Quantitative determination of antiviral nucleoside analog in DNA.

A technique for the analysis of the amount of an antiviral nucleoside analog incorporated into DNA, utilizing enzymatic digestion of DNA, followed by high-performance liquid chromatography is described. The cells or tissue samples were treated with perchloric acid to inactivate the nucleases, then digested with pronase in the presence of EDTA. DNA was purified by CsCl centrifugation followed by Sephadex chromatography and treatment with deoxyribonuclease 1 and venom phosphodiesterase. The deoxyribonucleoside monophosphates and the monophosphate of the nucleoside analog liberated from DNA were separated and quantitated by HPLC analysis and measurement of radioactivity. This assay is more sensitive, specific, and precise than the determination of DNA density shift. It is also applicable for nucleoside analogs which do not change the density of DNA either because of their structure or their very small degree of incorporation.

Partial characterization of protein kinase C and inhibitor activity of protein kinase C in rabbit peritoneal neutrophils.

Analysis of the cytosol fraction containing protein kinase C activity from rabbit peritoneal neutrophils by DEAE-cellulose chromatography identified protein kinase C activity in the fractions eluted with 0.08 M-0.14 M NaCl and protein kinase C inhibitor activity in the fraction eluted with 0.16 M-0.5 M NaCl. On further analysis by Sephadex G-150 gel filtration, one peak of protein kinase C and two peaks of inhibitor activity were identified. The peak of protein kinase C and two peaks of inhibitor activity were identified. The peak of protein kinase C activity eluted at Ve/Vo 1.6 corresponding to a Stokes radius of 35 A. The first peak of the inhibitor activity eluted at Ve/Vo 1.4 and the second peak of the inhibitor activity eluted at Ve/Vo 2.5. The peak of phosphoprotein phosphatase activity does not coincide with the peaks of inhibitor activity of protein kinase C.