamoyltransferase activity is located on a portion ( approximately 20,000 daltons) at one end of the polypeptide; (iii) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (iv) the mutations in Urd(-)A and the Urd(-)A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.

Citing Articles

Analysis of the Zebrafish perplexed mutation reveals tissue-specific roles for de novo pyrimidine synthesis during development.

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Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-alpha-pyrophosphate (PRPP) and UTP.

Simmons C, Simmons A, Haubner A, Ream A, Davidson J Biochem J. 2003; 378(Pt 3):991-8.

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Demonstration, by somatic cell genetics, of coordinate regulation of genes for two enzymes of purine synthesis assigned to human chromosome 21.

Patterson D, Graw S, Jones C Proc Natl Acad Sci U S A. 1981; 78(1):405-9.

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Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture.

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Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis.

Rumsby P, Campbell P, Niswander L, Davidson J Biochem J. 1984; 217(2):435-40.

PMID: 6365086 PMC: 1153234. DOI: 10.1042/bj2170435.

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