6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification, kinetics and immunological properties.
Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55,800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53,900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53,300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mumol.l-1).(ABSTRACT TRUNCATED AT 400 WORDS)
Hormone and Metabolic Research Unit, University of Louvain Medical School, Brussels, Belgium.
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Pyko
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Rider
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L
Hue
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Wegener
D000818:Animals; D005635:Fructosediphosphates / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D025481:Phosphofructokinase-2; D010766:Phosphorylation; D010770:Phosphotransferases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011893:Rana esculenta
D005635:Fructosediphosphates; C027652:fructose 2,6-diphosphate; D010770:Phosphotransferases; D011494:Protein Kinases; D025481:Phosphofructokinase-2