Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.
We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.
Sakata
J
J
Department of Veterans Affairs Medical Center, Dallas, Texas.
Abe
Y
Y
Uyeda
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics*; D009206:Myocardium / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013737:Testis / Q000201:enzymology*
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D004247:DNA; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases