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Because of the diverse nature of the organisms which are all classed as 'protozoa' (and because of the lack of detailed information on phospholipid metabolism about most of them), it will probably never be possible to generalize phospholipid metabolism to the degree that it has been possible to characterize a mammalian metabolism. Nonetheless, patterns have begun to emerge (i.e. the similarities among the ciliates Entodinium, Paramecium and Tetrahymena) and will not doubt be expanded upon in the future.

The phospholipids of control and lipid-modified Tetrahymena thermophila were identified and quantified, using 1-D and 2-D COSY proton NMR spectroscopy on intact lipids, before and after HPLC separation. The results are comparable to those obtained using classical lipid analytical techniques. The results indicate that the study of enzyme pathways and other metabolic processes involving phospholipids in Tetrahymena and related protozoa can be carried out using proton NMR spectroscopy as the investigating technique.

Over the past few years, my laboratory has been engaged in studies on phospholipid metabolism using the ciliate protozoan Tetrahymena thermophila as a model system for eukaryotic phospholipid metabolism (Smith, 1983; Smith, 1984; Smith, 1986b; Smith, et al, 1992b; Smith and O’Malley, 1978). Tetrahymena is characterized by having high concentrations of phosphonolipids — 2-aminoethylphosphonoglyceride and 2-aminoethylphos-phonoceramide — as well as phosphatidylcholine and phosphatidylethanolamine as the major phospholipids (Smith, 1985; Smith and Giegel, 1981; Smith and Giegel, 1982; Smith, et al., 1992; Smith and O’Malley, 1978; Thompson, 1972; Thompson, et al, 1971).

The methylation inhibitors 3-deazaadenosine and 3-deazaaristeromycin inhibited the methylation pathway for phosphatidylcholine formation in Tetrahymena. At the same time, the phosphatidylcholine levels within the cell were maintained by increased use of the phosphotransferase pathway. Cell growth was not affected at inhibitor concentrations up to 50 microM but was 50% inhibited at inhibitor concentrations of 100 microM. This growth inhibition was not reversed by the addition of choline to the growth medium. The added choline resulted in greater use of the phosphotransferase pathway, even in uninhibited cultures, but there was no effect on phosphatidylcholine levels, pointing to a much tighter control on phosphatidylcholine formation than exists in animal cells.

The phosphonolipid content of the protozoan Tetrahymena pyriformis was increased by growing the organism on a medium containing increasing amounts of 2-aminoethylphosphonic acid. With levels of 0, 1, 5 and 10 mM 2-aminoethylphosphonic acid, the phosphonolipid content was 23, 25, 31 and 37% of the total cellular phospholipids, respectively. This increase was accompanied by a reciprocal decrease in phosphatidylethanolamine. With 32Pi in the growth medium along with the 2-aminoethylphosphonic acid, the incorporation of the radioactivity into new molecules of 2-aminoethylphos phonic acid was almost totally inhibited, indicating a feedback control on phosphonic acid synthesis.

The phospholipid composition of the stalked and swarmer cell types of the differentiating, Gram-negative bacterium Caulobacter crescentus was determined. The phospholipid composition of the stalked cell type was 86.5% phosphatidylglycerol, 10.4% lysylphosphatidylglycerol, and 3.0% cardiolipin; that of the swarmer cell type was 84.1, 11.4, and 4.4%, respectively. Phosphatidylethanolamine, which is a major phospholipid component of most Gram-negative bacteria, was totally absent.

Dimethylaminoethylphosphonate (DMAEP) was incorporated into the phospholipids of the ciliate protozoan Tetrahymena thermophila at the expense of both phosphatidylethanolamine and phosphatidylcholine, but it had no effect on the levels of the 2-aminoethylphosphonolipid. The newly formed DMAEP-lipid accounted for almost 50% of the phospholipids of the organism. The DMAEP was incorporated into the phospholipids using both the ethanolaminephosphotransferase and cholinephosphotransferase pathways. The DMAEP-lipid was not methylated to the trimethyl derivative, confirming the lack of methylation of phosphonolipids by Tetrahymena.

A protein which contains 2-aminoethylphosphonic acid (AEP) has been isolated from the ciliate protozoan Tetrahymena thermophila . The protein contains about 30% carbohydrate with both N - and O -glycosidic linkages to the polypeptide and 8% AEP which is attached only to the O -linked glycoside. The amino group of AEP is unreactive to dansyl chloride as is the amino terminus of the protein. The polypeptide portion of the molecule, M r 22,500, contains 22% glycine, 5.5% hydroxyproline, and is quite acidic. The phosphoprotein is found in the cell membranes. Its synthesis is inhibited by tunicamycin to the same extent which the antibiotic inhibits cell division.

