THE MOLECULAR-BASIS OF CANINE PYRUVATE-KINASE DEFICIENCY

Inherited hemolytic anemia due to pyruvate kinase (PK) deficiency is a n autosomal recessive disease of the Basenji dog that closely resemble s human PK deficiency. Characterization of transcriptional and transla tional expression of PK isozymes and sequencing of DNA from normal and mutant dogs were performed to identify the genetic defect in Basenji dogs. Measurement of erythrocytic PK activity by ion exchange chromato graphy, substrate kinetics, immunologic reactivity, and electrophoreti c mobility suggests that M(2)-type PK is the major form of PK activity in erythrocytes of PK-deficient dogs, in contrast to normal dogs havi ng only R-type PK activity. Both R-type and M(2)-type PK mRNA are dete ctable in reticulocytes of PK-deficient dogs, suggesting that the aber rant isozyme expression is not due to a failure in the erythroid matur ational switch from M(2-) to R-type isozymes. Nucleotide sequence data from wild-type and mutant R-type PK cDNA identified a single nucleoti de deletion, Delta C-433, in the mutant cDNA. The deduced amino acid s equence predicts a truncated mutant protein devoid of all residues con tributing to the catalytic site of the wild-type protein. In the absen ce of R-type PK activity, there is anomalous compensatory expression o f M(2)-type PK in erythroid cells of PK-deficient Basenjis. The PK-def icient Basenji dog may be valuable in somatic cell gene therapy trials involving manipulation of hematopoietic stem cells.