Acetyl Coenzyme A Carboxylase: MOLECULAR FORMS AND SUBUNIT COMPOSITION OF BIOTIN CARBOXYL CARRIER PROTEIN

Abstract

Abstract Milligram quantities of the apparent native form of biotin carboxyl carrier protein (BCCP), a component of Escherichia coli acetyl-CoA carboxylase, have been purified to apparent homogeneity using a procedure which largely avoided dissociation or proteolysis. The isolated BCCP served as an effective carboxyl acceptor and donor in the biotin carboxylase and transcarboxylase half-reactions of acetyl-CoA carboxylase at concentrations which were 50- to 100-fold lower than those needed with either BCCP(9100) or BCCP(10400) (previously isolated smaller forms of BCCP with molecular weights of 9,100 and 10,400, respectively). The purified BCCP exhibited a single protein band upon electrophoresis in polyacrylamide gels in the presence or absence of 8 m urea or 1% sodium dodecyl sulfate (SDS), and upon isoelectric focusing in polyacrylamide gels. From the latter an isoelectric point of 4.5 was determined. BCCP was found to contain an average of 10.68 µg of biotin per mg of protein, or 1 mole of biotin per 22,900 g of protein. Amino acid analysis revealed 207 residues per residue of biotin, corresponding to a residue molecular weight of 22,156; 2 half-cystine residues were indicated. Only 1 of these half-cystine residues could be titrated with 5,5'-dithiobis(2-nitrobenzoic acid), and then only in the presence of denaturing agents such as 8 m urea or 2% SDS. The denaturation by urea was partially reversible. A comparison of the amino acid composition of BCCP with those of BCCP(9100) and BCCP(10400) lends support to the suggestion that these smaller forms were derived from native BCCP by proteolysis. The subunit molecular weight of BCCP was determined by (a) polyacrylamide gel electrophoresis in the presence of 1% SDS which, even without prior reduction, gave a single protein band of molecular weight 22,500 ± 1,200, as determined by comparison with the migration of standard proteins; (b) gel filtration along with standard proteins in the presence of 6 m guanidine hydrochloride which indicated an average molecular weight of 21,800. These values correlated well with the biotin content, assuming 1 residue of biotin per polypeptide chain. Various multimeric forms of BCCP were detected in vitro by the following. (a) Gel filtration of BCCP in buffer on a column calibrated with standard proteins revealed a dimer of approximate molecular weight 46,000, which under various mild conditions dissociated (apparently irreversibly) to a monomer of approximate molecular weight 24,000; o-phenanthroline largely prevented the dissociation, suggesting the involvement of a metalloprotein in this process. (b) Sedimentation velocity centrifugation of BCCP exhibited a single symmetrical peak with a sedimentation coefficient of 5.7 to 5.9 S; this species may represent a tetrameric form of BCCP; (c) prolonged dialysis of apparently homogenous BCCP resulted in a polydisperse mixture with molecular weight ranging from approximately 20,000 to greater than 200,000 as measured by sedimentation equilibrium. It is apparent that under appropriate conditions BCCP can undergo dissociation or aggregation. The evidence to date suggests that the apparent native form of BCCP is best represented by a dimeric structure (molecular weight 45,000) which is composed of 2 subunits (molecular weight 22,500), each containing one biotin prosthetic group.