Effect of Iron on Lipid Metabolism of Tetrahymena pyriformis

Increased synthesis of phosphonic analogues of phospholipids was observed in Tetrahymena pyriformis grown in media supplemented with iron. These compounds imparted resistance to phospholipase C hydrolysis. Iron supplementation also increased microsomal A-6-desaturase activity resulting in synthesis of a 4-6,9 isomer of linoleic acid. These findings are discussed in relation to the increased glyconeogenic capacity of iron-grown Tetrahymena. J. Nutr. 101: 1177-1184, 1971. Compounds with a carbonâ€”phosphorus bond are widely distributed in nature. They are synthesized by phytoplankton, the base of the food chain of the sea (1) and are readily incorporated into mammalian tis sues. The phosphonic analogue of phosphatidyl ethanolamine has been isolated from Tetrahymena pyriformis, sea anem one, snails, slugs, hydra, abalone and ru men protozoa. Although mammals are not known to synthesize 2-aminoethylphosphonic acid, phosphonolipids containing this compound have been detected in rat, goat liver, bovine kidney and milk and hu man erythrocytes. Present evidence suggests that 2-aminoethylphosphonic acid arises through a rear rangement of phosphoenolpyruvate (2-4). In Tetrahymena which synthesize large amounts of phosphonolipids phosphoenol pyruvate is also an important substrate for glyconeogenesis. Recently it was shown that iron-grown Tetrahymena deposited more glycogen than is usually found under glyconeogenic conditions (5). In addition iron produced other changes in cell metab olism which we considered to be related to an effect on membranous structures, par ticularly phospholipids. The phosphonic analogues of phospholipids are resistant to lipases and may introduce specific proper ties to cells. It therefore seemed of interest to investA-gate the synthesis, localization and effects on metabolism of phosphono lipids in Tetrahymena pyriformis grown in the presence of iron. METHODS Culturing. Tetrahymena pyriformis, strain E, was maintained in 500-ml Erlenmeyer flasks containing 100 ml of 2% sterile proteose peptone media3 prepared with deionized glass-distilled water. FeCl36HiO (5 Â?Â?g/ml) and additional constitu ents were added aseptically. Experimental cultures were started with a 1% inoculum of log-phase cells and grown in the dark at 25 to 27Â°under good aeration by shaking on a gyrorotary shaker4 at 150 rpm. Aliquots of cells were counted using a hemocytometer or counted and plotted for size distribution by an electronic particle counter5 calibrated with ragweed pollen. The correlation between the two counting methods was + 0.96. The cells were har vested at 4Â°by centrifugation at 600 X g for 15 minutes in a refrigerated centri fuge.6 The packed cells were washed twice and taken up in 0.9% (w/v) Nad. Mitochondria were isolated by suspend ing the cells in three volumes 0.25 M su crose and homogenizing in a PotterElevehjem Teflon homogenizer until few Received for publication January 15, 1971. i Supported in part by U. S. Public Health Service Grant no. GM 14033 and the Wisconsin Agricultural Experiment Station Project 1555. * Approved for publication by the Director of the Re search Division of the College of Agricultural and Life ScienceÂ«, University of Wisconsin. a Difco Laboratories. Inc., Detroit, Mich. 48201. See Difco manual for analysis of proteose peptone. Â« Model G-10 New Brunswick Scientific Inc., New Brunswick, N. J. 08903. Â» Model B, 200 ILorifice. Coulter Electronics, Hlaleah, Fla. 33010. Â« Model PR-2 International Equipment Co., Needham Heights, Mass. 02194. J. NuxmrnoN, 101: 1177-1184. 1177 by gest on July 1, 2011 jn.nition.org D ow nladed fom 1178 YEI-MEI PENG AND C. E. ELSON cells could be seen when the homogenate was examined with a microscope. The homogenate was spun twice at 600 X g for 10 minutes and the pellets were discarded to minimize mitochondrial contamination by unbroken cells and debris. Following centrifugation ' at 9,000 X g for 10 min utes, the mitochondrial pellet was sus pended and washed twice in 5 ml 0.25 M sucrose. The microsomes were in the supernate following centrifugation of the soluble frac tion at 20,000 X g for 10 minutes. The lipid layer and pellet were discarded and the supernate was centrifuged again at 20,000 X g for 10 minutes. This fraction, 20-SUP, was used for the assay of A-6-desaturase activity. Lipid analysis. Cellular and mitochon drial lipids were isolated by the Folch pro cedure (6). The phospholipids were separaated by thin-layer chromatography (TLC) on silica gel sheets ' using the two-dimen sional solvent system suggested by Kapoulas (7). Resolution of the phosphonic ana logues of phospholipids is achieved by this method. Reagent grade redistilled solvents