Ganglioside-modulated protein phosphorylation. Partial purification and characterization of a ganglioside-inhibited protein kinase in brain.
A novel protein kinase which could be inhibited specifically by gangliosides has been partially purified from the particulate fraction of guinea pig brain through extraction with nonionic detergent, ion-exchange chromatography, hydrophobic chromatography, hydroxylapatite chromatography, and gel filtration. The ganglioside-inhibited kinase activity was eluted with a Stokes radius of 29-30 A, corresponding to a globular protein of approximately 40,000 in molecular weight. Only gangliosides, especially polysialogangliosides, are potent inhibitors for this enzyme preparation. The modulatory action of the glycolipids on the kinase activity is not time-dependent, indicating that the mode of inhibition may not be mediated through a ganglioside-dependent proteolytic process. Calcium was not required for the inhibitory effects of the various gangliosides tested, suggesting that prior formation of Ca2+.ganglioside complexes are not necessary. The partially purified ganglioside-inhibited protein kinase can phosphorylate exogenous substrates such as a synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly. The optimal pH for this reaction occurred between 7.0 and 7.4. Mg2+ (5-10 mM) is required for the enzymic activity and cannot be substituted by Mn2+. Although the nature of the authentic substrates for this ganglioside-inhibited protein kinase is yet unknown, a search for other potential substrates revealed that the synthetic peptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val was the best phosphate acceptor tested so far. Other substrate specificity studies also showed that the ganglioside-inhibited protein kinase is distinct from either the ganglioside-stimulated protein kinase or protein kinase C. Thus, it is possible that gangliosides can act as bio-modulators which may confer a synchronistic action on these three different protein kinase systems.
Chan
K F
KF
Laboratory of Experimental Neuropathology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D005732:Gangliosides / Q000494:pharmacology*; D006168:Guinea Pigs; D006882:Hydroxyapatites; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D005732:Gangliosides; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D047428:Protein Kinase Inhibitors; D017886:Durapatite; D011494:Protein Kinases