Use of cultured lymphoblastoid cells for the study of abnormal enzymes: molecular abnormality of a phosphoglycerate kinase variant associated with hemolytic anemia.

A phosphoglycerate kinase (PGKase: ATP:3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3; X chromosome-linked) variant, PGKase-Tokyo, is associated with enzyme deficiency, nonspherocytic hemolytic anemia, and neurological disturbances. Because a sufficient amount of the patient's erythrocytes was not available, the variant enzyme was purified to homogeneity from the cultured lymphoblastoid cells of the patient. The enzyme activity of the variant lymphoblastoid cells was about 16% of that of the normal lymphoblastoid cells. PGKase-Tokyo, compared to the normal enzyme, had a lower specific activity (31% of normal in the backward reaction and 15% of normal in the forward reaction), higher than normal Michaelis constants for ATP and 3-phosphoglycerate, a more acidic pH optimum, and increased thermal instability. Microscale peptide mapping analysis revealed that the structural abnormality of PGKase-Tokyo is a single amino acid substitution from valine to methionine at position 266. Thus, the use of the cultured lymphoblastoid cells is proven to be useful for the study of structural and functional abnormalities of mutant enzymes.