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This dissertation, "Characterization of a Physiological 62-kDa Protein Substrate for Ganglioside-stimulated Protein Kinase in Central Nervous System Myelin" by Ka-wai, Chan, 陳嘉威, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Characterization of a Physiological 62-kDa Protein Substrate for Ganglioside-Stimulated Protein Kinase in Central Nervous System Myelin Submitted by Ka Wai CHAN for the degree of Master of Philosophy at The University of Hong Kong in August 2004 Protein phosphorylation has been shown to occur in central nervous system (CNS) myelin. Previous studies in our laboratory revealed that gangliosides could enhance the endogenous phosphorylation of a 62-kDa protein, presumably catalyzed by a 68-kDa ganglioside-stimulated protein kinase (PKJ), in purified CNS myelin. To learn more about this ganglioside-modulated protein phosphorylation pathway, the physicochemical properties of the 62-kDa protein were investigated. One- dimensional peptide mapping technique was used to verify whether the ganglioside- stimulated protein kinase and its 62-kDa protein substrate are similar in primary 32 structures. Limited proteolytic cleavages of P-labeled PKJ and 62-kDa protein bands using staphylococcal V8 protease resulted in completely different phospho- peptide maps, indicating that the two proteins are non-identical. Phospho-amino acid analysis revealed that the 62-kDa protein is phosphorylated mainly at serine residue(s). Endogenous phosphorylation of this protein in purified guinea pig brain 2+ myelin also could be achieved by using Mn as a cofactor. The maximal phosphorylation attained was 2- to 4.5-fold higher than those carried out in the 2+ presence of Mg (0.5-16mM). These results are in agreements with the requirement 2+ of Mn for catalytic activity of purified PKJ, indicating that the endogenous phosphorylation of the 62-kDa protein is indeed catalyzed by this enzyme rather than mediated though a substrate-directed effect, i.e., the binding of ganglioside to the 62- kDa protein somehow enhances its phosphorylation by some other protein kinases. This suggestion is further substantiated by heat-inactivation experiments. We found that pre-incubation of purified myelin at 50 C in the presence, but not the absence, of gangliosides could within 2-3 minutes rapidly reduce the 62-kDa protein phosphorylation to less than 20% of its original activity. These findings are in agreement with those published previously which indicated that purified PKJ is sensitive to heat-inactivation at 50 C in the presence, but not the absence, of gangliosides. By contrast, other well-known protein kinases such as protein kinase C do not exhibit such heat-sensitivity profiles. The phosphorylated form of the 62-kDa protein could not be extracted from myelin membrane by using acetic acid at pH 3.0, or by 100mM CHAPS, a zwitterionic detergent. Non-ionic detergents such as NP- 40(1%, v/v), Triton(R) X-100(1%, v/v), β-octylthioglucopyranoside (100mM) also were ineffective. However, both the 62-kDa protein and myelin basic protein (MBP) were extracted from purified myelin by using HCl at pH 2 or below. These findings indicate that the 62-kDa protein may exist as an extrinsic peripheral membrane protein, like MBP. Similar ganglioside-stimulated 62-kDa protein phosphorylation was also found in CNS myelin isolated from other animal species. These include rat, mouse, and chicken. One-dimensional peptide mapping and phospho-amino acid analysis revealed that the 62-kDa protein in all the