$YE§§CTURE AN?) FUNCTION OF QCEGLEPLASMA MEMBRANES - - EFFECTS OF LIPID CRMN {ENGTH AND CAROTENUID PEGMENTS {5%. Disswtohon {$09 fine Degree o§ pit». D. MICEHGAH STATE UNIVERSKTY Leaf Huang 1974 LIBRA R y Michigan Sta te nivcrsity 1' I I. r I l I QfiWASM MEMBRANES—- EFFECTS ”(3F LIPID CHAIN LENGTH AND CAROTENOI D P l GMENTS presented by LEAF HUANG has been accepted towards fulfillment of the requirements for Ph. D. degepin Biophysics 0880! [4: “QM/OI Alfred Haug COR bra Ple yi. th \«"1 sl ABSTRACT STRUCTURE AND FUNCTION OF ACHOLEPLASMA MEMBRANES-- EFFECTS OF LIPID CHAIN LENGTH AND CAROTENOID PIGMENTS By Leaf Huang The effects of lipid chain length and carotenoid pigment content on the structure and function of Acholeplasma laidlawii mem- branes were investigated. Two membrane preparations from the cells were obtained by sup- plementing the growth media with either arachidic (C20:0) or lauric (C12:0) acid. The cells grown with arachidic acid supplementation yielded membrane lipids greatly enriched with the arachidoyl group and those supplemented with lauric acid yielded membrane lipids enriched with lauroyl, myristoyl, and palmitoyl groups. The cell size (0.1 - 0.7u), the membrane thickness (70:14 R), and the cell shape (coccoid) showed no difference for these two preparations. The arachidoyl en- riched membrane had a greater buoyant density, a smaller permeability to glycerol, and a greater sensitivity to osmotic shock compared to the membrane enriched with shorter acyl groups. The spin label 12 N8 is less mobile in the arachidoyl enriched membrane than in the shorter acyl groups enriched membrane. This difference in membrane fluidity can account for the differences observed for the membrane properties. be va ing s neith membr fluid Chara and l tenoi more of so incre 20 g/ acid found ones, Spin- lipid in a arach lipid measu than thESe Leaf Huang Secondly, the carotenoid pigment content in the membrane could be varied by one order of magnitude by growing cells in media contain- ing sodium acetate or propionate (5 g/l). This alteration influenced neither the fatty acyl composition nor the lipid/protein ratio in the membrane. However, Spin-labeling experiments showed a greater lipid fluidity in carotenoid-poor membranes. Carotenoid-rich membranes were characterized by a higher buoyant density, higher osmotic fragility, and lower glycerol permeability. These results suggested that caro- tenoid pigments make the hydrophobic regions of Acholeplasma membranes more rigid. Thirdly, when cells were grown in a medium containing 20 g/l of sodium acetate, the membrane carotenoid pigment content could be increased by 57—fold as compared to cells grown in a medium containing 20 g/l of sodium propionate. Although the same amount of arachidic acid was added to both kinds of medium, less arachidoyl groups were found in lipids of carotenoid-rich membranes than in carotenoid-poor ones. The relative amount of unsaturated acyl groups also increased. Spin-labeling experiments demonstrated only a slight difference in lipid fluidity between the two types of membrane. Cells grown at 28°C in a medium with acetate (5 g/l) contained significantly less arachidoyl groups and more unsaturated acyl chains in the membrane lipids than cells grown in the same medium at 37°C. Spin-labeling measurements revealed that cells grown at 28°C had more fluid membranes than those grown at 37°C. At the respective growth temperatures of these cells, however, the membrane lipid fluidity were rather similar. Leaf Huang It is concluded that Acholeplasma laidlawii cells are capable of adjusting their lipid composition in order to maintain the membrane fluidity within a narrow range. STRUCTURE AND FUNCTION OF ACHOLEPLASMA MEMBRANES-- EFFECTS OF LIPID CHAIN LENGTH AND CAROTENOID PIGMENTS By Leaf Huang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1974 DEDICATION to my wife, Shilling, and to my parents, Professor and Mrs. P. C. Huang ii Ha; dis Pro Mr. man; Pro is ; Pro 1111 Con' ACKNOWLEDGMENTS The author wishes to express his gratitude to Professor Alfred Haug for his constant enthusiasm and guidance throughout this dissertation work. The helpful advice from the committee members, Professors E. M. Eisenstein, A. El—Bayoumi, D. T. A. Lamport and H. T. Tien are also acknowledged. The author also owes thanks to Mr. D. D. Jaquet, Dr. D. Hoy and other members in the laboratory for many technical assistances and discussions. Financial support by Professor B. Rosenberg in author's first two years of graduate study is greatly appreciated. Finally, the author also wishes to thank Professor A. Lang for enabling me to carry out these investigations in the MSU/AEC Plant Research Laboratory. This work was supported by the U.S. Atomic Energy Commission Contract No. AT (ll-l)-1338. iii LIST 01 LIST 0' ORGANI CHAPTE! I . II. TABLE OF CONTENTS LIST OF TABLES . . . . . . . LIST OF FIGURES ORGANIZATION OF DISSERTATION CHAPTER I. GENERAL INTRODUCTION II. EFFECT OF FATTY ACYL CHAIN LENGTH ON SOME STRUCTURAL AND FUNCTIONAL PARAMETERS OF ACHOLEPLASMA MEMBRANES . . . . Introduction . . . . . . . . . Materials and Methods . . . . Organism and Growth Membrane Buoyant Density . Osmotic Fragility . . Osmotic Swelling . . . Glycerol Permeability . . . . . Electron Microscopy . . . . Lipid Extraction and Determination of. Fatty Acyl Group Composition . Electron Paramagnetic Resonance Spectrosc0py (EPR) Other Chemical Methods Chemicals . . Results . . . Cell Growth . . Cell Morphology and Membrane Thickness Membrane Lipid Fatty Acyl Composition and Lipid/Protein Ratio . . . . . . . iv Page vii viii 11 12 12 12 13 13 13 _16 CHAR} Ill. CHAPTER Page Membrane Buoyant Density . . . . . . . . . . 20 Osmotic Fragility of Membranes . . . . . . 20 Osmotic Swelling and Glycerol Permeability . . . . 25 Spin-Labeling of Membranes . . . . . . . . . 25 Discussion . . . . . . . . . . . . . . . 30 III. CONTROL OF MEMBRANE LIPID FLUIDITY BY CAROTENOID PIGMENT CONTENT IN ACHOLEPLASMA LAIDLAWII . . . . . 36 Introduction . . . . . . . . . . . . . . 36 Methods . . . . . . . . . . . . . . . . 37 Organism and Growth . . . . . . . . . . . 37 Plasma Membrane Preparation . . . . 37 Determination of Fatty Acyl Composition and the Carotenoid Content in the Membrane Lipids . . . 38 Buoyant Density of the Membrane . . . . . . . 38 Spin- Labeling of the Membranes . . . . . . 38 Osmotic Swelling, Relative Glycerol Permea- bility and Osmotic Fragility of the Cells . . . 39 Results . . . . . . . . . . . . . . . . 39 Carotenoid Content in A. laidlawii Membranes . . . 39 Lipid Fatty Acyl Compogit1on and Membrane Lipid/Protein Ratio . . . . . . . . . . . 4O Membrane Lipid Fluidity . . . . . . . 4O Membrane Buoyant Density and Relative Glycerol Permeability . . . . . . . . . . 42 Osmotic Fragility of Cells . . . . . . . . . 47 Discussion . . . . . . . . . . . . . . . 47 Iv. REGULATION OF MEMBRANE LIPID FLUIDITY IN ACHOLEPLASMA LAIDLAWII . . . . . . . . . _. . . . . . 52 Introduction . . . . . . . . . . . . . . 52 Methods . . . . . . . . . . . . . . . . S3 Organism and Growth . . . . . . . . . . . 53 Preparation of Plasma Membranes . . . . . . . 53 Analysis of Fatty Acyl Composition and Determination of Membrane Carotenoid Content . . . . . . . . . . . S3 Spin- -Labeling of Membranes . . . . . . . . . S4 C'r E I B CHAPTER Page Results . . . . . . . . . . . . . . . . 54 Carotenoid Content of Membranes . . . . . . 54 Fatty Acyl Composition of Total Membrane Lipids in Carotenoid- Rich and Carotenoid- Poor Cells . . . . . 55 Membrane Lipid Fluidity of Carotenoid- Rich and Carotenoid- Poor Cells . . . . . . 57 Fatty Acyl Composition of Total Membrane Lipids and the Carotenoid Content in Cells Grown at Different Temperatures . . . . 61 Membrane Lipid Fluidity of Cells Grown at Different Temperatures . . . . . . . . 61 Discussion . . . . . . . . . . . . . . . 6S BIBLIOGRAPHY . . . . . . . . . . . . . . . . . 68 vi Table Table LIST OF TABLES Fatty Acyl Compositions of Total Membrane Lipids and Membrane Lipid/Protein Ratio of Acholeplasma laidlawii Cells Supplemented with TWO D1fferent Saturated Fatty Acids . . . . . . . . . Total Lipid Fatty Acyl Composition, Carotenoid Content, and Lipid/Protein Ratio of A, laidlawii Membrane Motion Parameters of the Spin Label 12NS Incorporated into Two Types of Membrane of A: laidlawii . . . . . . . . . Buoyant Densities and Relative Glycerol Permeabilities of A: laidlawii Membranes Total Lipid Fatty Acyl Composition and Carotenoid Content of A: laidlawii Membranes . Temperature Dependence of Motion Parameters of the Spin Label 12NS Incorporated into A: laidlawii Membranes . . Fatty Acyl Composition of Total Lipids and Membrane Carotenoid Content of A, laidlawii Cells Grown at Two Different Temperatures vii Page 19 41 45 46 S6 60 62 Figure LIST OF FIGURES Figure 1. Growth curves of A. laidlawii (oral strain) cells in tryptose brofh supplemented with 5 mg/l of arachidic acid (0), or lauric acid (A) . Electron micrograph of A. laidlawii (oral strain) cells grown in arachidic acid supplemented medium. Bar represents 0.5u, cells grown in lauric acid supplemented medium gave similar morphology, size and membrane thickness . Isopycnic centrifugation of A, laidlawii (oral strain) membranes in a linear sucrose density gradient (25 - 55%, W/W in 1:20 B-buffer) . Osmotic lysis of A, laidlawii (oral strain) cells grown in arachidic acid (0) or lauric acid (A) supplemented medium . . . . . Osmotic swelling of A, laidlawii (oral strain) cells grown in arachidic acid (0) or lauric acid (A) supplemented medium. Absorbances were measured one hour after cells swelled to equilibrium at room temperature in sucrose solutions of various concentration . . Temperature dependence of glycerol permeability in A. laidlawii (oral strain) cells grown in araEhidIc To), or lauric acid (A) supplemented medium. The initial swelling rate of cells in isotonic glycerol solution is prOportional to the passive permeability of glycerol to the cell membranes . . . . . . . . . Electron paramagnetic resonance Spectra of a fatty acid spin label, lZNS, incorporated into membranes of A. laidlawii (oral strain) cells grown in the-medium supplemented with the fatty acid indicated. Arrows indicate signals from unincorporated spin labels viii Page 15 18 22 24 27 29 32 Figure 10. 