Identification and cloning of yeast phosphofructokinase 2.
Fructose-6-phosphate 2-kinase ('phosphofructokinase 2') was purified from a strain of Saccharomyces cerevisiae lacking fructose-6-phosphate 1-kinase. After chromatography on DEAE-Sephacel, Sephacryl blue, CM-Sephadex and rechromatography on CM-Sephadex with fructose-6-phosphate elution, the specific activity was 1.6 U/mg protein. Although the latter value is high for fructose-6-phosphate 2-kinase, as was the purification factor of 3 x 10(4), staining with Coomassie blue showed the fraction to still contain many proteins. Incubation with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase gave a further increase in specific activity and labeling of, only, 96-kDa and 93-kDa polypeptides. Antiserum raised against these polypeptides recognized them in an immunoblot and could be used to remove the enzyme activity from crude extracts. Tryptic peptide profiles were obtained from about 10 pmol of the 96-kDa and 93-kDa polypeptides. The profiles were similar and sequencing allowed construction of mixed probes and identification of a putative single structural gene. Returned to yeast on a multicopy plasmid, phosphofructokinase 2 activity was considerably above the wild-type level, as was polypeptide revealed by immunoblotting.
Kretschmer
M
M
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
Tempst
P
P
Fraenkel
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DG
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D025481:Phosphofructokinase-2; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin
D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D014357:Trypsin