Abstract
Rat liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated by [gamma-32P]ATP plus fructose-6-P or [2-32P]fructose-2,6-P2. The radioactivity co-migrated with homogeneous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase during sodium dodecyl sulfate-disc gel electrophoresis and the phosphoenzyme was acid-labile and base-stable. Hydrolysis of the phosphate group from phosphoenzyme prepared with either donor and the hydrolysis of their tryptic phosphopeptides depended on pH similarly. The pH dependence suggested that phosphate was linked to the N3 of a histidine residue. Co-electrophoresis and co-chromatography of alkaline hydrolysates of the labeled phosphoenzyme prepared from either substrate allowed the definitive identification of 3-phosphohistidine in the degradation products. Modification of the enzyme with diethylpyrocarbonate inactivated both the phosphotransferase and phosphohydrolase activities and suppressed the phosphorylation of the enzyme by ATP and fructose-6-P or by fructose-2,6-P2. Trypsin digestion of the phosphoenzyme formed upon incubation with ATP or fructose-2,6-P2 yielded an identical phosphopeptide after high pressure liquid chromatography. All the above data are consistent with this enzyme catalyzing both phosphohydrolase and phosphotransferase reactions with the mediation of phosphohistidine in the reaction scheme(s).
Highlights
From the $Department of Physiology, Vanderbilt School of Medicine and the §Departmentof Chemistry, Vanderbilt School of Medicine, Nashville, Tennessee 37232
All the above data are consistent with this enzyme catalyzing both phosphohydrolase and phosphotransferase reactions with the mediation of phosphophosphorylated derivatives were separated by DEAE-Sephadex chromatography a t pH 8.2 using a 20-500 m~ TEA, HC03 gradient and characterized as to phosphate and histidine content by reaction with the Pauly reagent asdescribed by DeLuca et al [8].Histidine and its derivatives were located by following UV absorption at 235 pm
The radioactivity migrated with a relative mobility of 0.47 and was coincident with the Coomassie blue staining band (M,= 55,000) which represented the 6-phosphofructo-2-kinase/fructose2.6-bisphosphaenzyme with [y3'P]ATP was shownto depend on various tase
Summary
Simon J. Elkis$,Mark WalderhaugS, Ken Murray#, AlbertBeth#, SindhaghattaD. Venkataramug, Jo FilkisS, and M. Raafat El-Maghrabit From the $Department of Physiology, Vanderbilt School of Medicine and the §Departmentof Chemistry, Vanderbilt School of Medicine, Nashville, Tennessee 37232 Rat liver 6-phosphofructo-2-kinase/fructose2,6-bis- phohydrolase and phosphotransferase reactions at one or phosphatase was phosphorylated by [y-32P]ATPplus more catalytic sites with a phosphoryl enzyme intermediate fructose-6-Por [2-S2P]fructose-2,6-PTz.he radioactivity being involved. We report here physical and chemical studies co-migrated with homogeneous 6-phosphofructo-2-ki- on aphosphorylated intermediate of this enzyme. nase/fructose 2,6-bisphosphataseduringsodiumdodecylsulfate-disc gel electrophoresisand the phos-
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