Vol. 129, No. 2
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 707-713 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Membrane-Bound Thioesterase Activity in Mycoplasmas S. ROTTEM, S. L. TROTTER, AND M. F. BARILE* Biomembrane Research Laboratory, Department ofClinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel, and Division ofBacterial Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014*
Received for publication 8 August 1976
Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity ofAcholoplasma laidlawii is confined to the cell membrane. The enzyme could not be released from the membrane by either low- or high-ionic-strength solutions, with or without ethylenediaminetetraacetic acid, nor solubilized by detergents. The enzyme has a general specificity for long-chain saturated and unsaturated fatty acid thioesters. The preferred substrates among the saturated fatty acyl derivatives are the myristyl and palmityl derivatives. Arrhenius plots of thioesterase activities in A. laidlawii membranes enriched with elaidic or palmitic acids showed discontinuities at 12 and 1800, respectively. The possible regulatory significance of the thioesterase activity for the fatty acid synthetase and the possibility that the activity of the enzyme is controlled by the physical state of membrane lipids are discussed. Thioesterase activity has been found in the cytoplasmic fluids of several bacterial systems (3, 4, 22), and its possible role in regulating the activity and specificity of the fatty acid synthetase has been discussed (3, 22). Since the ability to synthesize long-hain fatty acids from acetate is a major physiological feature distinguishing Acholeplasma from Mycoplasma species (13), it was of interest to determine the presence ofthioesterase activity in mycoplasma and to correlate the level of activity in the various species with their ability to synthesize fatty acids. In this communication, the activity of a membrane-bound long-chain fatty acylcoenzyme A (CoA) thioesterase in mycoplasmas is described. The absence of a cell wall in mycoplasmas (13) and the ability to vary the fatty acid composition of membrane lipids (10) enabled us to study the disposition of the enzyme on the outer and inner surfaces of the cytoplasmic membrane and to study the effect of the physical state of membrane lipids on its activity. The partial characterization of the enzyme is presented and discussed in light of the possible role of this enzyme in controlling lipid metabolism. MATERIALS AND METHODS Organisms and growth conditions. Acholeplasma and Mycoplasma species obtained from the National Institute of Allergy and Infectious Diseases Research Reference Reagent collection, Bethesda, Md., included Acholeplam laidiavii, PG 8; A. axan-
thum, S. 743; A. granularum, BTS 39; Mycoplasma neurolyticum, type A; M. anatis, 1340; M. conjunctivae, 581; M. orale, CH 19299; M. arginini, G 230; M. hominis, PG 21; M. gallisepticum, PG 31; M. pulmonis, ASH, and M. pneumoniae, FH. M. capricolum 14 was provided by J. G. Tully, National Institute of Allergy and Infectious Diseases, Bethesda, Md..Most of the work to be described was done with A. laidlawii and M. neurolyticum. The organisms were grown in 100- to 200-ml volumes of a modified Edward medium (2) supplemented with 10% fresh yeast extract and 2% PPLO serum fraction (Difco) at 37°C for 18 to 24 h. The organisms were harvested by centrifugation at 10,000 x g for 20 min, washed twice, and suspended in 0.25 M NaCl. Preparation of membranes. Membranes of A. laidlawii and M. neurolyticum were prepared by osmotic lysis (12). Cell suspensions were diluted 1:20 with deionized water and incubated at 37°C for 10 min. Unbroken cells were removed by centrifugation at 4,000 x g for 10 min, followed by gentle decantation of supernatants. Membranes were harvested from the supernatant fluids by centrifugation at 31,000 x g for 20 to 30 min, washed twice, and suspended in deionized water. The amounts of protein in whole cells, cytoplasmic fluid, and membrane preparations were determined according to the method of Lowry et al. (7) using crystalline bovine serum albumin as standard. All preparations were stored at -200C for a period no longer than 2 weeks before use. Solubilization of membranes. Washed membranes (4 mg of membrane protein per ml) were treated for 15 min at 37C with (per ml) 0.1 to 10 mg of Triton X-100, sodium deoxycholate, or sodium 707
