title: Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources.
abstract: Two cDNA clones, PK(p)alpha and PK(p)beta, for the leucoplast isozyme of pyruvate kinase have been isolated and characterized. A Southern blot of castor (Ricinus communis) DNA probed with PK(p)alpha indicates the presence of a single gene for PK(p). Most (1610 base pairs) of the sequence of both cDNAs is identical. These 1610 base pairs begin with an ATG translation initiation codon, and have 248 base pairs of 3'-untranslated and 1362 base pairs of coding sequence. The sequences of the two clones 5'- to the identical regions are different but both encode peptides with a high percentage of hydrophobic amino acids. The derived sequence of PK(p)alpha encodes eight amino acid residues which have been identified as the amino-terminus of one subunit of PK(p) from castor seed leucoplasts when the enzyme is purified in the absence of cysteine endopeptidase inhibitors. The sequence upstream of these amino acids is possibly the transit peptide for this protein. When PK(p) is extracted under conditions that eliminate its proteolytic degradation, its alpha-subunit has a relative molecular weight equal to the full-length coding sequence of PK(p)alpha. The data indicate that the transit peptide for the subunit of leucoplast pyruvate kinase encoded by PK(p)alpha is not cleaved until the protein is released from the plastid. The derived amino acid sequences of PK(p)alpha and PK(p)beta are most closely related to Escherichia coli pyruvate kinase. Although the residues involved in substrate binding are conserved in leucoplast pyruvate kinase, there is no phosphorylation site and only 5 of 15 amino acids in the E. coli fructose-1,6-bisphosphate binding site are conserved.
authors: Department of Biology, Queens University, Kingston, Ontario, K7L 3N6 Canada.
authors: S D
authors: SD
authors: Blakeley
authors: W C
authors: WC
authors: Plaxton
authors: D T
authors: DT
authors: Dennis