Open Collections

Abstract

Semliki Forest (SF) virus caused an inhibition of 77% in incorporation of [³H] choline into phosphatidylcholine (PC) of Baby Hamster Kidney -21 (BHK) cells, at 6½-7½ hours post infection (p.i.). Choline uptake, enzyme activities, and pool sizes were measured to clarify the mechanism of inhibition and to understand the regulation of PC synthesis. Choline uptake has a K[sub m] of 17 μM and V[sub max] of 381 pmoles min⁻¹ mg cell protein⁻¹ in mock-infected (control) cells. Uptake is inhibited in infected cells, although such inhibition only partly accounts for incorporation inhibition. Maximal velocities of the enzymes of de novo PC synthesis in nmoles min⁻¹ g cells⁻¹, from control cells, were: choline kinase - 7.3; cytosolic phosphocholine cytidylyltransferase (cytosolic CT) -17.3; microsomal CT - 14.6; and cholinephos-photransferase (CPT) - 47.6. In infected cells, the respective activities were less: 5.2, 12.1, 4.2, and 19.8, at 7 hours p.i.. Choline, phosphocholine, and CDP-choline were separated by ion exchange and charcoal chromatography. Phosphocholine and CDP-choline were hydrolyzed to choline, which was measured enzymically. Diglyceride was hydrolyzed to glycerol, which was measured enzymically. CTP was measured by a new enzymic technique which uses rat liver CT. ATP was measured by its absorbance after high pressure liquid chromatography. PC was measured by lipid phosphorus analysis. Pool sizes in nmoles/g cells, from control (and infected) cells were: choline - 146 (68); phosphocholine - 34 (120); CDP-choline - 6.1 (15.7); diglyceride - 47 (43); CTP - 149 (79); and ATP - 1800 (1080), all at 6½-7½ hours p.i.. Increases in phosphocholine and CDP-choline, and decreases in CTP and ATP, were all significant (p