708
ROTTEM, TROTTER, AND BARILE
dodecyl sulfate. The solubilized membrane material was separated from the insoluble residue by centrifugation at 100,000 x g for 1 h at 4°C. Both fractions were assayed for protein and thioesterase activity. Proteolytic digestion of cells and membranes. Digestion of A. laidlawii cells or membrane preparations (2 mg of membrane protein per ml) by an insoluble protease preparation derived from Streptomyces griseus (Sigma, St. Louis, Mo.) was carried out with 1 to 10 enzyme units at 37°C with continuous shaking for 1 h in a final volume of 1 ml. To stop the digestion, the samples were diluted with 9 volumes of ice-cold 0.25 M NaCl solution and centrifuged in the cold at 34,000 x g for 30 min. When intact cells were digested, the supernatant fluid was separated, and the pellet was suspended in 1 ml of 0.25 M NaCl solution and osmotically lysed. The membranes were collected as described above. Assay procedures. Thioesterase activity was determined by spectrophotometrically monitoring the production of 2-nitro-5-thiolbenzoate formed by the reaction of enzymatically cleaved CoA with dithionitrobenzene (DTNB). Assays were performed by adding 0.08 to 0.2 mg of cellular or membrane protein to 1.0 ml of a reaction mixture composed of 0.1 ,mol of fatty acyl-CoA substrate, 5 ,umol of MgCl2, 1 mg of bovine serum albumin, and 1 j,mol of DTNB in 0.05 M tris(hydroxymethyl)aminomethane-maleate buffer, pH 7.6. Reactions were carried out at 37°C, and the change in absorbance at 413 nm was continuously monitored for 5 min by a Bausch & Lomb Spectronic 88 equipped with a Fisher Recordall 5000. The initial rate of increase in absorbance was linear with respect to the amount of protein employed. Results are expressed as nanomoles of CoA released per minute per milligram of protein. Thioesterase activities were calculated by assuming a millimolar extinction coefficient for DTNB of 13.6 cm-' at 413 nm. Reduced nicotinamide adenine dinucleotide (NADH) oxidase (EC 1.6.99.3) activity was measured spectrophotometrically (9). Results were expressed as decrease in absorbance at 340 nm per minute per milligram of protein. Lipid analyses. Lipids were extracted from membrane preparations by two successive extractions with chloroform-methanol (2:1, vol/vol) as described previously (17). Methyl esters of fatty acids were prepared by heating the lipid sample for 15 min at 80°C in BF3-methanol solution (Supelco Inc., Bellefonte, Pa.). The resultant methyl esters were extracted with hexane and examined by capillary gasliquid chromatography (10) in a Perkin Elmer model 990 apparatus using a polar column (150 feet by 0.01 inch [46 m by 0.025 cm]) coated with Carbowax K-20 + V-93 (99:1, wt/wt). For detecting the distribution of radioactivity in reaction mixtures containing [1'4C]palmitoyl-CoA (50 Ci/mol, New England Nuclear Corp., Boston, Mass.), lipids obtained by a chloroform-methanol (2:1, vol/vol) extraction of freeze-dried reaction mixtures were chromatographed on Silica Gel G plates developed according to Freeman and West (6). Lipid spots, detected by iodine vapor, were scraped offthe plates into scintillation vials containing 10 ml of a dioxane-toluene liquor (18), and radioactivity was measured in a Tri-
J. BACTZRIOL.
Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Gove, Ill.). Paramagnetic resonance spectroscopy. Membranes were spin-labeled with 12-nitroxystearate (Syva, Palo Alto, Calif.) by exchange from bovine serum albumin, as previously described (15). Electron paramagnetic resonance spectra of the spinlabeled membranes were measured at various temperatures with a Varian E-3 spectrometer equipped with a temperature control unit. The molecular motion is reported as x0, an empirical parameter calculated from the formula (11) T0 = 6.5 10-10 * WO (h 1) - 1], where W. is the line width of the mid-field line and ho and h_, are the heights of the mid- and high-field lines on a first-derivative absorption spectrum. Greater freedom of motion is associated with smaller values of T0.
