Enzymatic Studies on Hereditary Pyruvate Kinase Deficiency

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It has been shown that three unique types of PK, namely L, M1 and M2, are present in both rat and human tissues. In this report, the relationship of enzyme abnormality due to hereditary PK deficiency to the gene expression was investigated by using PK isozymes as a marker. Acrylamide gel electrophoresis, immunological study using anti-rat M1 and M2 sera (since both sera perform cross reaction with M1 and M2 PKs of human tissues), and kinetic studies were made on six cases with PK deficiency. In two cases of them (Y. K. and F. K.), the same experiments were carried out about not only PKs from erythrocyte but also those from biopsy samples (liver, muscle and adipose tissue) taken during splenectomy. In a normal erythrocyte, the activity level of PK is 4-5 U/1010 cells, and Km for PEP in the presence and the absence of FDP are 0.11 and 0.53 mM, respectively. The six cases were able to be classified into three groups on the basis of activity level and kinetic properties of erythrocyte PK (R. B. C.-PK). In the first group (T. O. and T. S.), the activity level is very low, however, Km for PEP in the presence or the absence of FDP is within the normal range. In the second (Sapporo and Nagasaki-types), the activity level is normal or rather higher than then normal, however, Km for PEP in the presence or the absence is much higher than that of the normal. In the third (A. N., and Y. K., F. K. sisters), both the activity level and the Km for PEP are abnormal (very low and very high, respectively). The present investigation was focussed on the third group, especially Y. K. and F. K. sisters.(1) Zymogram of PK from the normal tissues showed that R. B. C.-PK was separated from L-type PK, migrating to the anode more slowly than that. It has been thought that R. B. C.-PK is L-type, however, this suggests a possibility that normal R. B. C.-PK is a heterogeneous hybrid of M2- and L-type. In two deficient cases(Y. K. and F. K.), R. B. C.-PK from them migrated to the anode more slowly than the normal one, and was separated. PK isozymes from other tissues, muscle (M1-type), adipose tissue and spleen (M2-type) of the patients were not distinguishable electrophoretically from those of the normal. In contrast, a comparative zymogram of PK from the patients' and the normal liver showed that L-type was not detected in the patients liver and the activity band corresponding to L-type was separated from normal L-type migrating to the anode at a slower rate, while M2-type as a minor component in liver was not different from each other. Then, L-type in the patient's liver was designated as L'-type.(2) Kinetic properties of PKs from R. B. C. and liver of the patients were different from those of the normal, especially Km for PEP in the presence and the absence of FDP were higher than those of the normal. On the other hand, M1 (muscle) and M2-type (spleen and adipose tissue) were not different kinetically from each other as well as the electrophoretic results.(3) Immunoreactivity of R. B. C.-PK from both the patient and a normal subject with anti-M2 serum greatly differed; the amount of neutralization of the patient R. B. C.-PK by anti-M2 serum was very low (0-15%), on the other hand, that of normal R. B. C.-PK was very high (50-60%). Both the PKs were not neutralized by anti-M1 serum. Both the M1 and M2-types of PK from the patients and the normal did not differ qualitatively. Therefore, if one assumes that R. B. C.-PK is a hybridized form of L-and M2-type subunits, then one would expect that the qualitative abnormality of the enzyme (L'-type) due to mutation of structure gene concerned in L-type subunit formation occurs in R. B. C. and liver of PK deficient subjects, and that, as a result, R. B. C.-PK of the patients is a L'-M2 type hybrid
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Enzymatic Studies on Hereditary Pyruvate Kinase Deficiency

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