The activity of phosphatidylethanolamine N -methyltransferase is less than 10% of control levels in microsomes prepared from the ciliate protozoan Tetrahymena thermophila whose phospholipid composition had been altered by being cultured on media containing phosphonic acids. The primary modification obtained is decreased levels of phosphatidylethanolamine ( J. D. Smith and D. A. Giegel, Arch. Biochem. Biophys. , 206 , 420–423 (1981) and 213 , 595–601 (1982)). The enzyme protein is present in these cells at normal levels since addition of the substrate phosphatidylethanolamine to the assay system restores enzyme activity of the lipid-modified microsomes to control levels, while the enzyme from control microsomes is not affected by added phosphatidylethanolamine. The microsomal enzyme is inhibited by the anionic phospholipids cardiolipin, phosphatidylglycerol, and phosphatidylinositol and by lysophosphatidylethanolamine while it is activated only by phosphatidylserine in addition to the substrates phosphatidylethanolamine and phosphatidyldimethylethanolamine. The added phosphatidylethanolamine acts directly as a substrate for the methyltransferase rather than acting by merely stimulating utilization of endogenous lipid since added phosphatidyl[ 14 C]ethanolamine is directly converted to phosphatidylcholine. The results suggest that the technique of phosphonic acid-induced modification of lipid composition will be useful for the study of other membrane-bound enzymes.

When Tetrahymena thermophila is grown on a medium containing up to 5 m m 3-aminopropylphosphonate, up to 90% of the ethanolamine phosphate in phosphatidylethanolamine is replaced by the 3-aminopropylphosphonate. No accompanying alteration of the phospholipid composition of Tetrahymena is observed. This contrasts with the results obtained when 2-aminoethylphosphonate, the naturally occurring compound, is added to the growth medium ( Biochim. Biophys. Acta 528 , 394–398, 1978); the 2-aminoethylphosphonate causes a substantial increase in the 2-aminoethylphosphonolipid and a reciprocal decrease in phosphatidylethanolamine. Thus, there is apparently a one-way control system in Tetrahymena whereby 2-aminoethylphosphonate and its phosphonolipid may influence the level of phosphatidylethanolamine in the cell, but ethanolamine phosphate, as represented by its isosteric analog, does not influence the phospholipid levels. There is no effect of the 3-aminopropylphosphonate on de novo 2-aminoethylphosphonate biosynthesis indicating a strict specificity for 2-aminoethylphosphonate as its own feedback inhibitor.

Phosphatidylserine formation and decarboxylation are decreased in Tetrahymena in which phosphatidylethanolamine has been replaced by its isosteric analog 3-aminopropylphosphonolipid (1,2-diacylglyceryl-3- O -(3-aminopropylphosphonate)). The combined activity of the phosphatidylethanolamine: serine phosphatidyltransferase/ phosphatidylserine decarboxylase complex in isolated mitochondria from lipid-altered cells [J. D. Smith and D. A. Giegel (1981) Arch. Biochem. Biophys. 206 , 420–423] is about 20% of the activity in mitochondria from control cells. The enzyme activity in the lipidaltered mitochondria is stimulated by the addition of exogenous phosphatidylethanolamine to the assay system while the enzymes of the control mitochondria are not. In vivo the lipid-altered cells are able to incorporate radioactivity from [3- 14 C]- or [3- 3 H]serine into phosphatidylserine and phosphatidylcholine in amounts comparable to normal cells. Thus, under conditions of “stress” (e.g., the depletion of phosphatidylethanolamine), the phosphatidyltransferase is apparantly capable of utilizing other phospholipids besides its normal substrate phosphatidylethanolamine.

1. 1. Phosphatidylcholine biosynthesis in Tetrahymena pyriformis was investigated. Experiments in vivo using [Me-14C]methionine showed incorporation of radioactivity into the choline portion of phosphatidylcholine. No methylation of 2-aminoethylphosphonic acid was detected. Incorporation of the intact choline molecule into lecithin was demonstrated when the cells were grown on [Me-14C]choline. 2. 2. The transfer of the methyl group of [ Me- 14 C ] S - adenosyl - l - methionine to form lecithin was demonstrated in cell-free systems. The activity was localized in the microsomal cell fraction. The enzyme system accepted exogenous phosphatidylmonomethylethanolamine as substrate but not phosphatidylethanolamine or phosphatidyldimethylethanolamine. 3. 3. CDP-choline: diglyceride phosphocholinetransferase activity was localized in the mitochondria. The enzyme was twice as active with the optimal concentration of Mn2+ as Mg2+; the enzymatic activity was inhibited by low concentrations of Ca2+ or Hg2+.

