Protein phosphorylation in intact superior cervical ganglion during regeneration.
The incorporation of radioactive phosphate into proteins of both normal and regenerating superior cervical ganglion nerve of the rat is reported. Incorporation studies carried out by in vitro and in vivo methods are compared. In the in vitro method, excised intact ganglia or their homogenates were incubated in the presence of inorganic phosphate or ATP, respectively, under various conditions. Proteins were analyzed by gel electrophoresis followed by autoradiography, in which quantitative but not qualitative differences between regenerating and control cases were apparent. In the in vivo procedure, inorganic phosphate was injected into the living animal 4 h before removal of ganglia. At least fivefold more proteins became labeled in vivo than in vitro, whereas no similarity in the pattern of labeling between the two methods was observed. For example, the most heavily labeled protein in the in vivo method, tentatively identified as microtubule-associated protein-2, was not detected on autoradiograms of proteins labeled by the in vitro method. In this latter method, an 85-kDa species and growth-associated protein-43 were always labeled, and the extent of their phosphorylation was enhanced by the additional presence of phosphatidylserine and Ca2+, a result indicating that these labeled species are substrates of protein kinase C. The in vitro conditions also led to the labeling of proteins identified as alpha- and beta-tubulin. Comparison of the methods suggests that removal of the ganglion interferes with the function of protein phosphorylation systems and that this effect involves elements of the cytoskeleton.
Watterson
J G
JG
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Good
R
R
Hearn
M T
MT
Austin
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D019922:GAP-43 Protein; D005728:Ganglia, Sympathetic / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008562:Membrane Glycoproteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008970:Molecular Weight; D009416:Nerve Regeneration / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D014404:Tubulin / Q000378:metabolism
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D010710:Phosphates; D010750:Phosphoproteins; D014404:Tubulin; D000255:Adenosine Triphosphate