Doctoral Dissertations

Date of Award

8-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Fred Snyder

Committee Members

Francis T Kenney, John S. Cook, R. Julien Preston

Abstract

The synthesis of platelet activating factor (PAF), a potent, bioactive phospholipid, occurs via two enzymatic pathways: a) the acetylation of l-alkvl-2-lyso-sn-glycerophosphocholine by a specific acetyltransferase (Wykle et al., 1980) or b) the transfer of the phosphocholine base group from CDP-choline to 1-alkyl-2-acetyl-sn-glycerol by a cholinephosphotransferase (Renooij amd Snyder, 1981a). Synthesis of PAF was shown to be catalysed in rat kidney inner medulla microsomes by a DTT-insensitive cholinephosphotransferase. The DTT-insensitive cholinephosphotransferase differed from the diacylglycerol cholinephosphotransferase (sensitive to 5 nM DTT) that forms phosphatidylcholine; greater than 90% inhibition of the diacylglycerol cholinephosphotransferase activity occurred in the presence of DTT. This inhibition was specific for DTT since cysteine, reduced glutathione, or 2-mercaptoetheinol did not cause comparable inhibition. The two cholinephosphotransferase activities also differed in their response to microsomal preincubation temperatures. The DTT-insensitive cholinephosphotransferase retained 70% of control activity at a preincubation temperature of 55°C, while the DTT-sensitive activity was inhibited by 60% at 55°C. Deoxycholate at low concentrations caused stimulation of the DTT-sensitive cholinephosphotransferase, probably due to modification of the enzyme-substrate interaction, but had no effect on the DTT-insensitive cholinephosphotransferase. High concentrations of deoxycholate inhibited both activities. The two activities responded similarly to requirements for magnesium or manganese, inhibition by calcium, and the requirement for CDP-choline as a co-factor; both activities were located on the cytoplasmic face of microsomal vesicles. Attempts to solubilize the enzymes from microsomes produced an enriched enzyme preparation, but were not successful in solubilizing either enzyme activity. Substrate competition studies suggested that two independent forms of cholinephosphotransferase occur in the rat kidney medulla; the UTT-insensitive form responsible for the synthesis of PAF and the DTT-sensitive form responsible for the synthesis of phosphatidylcholine. The concept of two separate enzymes for PAF and phosphatidylcholine synthesis is further substantiated by the differences in pH optima, substrate specificities, effect of detergents and ethanol, and response to microsomal preincubation temperature.

The specific activity of the cholinephosphotransferase enzyme in rat kidney inner medulla microsomes is 100-fold higher than the acetyltransferase (12.1 + 3.7 versus 0.1 + 0.0 nmol/min/rng protein). This observation suggests that the cholinephosphotransferase activity is the predominant pathway for the synthesis of PAF in the kidney inner medulla. Study of the substrate specificitiy of the DTT-insensitive cholinephosphotransferase showed that the enzyme prefers a lipid substrate with an sn-2 short chain ester (acetate or propionate) and that CDP-choline is the prefered water-soluble substrate (versus CDP-ethanolamine). Utilization of endogenous neutral lipid substrates by the DTT-insensitive cholinephosphotransferase demonstrated that this enzyme has the capacity to synthesize physiological quantities of PAF from endogenous neutral lipid precursors present in the microsomes.

These studies have characterized the properties of the membrane-bound DTT-insensitive cholinephosphotransferase from rat kidney inner medulla. This enzyme represents the final step in the synthesis of platelet activating factor by the de novo pathway; it has the potential of being another important enzyme that contributes to the physiological regulation of blood pressure.

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