Phospholipid biosynthesis in the anaerobic protozoon Entodinium caudatum

Abstract

1. The anaerobic rumen protozoon Entodinium caudatum was incubated either intact or with various radioactive precursors of phospholipids after ultrasonication. 2. Pulse-chase experiments showed a rapid turnover of phosphatidylinositol and much slower turnovers of phosphatidylethanolamine and phosphatidylcholine. 3. E. caudatum imbibed choline very rapidly; this was immediately and exclusively converted into phosphatidylcholine which was shown by radioautography after 10 min to be distributed throughout the cell membranes. 4. Phosphatidylcholine was synthesized through a phosphorylcholine-CDP-choline pathway, the methylation or base-exchange pathways not being present. 5. Under suitable conditions [Me-14C]choline can be substantially (50-60%) converted into CDP-choline by sonicated E. caudatum and this provides an excellent method of preparing this biosynthetic intermediary. 6. [2-14C]Ethanolamine was taken up much less readily than choline. The former was incorporated into phosphatidylethanolamine by the CDP-ethanolamine pathway. 7. Doubly labelled [32P]phosphatidyl[2-3H]ethanolamine was converted into ceramide phosphorylethanolamine and N-(1-carboxyethyl)phosphatidyl-ethanolamine, without change in the isotopic ratio. Ceramide phosphoryl [2-14C]-ethanolamine was converted into phsophatidylethanolamine. 8. Palmitic acid, oleic acid and linoleic acid were taken by E. caudatum cells and incorporated into phospholipids. By contrast, although stearic acid was taken up it was hardly incorporated into phospholipids.

The Rumen Microbial Ecosystem

“…Fatty acids esterified in the main phospholipid groups were predominantly octadecenoic acid and palmitic acid; stearic Dawson (1975). " A novel phospholipid, phosphatidyl-N-(2hydroxyethyl)alanine (N-(l-carboxyethyl)phosphatidylethanolamine), first detected in the lipids of mixed rumen protozoa (Kemp and Dawson, 1969a,b), is probably unique to E. caudatum, where it accounted for 5-7% of total lipid P (Dawson and Kemp, 1967;Broad and Dawson, 1975). acid was virtually absent.…”

“…Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids (Table 9.8). Fatty acids esterified in the main phospholipid groups were predominantly octadecenoic acid and palmitic acid; stearic Dawson (1975). " Fatty acids esterified in the main phospholipid groups were predominantly octadecenoic acid and palmitic acid; stearic Dawson (1975). "…”

Digestion of a dehydrated lucerne/barley diet (70:30) in defaunated, Isotricha-monoinoculated and mixed-fauna-inoculated rumen in sheep

“…The corresponding biomass was calculated from the estimations of Leng et al (1981) and Broad and Dawson (1975) Van Soest and Wine (1967).…”

Incorporation of choline and ethanolamine into phospholipids in germinating soya bean

“…Similarly, the uptake of [Me-14C]choline was not affected by ethanolamine. Broad & Dawson (1975) also found no effect of ethanolamine on (Harwood, 1975;Hoelzl & Wagner, 1966,1971 (Hoelzl & Wagner, 1971 Table 5. There was excellent agreement between the two methods for choline, but the isotope-dilution method for ethanolamine gave a high value for the pool size.…”

“…Cycloheximide, which inhibits microsomal protein synthesis (Marcus & Freeley, 1966), and chloroamphenicol, which inhibits chloroplast (protoplast) and mitochondrial protein synthesis (App & Jagendorf, 1963), were reported to have little effect on the labelling of phosphatidylcholine and phosphatidylethanolamine from [1-14C]acetate (Harwood, 1975 (Broad & Dawson, 1975) and hence is caused indirectly by choline kinase activity, then the absence of an inhibition by cycloheximide of the labelling of the aqueous-soluble pool would place the effect of the inhibitor at either the second or third step of the pathway. It is possible also that transport ofcholine to the site of phospholipid synthesis is dependent on protein synthesis, and thus the effect of cycloheximide is indirect.…”