In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.
A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.
D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000096:biosynthesis* / Q000302:isolation & purification; D004792:Enzyme Precursors / Q000096:biosynthesis*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010770:Phosphotransferases / Q000096:biosynthesis*; D011132:Polyribosomes / Q000201:enzymology; D014176:Protein Biosynthesis; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism
D004792:Enzyme Precursors; D010770:Phosphotransferases; D002222:Carbamoyl-Phosphate Synthase (Ammonia)