Hynes, Andrew Mark; (1997) Rx-kinase and protein kinase C in superoxide production from neutrophils. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The phorbol ester TPA activates PKC in vitro at low (nM) concentrations, whilst a related compond, Rx, only activates PKC at high (μM) concentrations. Another kinase, Rx-kinase, can be activated by low (nM) concentrations of Rx, but is unaffected by TPA. The aim of the project was to purify and characterise this novel Rx-kinase. However, screening of known sources revealed that Rx-kinase activity was sporadic and it was concluded that the enzyme was inducible. Human neutrophils were chosen for further studies, because of their ready availability and easy isolation. Using a 32P PKC assay and anti-sera raised against specific PKC isoforms, neutrophils were shown to contain β1- PKC, but not α-, β2-, δ-, ϵ-, ζ-, η-, or &thetas;-PKC. A micro-titre based cytochrome c-reduction assay was developed to measure the phorbol ester stimulated superoxide production from neutrophils. The micro-titre format allowed screening of large numbers of samples, over a prolonged time period. Cells responded to low (nM) concentrations of TPA, but showed two, distinct, sensitivities to Rx. Cells responding to low (nM) concentrations of Rx, insufficient to stimulate PKC, were found to contain Rx-kinase and related protamine sulfate kinase activity. However, cells that only responded to high (?M) concentrations of Rx did not contain such activity. T.L.C. analysis of the purchased Rx revealed contamination with Ro, a known activator of PKC, and accounted for the response of cells to high concentrations of Rx. Indeed, analysis of FPLC fractions from neutrophils revealed that Ro stimulated β1-PKC. However it was concluded that the response of cells to low concentrations of Rx was mediated by the Rx/protamine kinases, and not PKC. The superoxide assay was used to screen for compounds capable of inducing a high sensitivity state in neutrophils (i.e. responding to low Rx concentrations), but substance P, lipopolysaccharide, muramyldipeptide, platelet aggregating factor, plumbagin, GM-CSF and overnight incubation all failed to do so. The results suggest that experiments successfully inducing high sensitivity to Rx, as measured by phagocytic cell superoxide production, thereby induce a family of related protamine sulfate/Rx kinases in these cells. Elucidation of such mechanisms of induction would be highly rewarding in unravelling the biochemical basis of the respiratory burst.

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