93 MOLECULAR ASPECT OF MYOGENIC HYPERURICEMIA: CLONING OF HUMAN MUSCLE PHOSPHOFRUCTOKINASE cDNA

Abstract

Our recent investigations revealed that myogenic hyperuricemia (Mineo et al.) is a common pathologic feature of glycogenosis types III, V and VII, in which muscle glycolysis is impaired. To clarify the molecular defect in glycogenosis type VII (human muscle phosphofructokinase (HMPFK) deficiency), we began performing the molecular cloning of HMPFK cDNA. A human muscle cDNA library was prepared by Okayama and Berg method and screened by colony hybridization with a rabbit muscle PFK cDNA (a gift from Dr. S. D. Putney of the Repligen Corp. MA, U.S.A.). A partial HMPFK cDNA clone was isolated. The clone was primer-extended and two other overlapping clones to cover the full-length were isolated. Complete primary structure of the enzyme was determined through the sequence analysis. HMPFK was 85,050 Da in size with 779 amino acid residues. Internal homology between N- and C- halves of the peptide was observed as in rabbit muscle PFK. Sequence homologies between rabbit and human muscle PFK were 96% in amino acids and 89% in nucleotides. Cloning of HMPFK cDNA will help analyzing the pathological regulatory mechanism of muscle glycolysis responsible for the development of myogenic hyperuricemia at the molecular level.