RESULTS Distribution of thioesterase activity in mycoplasmas. Acholeplasma species hydrolyzed palmityl-CoA at rates 2- to 30-fold higher than the sterol-requiring Mycoplasma species (Table 1). The only Mycoplasma exhibiting enzymatic activity similar to the Acholeplasma species was M. neurolyticum. A. laidlawii was chosen for most of the studies described because of the high thioesterase activity. M. neurolyticum preparations were used for comparison with the sterol-requiring Mycoplasma species. The thioesterase activity of A. kaidlawii was highest when the cells were harvested during the midexponential phase of growth (Fig. 1). However, old cultures (42 h) still possessed 50% of the activity of exponential-phase cells (16 h). TABLE 1. Thioesterase activity of various mycoplasma speciesa Organism Sp act' % Activityc Acholeplasma laidlawii 72.8 100 A. axanthum 59.2 81 A. granularum 55.8 75 Mycoplasma neurolyticum 43.2 60 M. pneumoniae 22.4 31 M. anatis 13.6 18 M. conjunctivae 12.8 17 M. orale 12.8 17 M. capricolum 4.0 5 M. arginini 4.0 5 M. hominis 4.0 5 M. gallisepticum 3.2 4 M. pulmonis 2.4 3 aOrganisms were grown in a modified Edward medium (2) and harvested at the mid-exponential phase of growth. Data represent an average of three to six experiments with different batches of cells. b Expressed as nanomoles of CoA released per minute per milligram of cell protein. c Compared with that of A. laidlawii.
THIOESTERASE IN MYCOPLASMAS
VOL. 129, 1977
709
above these values. When [1-_4C]palmityl-CoA served as a substrate, the radioactivity in the ° 300 lipid fraction obtained after chloroform-metha0 o/~~~~~ In Inol (2:1, vol/vol) extraction of a reaction mix0. w 0.2 250 > ture containing A. laidlawii membranes was c 2. C-)c exclusively recovered from thin-layer chroma200 tography plates in the free fatty acid fraction. w thioesterase of A. laidlawii exhibited a The 50 cr preference for thioesters of long-chain fatty o1D 0.1,1
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 707-713 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Membrane-Bound Thioesterase Activity in Mycoplasmas S. ROTTEM, S. L. TROTTER, AND M. F. BARILE* Biomembrane Research Laboratory, Department ofClinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel, and Division ofBacterial Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014*
Received for publication 8 August 1976
Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity ofAcholoplasma laidlawii is confined to the cell membrane. The enzyme could not be released from the membrane by either low- or high-ionic-strength solutions, with or without ethylenediaminetetraacetic acid, nor solubilized by detergents. The enzyme has a general specificity for long-chain saturated and unsaturated fatty acid thioesters. The preferred substrates among the saturated fatty acyl derivatives are the myristyl and palmityl derivatives. Arrhenius plots of thioesterase activities in A. laidlawii membranes enriched with elaidic or palmitic acids showed discontinuities at 12 and 1800, respectively. The possible regulatory significance of the thioesterase activity for the fatty acid synthetase and the possibility that the activity of the enzyme is controlled by the physical state of membrane lipids are discussed. Thioesterase activity has been found in the cytoplasmic fluids of several bacterial systems (3, 4, 22), and its possible role in regulating the activity and specificity of the fatty acid synthetase has been discussed (3, 22). Since the ability to synthesize long-hain fatty acids from acetate is a major physiological feature distinguishing Acholeplasma from Mycoplasma species (13), it was of interest to determine the presence ofthioesterase activity in mycoplasma and to correlate the level of activity in the various species with their ability to synthesize fatty acids. In this communication, the activity of a membrane-bound long-chain fatty acylcoenzyme A (CoA) thioesterase in mycoplasmas is described. The absence of a cell wall in mycoplasmas (13) and the ability to vary the fatty acid composition of membrane lipids (10) enabled us to study the disposition of the enzyme on the outer and inner surfaces of the cytoplasmic membrane and to study the effect of the physical state of membrane lipids on its activity. The partial characterization of the enzyme is presented and discussed in light of the possible role of this enzyme in controlling lipid metabolism. MATERIALS AND METHODS Organisms and growth conditions. Acholeplasma and Mycoplasma species obtained from the National Institute of Allergy and Infectious Diseases Research Reference Reagent collection, Bethesda, Md., included Acholeplam laidiavii, PG 8; A. axan-
