Molecular cloning of cDNA sequences for rat M2-type pyruvate kinase and regulation of its mRNA.

Abstract

Rat M2-type pyruvate kinase mRNA was enriched from total polysomes isolated from AH-130 Yoshida ascites hepatoma cells, which contain a very high concentration of the M2-type enzyme, by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to M2-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-arrested translation, and hybrid-selected translation. A partial restriction map was constructed covering about 1.44 kilobase pairs. The cloned region of the M2-type mRNA showed a high degree of sequence homology with the M1-type mRNA and some homology with the L-type mRNA as determined by dot blot hybridization. The molecular size of the M2-type mRNA, which was estimated to be 2.35-2.65 kilobases on denaturing gel, was the same as that of the M1-type mRNA. The level of hepatic M2-type pyruvate kinase mRNA measured by hybridization assay using cloned cDNA as a probe was increased 2.5-fold 1 day and 3.9-fold 2 days after partial hepatectomy and then started to decrease. This induction was followed by similar changes in the enzyme activity. AH-130 hepatoma cells contained 100-150 times more M2-type isozyme mRNA than regenerating liver. These results suggest that the increased levels of M2-type isozyme in regenerating liver and hepatoma cells are primarily due to elevation of hybridizable M2-type mRNA concentration.