The antiviral agent phosphonoacetic acid inhibits growth of Tetrahymena thermophila at concentrations comparable to those inhibiting growth of other eukaryotic cells, with 50% inhibition at 0.5 mM phosphonoacetic acid. The compound is cytotoxk to Tetrahymena at concentrations greater than 2.0 mM. When a culture of Tetrahymena the growth of which was totally inhibited by 2.0 mM phosphonoacetic acid was diluted with fresh medium, growth resumed in an exponential, rather than synchronous, fashion. [2–14C]phosphonoacetic acid is not metabolized by Tetrahymena.

When Tetrahymena thermophila is grown on a medium containing increasing concentrations of N,N,N-trimethyl-2-aminoethylphosphonate (TMAEP), up to 60% of the choline phosphate in phosphatidylcholine is replaced by the phosphonic acid. There is an increase in the relative amount of quaternary ammonium-containing lipid (phosphatidylcholine plus TMAEP-lipid) at the expense of phosphatidylethanolamine. There is no effect of the TMAEP on either 2-aminoethylphosphonolipid levels or on de novo 2-aminoethylphosphonate synthesis. Higher levels of TMAEP in the medium (25 and 50 m m ) lead to decreased growth of Tetrahymena and to an abnormal cell morphology.

A protein which has a high affinity for S-adenosylmethionine (SAM) has been partially purified from rat liver. This binding protein stimulates both the rate and extent of product formation when added to both a lipid methylating system, phosphatidylethanolamine: SAM-N-methyltransferase, and an RNA methylating system, the t-RNA methylase complex from rat liver. The S-adenosylmethionine binding protein by itself has no enzymatic activity in either transmethylation system.

The relative contributions of the two pathways of phosphatidylcholine biosynthesis, phosphatidylethanolamine N -methyltransferase (EC 2.1.1.17) and diacylglycerol:CDP-choline cholinephosphotransferase (EC 2.7.8.1), are altered in the ciliate protozoan Tetrahymena thermophila whose phospholipid composition has been modified by culturing the organism in the presence of one of several aminophosphonic acids, as determined by measuring the incorporation of [ methyl - 3 H]choline and [ methyl - 14 C]methionine into phosphatidylcholine in vivo . In control cells the phosphotransferase pathway provides about 40% of the phosphatidylcholine, while in cells grown with 2-aminoethylphosphonate (AEP), 3-aminopropylphosphonate (APP), and N,N,N -trimethylaminoethylphosphonate (TMAEP) the contribution of the phosphotransferase pathway to phosphatidylcholine formation is 75, 90, and 26%, respectively. In AEP- and APP-grown cells, in which 80% of the phosphatidylethanolamine has been replaced by the corresponding phosphonolipid, the methyltransferase is less active since the level of the substrate phosphatidylethanolamine is reduced and neither of the phosphonolipids is a substrate for the enzyme. In TMAEP-grown cells, TMAEP competes with and reduces the incorporation of phosphocholine by the phosphotransferase pathway, leading to a smaller contribution of the pathway to phosphatidylcholine biosynthesis. The relative amounts of the two different radioactive labels incorporated into diacylphosphatidylcholine vs alkylacylphosphatidylcholine are also altered in the phosphonate-grown cells. The exogenous AEP induces a change in the glyceryl ether content of the 2-aminoethylphosphonolipid—33% in the AEP-grown cells compared to 70% in the control cells—indicating that the exogenous AEP is entering the phospholipids by the ethanolamine-phosphotransferase pathway rather than by the route of the endogenous AEP.

The lipid content of Blastocladiella emersonii zoospores is 5 pg/cell or about 13% of dry weight. Within the first few minutes of germination 60–70% of total zoospore lipid is lost, with neutral lipid, glycolipid and phospholipid fractions decreasing to about the same extent. These changes in lipid content precede the breakdown during germination of the complex and extensive membrane system of zoospores. During growth, which immediately follows germination, net phospholipid synthesis resumes so that total lipid is maintained at 6% of dry weight, but net synthesis of neutral and glycolipid does not begin until induction of sporulation. During sporulation the phospholipid level decreases so that the distribution of lipid among the three fractions approaches that found in zoospores. These changes in lipid content suggest that zoospore membranes containing neutral and glycolipids are synthesized de novo during spore formation.