thum, S. 743; A. granularum, BTS 39; Mycoplasma neurolyticum, type A; M. anatis, 1340; M. conjunctivae, 581; M. orale, CH 19299; M. arginini, G 230; M. hominis, PG 21; M. gallisepticum, PG 31; M. pulmonis, ASH, and M. pneumoniae, FH. M. capricolum 14 was provided by J. G. Tully, National Institute of Allergy and Infectious Diseases, Bethesda, Md..Most of the work to be described was done with A. laidlawii and M. neurolyticum. The organisms were grown in 100- to 200-ml volumes of a modified Edward medium (2) supplemented with 10% fresh yeast extract and 2% PPLO serum fraction (Difco) at 37°C for 18 to 24 h. The organisms were harvested by centrifugation at 10,000 x g for 20 min, washed twice, and suspended in 0.25 M NaCl. Preparation of membranes. Membranes of A. laidlawii and M. neurolyticum were prepared by osmotic lysis (12). Cell suspensions were diluted 1:20 with deionized water and incubated at 37°C for 10 min. Unbroken cells were removed by centrifugation at 4,000 x g for 10 min, followed by gentle decantation of supernatants. Membranes were harvested from the supernatant fluids by centrifugation at 31,000 x g for 20 to 30 min, washed twice, and suspended in deionized water. The amounts of protein in whole cells, cytoplasmic fluid, and membrane preparations were determined according to the method of Lowry et al. (7) using crystalline bovine serum albumin as standard. All preparations were stored at -200C for a period no longer than 2 weeks before use. Solubilization of membranes. Washed membranes (4 mg of membrane protein per ml) were treated for 15 min at 37C with (per ml) 0.1 to 10 mg of Triton X-100, sodium deoxycholate, or sodium 707
708
ROTTEM, TROTTER, AND BARILE
dodecyl sulfate. The solubilized membrane material was separated from the insoluble residue by centrifugation at 100,000 x g for 1 h at 4°C. Both fractions were assayed for protein and thioesterase activity. Proteolytic digestion of cells and membranes. Digestion of A. laidlawii cells or membrane preparations (2 mg of membrane protein per ml) by an insoluble protease preparation derived from Streptomyces griseus (Sigma, St. Louis, Mo.) was carried out with 1 to 10 enzyme units at 37°C with continuous shaking for 1 h in a final volume of 1 ml. To stop the digestion, the samples were diluted with 9 volumes of ice-cold 0.25 M NaCl solution and centrifuged in the cold at 34,000 x g for 30 min. When intact cells were digested, the supernatant fluid was separated, and the pellet was suspended in 1 ml of 0.25 M NaCl solution and osmotically lysed. The membranes were collected as described above. Assay procedures. Thioesterase activity was determined by spectrophotometrically monitoring the production of 2-nitro-5-thiolbenzoate formed by the reaction of enzymatically cleaved CoA with dithionitrobenzene (DTNB). Assays were performed by adding 0.08 to 0.2 mg of cellular or membrane protein to 1.0 ml of a reaction mixture composed of 0.1 ,mol of fatty acyl-CoA substrate, 5 ,umol of MgCl2, 1 mg of bovine serum albumin, and 1 j,mol of DTNB in 0.05 M tris(hydroxymethyl)aminomethane-maleate buffer, pH 7.6. Reactions were carried out at 37°C, and the change in absorbance at 413 nm was continuously monitored for 5 min by a Bausch & Lomb Spectronic 88 equipped with a Fisher Recordall 5000. The initial rate of increase in absorbance was linear with respect to the amount of protein employed. Results are expressed as nanomoles of CoA released per minute per milligram of protein. Thioesterase activities were calculated by assuming a