Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle.
Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.
Thalhofer
H P
HP
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
Daum
G
G
Harris
B G
BG
Hofer
H W
HW
D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000032:analysis* / Q000302:isolation & purification; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000302:isolation & purification
D007527:Isoenzymes; D000242:Cyclic AMP; D011494:Protein Kinases; D010732:Phosphofructokinase-1; D006152:Cyclic GMP