Abstract
The gene product of an open reading frame of the chloroplast genome, accD, that has sequence similarity with a subunit of acetyl-CoA carboxylase from Escherichia coli was detected immunochemically in pea chloroplasts. The apparent molecular mass of the accD protein was 87 kDa on SDS-polyacrylamide gel electrophoresis. The protein was acidic and had less mobility than the calculated value, 67,116. Acetyl-CoA carboxylase activity solubilized from pea chloroplasts was inhibited by antibodies against recombinant accD protein. The antibodies precipitated a polypeptide of 35 kDa containing biotin and a polypeptide of 91 kDa together with the 87-kDa-accD protein. The accD protein formed a complex with the molecular mass of about 700 kDa, probably with the 35- and 91-kDa proteins. These results indicate that the chloroplast-encoded polypeptide, accD protein, is a component of a functional acetyl-CoA carboxylase in chloroplasts and this enzyme is a multi-subunit complex, like that from E. coli. The synthesis of accD protein was not induced by light.
Highlights
AccD protein in E . coli is a B subunit with the molecular mass ‘The abbreviations used are: ORFs, open reading frames; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; dpm, disintegrations/min
A bacterial type of enzyme has been found in spinach chloroplasts,but the enzyme was not been purified, andthereis no evidence that the purified acetyl-coA carboxylase containsachloroplast-encoded subunit.Inthisreport we show immunochemically evidence that therewas accD geneproduct in pea chloroplasts and that thpirsotein was a subunitof acetyl-coA carboxylase
Cycles of denaturation at 94 "C for 1 min, primer annealing a protein with amolecular mass of 87kDa in the soluble fraction andhigh-salt extract from pea chloroplasts (Fig. lb)
Summary
AccD protein in E . coli is a B subunit with the molecular mass ‘The abbreviations used are: ORFs, open reading frames; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; dpm, disintegrations/min. Of 31.5 kDa that associates with LY subunits of 35 kDa to form carboxyltransferase (130 kDa), composed of azBz[7]. In mammals, this enzyme is composed of one kind of polypeptide containing three functional domains [9]. Several acetyl-coA carboxylases have been purified from plant sources, and these enzymes are not multi-subunit enzymes like those from bacteria, but aresingle oligomers [10]. A bacterial type of enzyme has been found in spinach chloroplasts (ll),but the enzyme was not been purified, andthereis no evidence that the purified acetyl-coA carboxylase containsachloroplast-encoded subunit.Inthisreport we show immunochemically evidence that therewas accD geneproduct in pea chloroplasts and that thpirsotein was a subunitof acetyl-coA carboxylase
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