The protein kinases induced in cells infected with herpesviruses

[摘要] The subject of this thesis is a protein kinase that had previously been detected in cellular cytoplasmic extracts of cells infected with the alpha-herpesvirus, pseudorabies (PRV). The object of the work undertaken here was to purify the enzyme to allow its proper characterisation, and to determine its genetic origin. The enzyme induced by infection of hamster fibroblasts with PRV was purified to apparent homogeneity by a combination of DEAE-cellulose, high-performance hydrophobic, threonine-sepharose, protamine-agarose and high-performance gel-exclusion chromatography. This purification resulted in an enzyme with a specific activity in excess of 1,000 nmol units/mg, a figure comparable with other homogeneous protein kinases. Polyacrylamide gel electrophoresis of the purified enzyme under denaturing conditions revealed a single stained band at a position of migration corresponding to an apparent molecular weight of 38,000. Polyacrylamide gel electrophoresis of the purified enzyme after incubation with [gamma32P]ATP in the absence of added substrate resulted in incorporation of 32P into this protein band, consistent with the 38,000 molecular weight protein being a protein kinase with a capacity for autophosphorylation. The isoelectric point of the phosphorylated form of the enzyme was approximately 4.9. The molecular weight of the native enzyme was determined by gel-exclusion chromatography and found to be approximately 70,000. It is therefore likely that the protein kinase is homodimeric. When hamster fibroblasts or Vero cells were infected with Herpes simplex type-1 (HSV-1) a new protein kinase activity was also detected. The protein kinases from cells infected with HSV-1 and PRV had similar catalytic properties. Both catalysed the transfer of phosphate from ATP (but not GTP) to basic (but not acidic) proteins, protamine being the preferred substrate in vitro, and they appear to be independent of known regulatory molecules. These protein kinases were also active at 1M KC1, a particularly striking characteristic. The protein kinase activity from cells infected with HSV-1 was partially purified using the methods used for the enzyme from cells infected with PRV. The final purified preparation contained two major species of apparent molecular weights 68,000 and 61,000 when analysed by polyacrylamide gel electrophoresis under denaturing conditions. In addition, when incubated with [gamma32P]ATP, the purified enzyme preparation phosphorylated a protein species of molecular weight 68,000 and isoelectric point 5.6. It is probable that this represents autophosphorylation of the HSV-1 protein kinase, which may be one of the two major species observed in the purified preparation. The molecular weight of the native enzyme was investigated by gel-exclusion chromatography and was found to be in the range of 150,000 - 200,000. This suggested that the enzyme may be a homodimer like the PRV protein kinase.

[发布机构] University:University of Glasgow

[关键词] Biochemistry, Molecular biology, Virology