were employed in all steps. The lipid frac tions were located by brief exposure to io dine vapor or by ninhydrin spray. For de termination of phospholipid concentrations scrapings from the thin-layer plates were transferred to Teflon-capped test tubes and extracted with 5 ml of l N HC1in redistilled methanol for 0.5 hour at 50 to 70Â°.The ex tracts were filtered directly into Kjeldahl digestion flasks and evaporated to dryness. Phosphorus was measured by the method of Bartlett (8) modified by digestion with perchloric acid according to Marinetti (9). To measure "POÂ«incorporation, frac tions were cut from the thin-layer sheets and radioactivity was determined by an end window counter.' Methyl esters of fatty acids from the cells, mitochondria and individual frac tions were prepared using BFs-MeOH fol lowing hydrolysis with 0.5 N NaOH in methanol ( 10). No spots other than methyl esters of fatty acids were noted when the isolated esters were chromatographed on TLC plates. Gas-liquid chromatography was performed using a gas Chromato graph 10equipped with a 0.32 by 183 cm glass column packed with 17% diethylene glycol succhiate polyester on 60-80 mesh chromosorb W. The instrument was oper ated isothermally at 165Â°. The methyl esters were identified by comparing reten tion time with standards (NIH mixture D) and by calculating equivalent chain lengths (ECL). Quantitation was done by triangulation. Linearity and accuracy of the detector response were checked by com parison with graded injections of stan dards. The methyl ester of the major unidentified fatty acid was collected by di verting 99% of the column effluent into chilled capillary tubes. The fatty acid methyl esters were hydrogenated according to Farquhar et al. (Il) and chromatographed. No cyclopropane fatty acids were present when the esters were examined according to Brian and Gardner (12). Oxidative cleavage of the unidentified fatty acid methyl esters was done by periodate-permanganate oxida tion. The products were methylated and analyzed by gas-liquid chromatography with the temperature programmed from 50 to 200Â°at 10Â°per minute. Assay of A-6 desaturase. Desaturation of linoleate by the 20-SUP fraction was as follows: the reaction mixture containing 5 to 30 mg protein, 350,000 cpm 14C-U-linoleate in 0.1 ml ethanol, 1 ml 0.08 M KsPOÂ« buffer (pH 7.4) containing 7.5 Â?unolesATP, 1.5 fimoles NADPH, 0.2 Â?onolescoenzyme A and 5 /Â?molesMgCls was incubated for 1.5 hours at 30Â°on a Dubnoff bath (13). The reactions were stopped by the addition of 10 ml of methanol containing 5 mg of carrier fatty acids (palmitic acid) and 0.5 /ig of hydroquinone. Methylation of the lipids was carried out as above. Mercuric adducts of the unsaturated fatty acid methyl esters were prepared by the pro cedure of Mangold (14). Saturated and the acetoxymercuricmethoxy derivatives of un saturated lipids were separated by thinlayer chromatography on silica gel sheets with solvent system: petroleum-diethyl ether 4:1 (v/v) followed by n-propanolâ€” glacial acetic acid 100:1 (v/v) in the same 7 Model B-20 International Equipment Co., Needham Heights. Mass. 02194. Â»Eastman Chromagram, Distillation Products Indus tries, Rochester, N. Y. 14603. â€¢ Model C-110B, Nuclear-Chicago Corp., Des Plaines, 111.60018. Â»o Model 400, F and M. Scientific Division, Hewlett Packard. Avondale, Pa. 19311. by gest on July 1, 2011 jn.nition.org D ow nladed fom LIPID METABOLISM IN T. PYRIFORMIS 1179 direction. Fractions were monitored with a rate meter " and identified by comparison with standard labeled saturated fatty acid methyl esters and mercuric adducts of unsaturated fatty acid methyl esters chromatographed on the same plate. The dis tribution of 14Clabel among the classes of fatty acids was determined by liquid scin tillation " counting. Determination of membrane resistance to phospholipase C. Cells suspended in pH 5.1 phosphate buffer were incubated with phospholipase Ca and cell counts were made at 15 minute intervals with an electronic particle counter.14 For these measurements, the lower threshold of the instrument was increased so that cells larger than 2840 ^ in volume would be counted. RESULTS The growth pattern of Tetrahymena re sembles that of many other microorga nisms. A short lag period is followed by an exponential growth period (log phase), a maximum stationary period and an ex ponential death period. During exponential growth, the cell population doubles in 4 hours or less depending on environmental factors. As these factors are depleted, the doubling time increases until the rate cells divide equals the rate cells die and the maximum stationary phase develops. Dur ing the exponential death phase, cell deaths exceed cell division. In a previous com-