Evaluation of phosphoenolpyruvate as a phosphoryl group donor for phosphoproteins in skeletal muscle.
The possible existence of a phosphoenolpyruvate-dependent protein kinase activity in muscle first reported by Khandelwal et al. (FEBS Lett. 162, 127-132, 1983) was further examined in this study. [32P]Phosphoenolpyruvate was used to assay rabbit muscle extract that was first dialyzed and then passed over a gel filtration column. Fractions from the column were assayed for activity (as shown by counts incorporated into an acid-precipitable product), which was observed only when two distinct and resolved fractions were combined. The approximate masses of these two components, as estimated by nondenaturing gel filtration chromatography, were 30 and 160 kDa. The activity showed time and concentration dependence and was inactivated by heat and proteolysis. [gamma-32P]ATP could not substitute for [32P]phosphoenolpyruvate in the activity assays, nor was there evidence for the formation of ATP during the assays. Elution of the reaction mixture from a sizing column revealed several radioactive peaks. Other tissues (heart, kidney, liver, lung, spleen, and back skeletal muscle) from rabbit and rat were screened; the total activity was detected in all tissues examined, but was highest in the skeletal muscle of both species, with that from rabbit being twice that from rat.
Jeyasingham
M D
MD
Department of Biochemistry, University of Tennessee, Memphis 38163, USA. mdj@rri.sari.ac.uk
Carlson
G M
GM
D000818:Animals; D005591:Chemical Fractionation; D004789:Enzyme Activation; D005260:Female; D018482:Muscle, Skeletal / Q000378:metabolism*; D009928:Organ Specificity; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats
D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D010728:Phosphoenolpyruvate