millimolar extinction coefficient for DTNB of 13.6 cm-' at 413 nm. Reduced nicotinamide adenine dinucleotide (NADH) oxidase (EC 1.6.99.3) activity was measured spectrophotometrically (9). Results were expressed as decrease in absorbance at 340 nm per minute per milligram of protein. Lipid analyses. Lipids were extracted from membrane preparations by two successive extractions with chloroform-methanol (2:1, vol/vol) as described previously (17). Methyl esters of fatty acids were prepared by heating the lipid sample for 15 min at 80°C in BF3-methanol solution (Supelco Inc., Bellefonte, Pa.). The resultant methyl esters were extracted with hexane and examined by capillary gasliquid chromatography (10) in a Perkin Elmer model 990 apparatus using a polar column (150 feet by 0.01 inch [46 m by 0.025 cm]) coated with Carbowax K-20 + V-93 (99:1, wt/wt). For detecting the distribution of radioactivity in reaction mixtures containing [1'4C]palmitoyl-CoA (50 Ci/mol, New England Nuclear Corp., Boston, Mass.), lipids obtained by a chloroform-methanol (2:1, vol/vol) extraction of freeze-dried reaction mixtures were chromatographed on Silica Gel G plates developed according to Freeman and West (6). Lipid spots, detected by iodine vapor, were scraped offthe plates into scintillation vials containing 10 ml of a dioxane-toluene liquor (18), and radioactivity was measured in a Tri-
J. BACTZRIOL.
Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Gove, Ill.). Paramagnetic resonance spectroscopy. Membranes were spin-labeled with 12-nitroxystearate (Syva, Palo Alto, Calif.) by exchange from bovine serum albumin, as previously described (15). Electron paramagnetic resonance spectra of the spinlabeled membranes were measured at various temperatures with a Varian E-3 spectrometer equipped with a temperature control unit. The molecular motion is reported as x0, an empirical parameter calculated from the formula (11) T0 = 6.5 10-10 * WO (h 1) - 1], where W. is the line width of the mid-field line and ho and h_, are the heights of the mid- and high-field lines on a first-derivative absorption spectrum. Greater freedom of motion is associated with smaller values of T0.
RESULTS Distribution of thioesterase activity in mycoplasmas. Acholeplasma species hydrolyzed palmityl-CoA at rates 2- to 30-fold higher than the sterol-requiring Mycoplasma species (Table 1). The only Mycoplasma exhibiting enzymatic activity similar to the Acholeplasma species was M. neurolyticum. A. laidlawii was chosen for most of the studies described because of the high thioesterase activity. M. neurolyticum preparations were used for comparison with the sterol-requiring Mycoplasma species. The thioesterase activity of A. kaidlawii was highest when the cells were harvested during the midexponential phase of growth (Fig. 1). However, old cultures (42 h) still possessed 50% of the activity of exponential-phase cells (16 h). TABLE 1. Thioesterase activity of various mycoplasma speciesa Organism Sp act' % Activityc Acholeplasma laidlawii 72.8 100 A. axanthum 59.2 81 A. granularum 55.8 75 Mycoplasma neurolyticum 43.2 60 M. pneumoniae 22.4 31 M. anatis 13.6 18 M. conjunctivae 12.8 17 M. orale 12.8 17 M. capricolum 4.0 5 M. arginini 4.0 5 M. hominis 4.0 5 M. gallisepticum 3.2 4 M. pulmonis 2.4 3 aOrganisms were grown in a modified Edward medium (2) and harvested at the mid-exponential phase of growth. Data represent an average of three to six experiments with different batches of cells. b Expressed as nanomoles of CoA released per minute per milligram of cell protein. c Compared with that of A. laidlawii.
THIOESTERASE IN MYCOPLASMAS
VOL. 129, 1977
709
above these values. When [1-_4C]palmityl-CoA served as a substrate, the radioactivity in the ° 300 lipid fraction obtained after chloroform-metha0 o/~~~~~ In Inol (2:1, vol/vol) extraction of a reaction mix0. w 0.2 250 > ture containing A. laidlawii membranes was c 2. C-)c exclusively recovered from thin-layer chroma200 tography plates in the free fatty acid fraction. w thioesterase of A. laidlawii exhibited a The 50 cr preference for thioesters of long-chain fatty o1D 0.1,1
