| Abstrakt: |
Propionyl-CoA carboxylase [PCC, propanoyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.3] is a biotin-dependent enzyme involved in the degradation of branched-chain amino acids, fatty acids with odd-numbered chain lengths, and other metabolites. Inherited deficiency of the enzyme results in propionic acidemia, an autosomal recessive disorder showing considerable clinical heterogeneity. To facilitate investigations of enzyme structure and the nature of mutation in propionic acidemia, we have isolated cDNA clones coding for the alpha and beta polypeptides of human PCC. Sequences of two peptides derived from human liver PCC were used to specify oligonucleotide probes that were then used to screen a human fibroblast cDNA library. Two classes of cDNA clones were thus identified. One class contained the anticipated Ala-Met-Lys-Met sequence, corresponding to the biotin binding site found in several biotin-dependent carboxylases, thus confirming the alpha-chain assignment of these clones. In addition, they contained the deduced amino acid sequence of two of the sequenced peptides, including that of one of the oligonucleotide probes. The second class, coding for the beta polypeptide, contained the sequences of four peptides, including the sequence corresponding to the other oligonucleotide probe. Blot hybridization of RNA from normal human fibroblasts revealed a single mRNA species of 2.9 kilobases coding for the alpha polypeptide and two species of 4.5 and 2.0 kilobases detected for the beta polypeptide. By use of a panel of somatic mouse-human hybrids, the human gene encoding the alpha polypeptide (PCCA) was localized to chromosome 13, while the gene encoding the beta polypeptide (PCCB) was assigned to chromosome 3. Restriction fragment length polymorphisms were identified, at both PCCA and PCCB, that should prove useful to individual families at risk for propionic acidemia. |