11. Figure 10. 11. Page Typical electron paramagnetic resonance Spectrum of the spin label 12NS incorporated into A, laidlawii membrane sample at 8°C. Arrows indicate signals from unincorporated Spin labels. Magnetic field strength increases from left to right . . . . . . . . . . . . 44 Osmotic fragility of A. laidlawii cells. Cells were grown in an arachidic acId.supplemented medium containing sodium prOpionate or acetate (5 g/l) . . . . . . . . . . . . . 49 Electron paramagnetic resonance spectra of the Spin label lZNS incorporated into isolated membranes from A, laidlawii. Cells were grown in a medium containing either sodium acetate or sodium propionate (20 g/l). Spectra were recorded at 20°C. Arrows indicate signals from unincorporated spin labels. Magnetic field strength increases from left to right . . . 59 Temperature dependence of the hyperfine splitting 2Th,of the spin label SNS incorporated into A, laidlawii membranes. Cells were gorwn either at 37°C (A), or at 28°C (0). Arrows indicate the growth temperatures . . . . . . . . . . . . . . 64 ix I is pres referer for put ORGANIZATION OF DISSERTATION The main body of this dissertation, Chapters II, III and IV, is presented individually in the format of a scientific paper. The references are, however, combined at the end of the dissertation. Materials in Chapters II, III and IV are to be submitted for publication. process: divisio: DurINg . and pla many in relatIO' bIOIOgi several able. A. CHAPTER I GENERAL INTRODUCTION BiOIOgical Inembranes play an important role in cellular processes, such as neural signal transmission, cell and nuclear division, transport, energy metabolism, and macromolecular synthesis. During differentiation membranes participate in cell-cell interactions and play also a role in carcinogenesis. In the past several decades, many investigators worked to elucidate membrane structure and the relationship to physiological functions. The exact three-dimensional arrangement of molecules in a biological membrane is far from clear at present time. However, several models describing the gross structure of membrane are avail- able. They will be briefly reviewed here. A. Models related to lipid bilayers: l. Davson-Danielli-Robertson unit membrane model (1,2): In this model, lipids are arranged in two layers with hydrophobic chains interacting at the interior and hydro- philic head groups facing out. At both sides of the bilayer, proteins are distributed either in a globular or extended configuration. Lipid-globular protein mosaic model (3): A basic lipid bilayer is assumed in the model. However, proteins are believed to be inserted into the bilayer in a mosaic fashion. Some proteins could merge into the bilayer, some could penetrate all the way through. An important feature of this model is that proteins can migrate laterally in the plane of membrane, provided the lipid bilayer is sufficiently fluid. Lipid-Protein association model (4): In this model, lipids are present as a bilayer, but each phOSpholipid has one chain interacting with a hydrOphobic bonding site on membrane protein, and the other chain directed into the non-polar membrane interior. B. Models unrelated to lipid bilayer: l. Spherical micelle model (5,6): Lipids are closely packed into globular micelles within the membrane, with hydro- carbon chains lying inside and polar head groups facing out. This configuration is perhaps essential for the fusion of two membranes. Lipoprotein subunit model (7): In this model, lipids of membrane "subunits" are bound hydrOphobically to the interior of proteins, with negatively charged groups on the surface. The membrane is the result of a two- dimensional aggregation of these lipoprotein subunits. The model was initially proposed for the chloroplast membrane. some neg protein physical b1010gic thorougl proposed One has membrane gOIng in this org (12) are fOllowin f ”hiCh art Pleuro.pr terized t 3. Repeating lipoprotein subunit model (8): The role of protein in constructing the membrane is strongly emphasized in this model. The membrane is essentially a continuous array of protein units, with lipids inserted in between. Many important functions such as energy transduction and protein tranSport were explained on the basis of this model. All these models have their supporting evidence as well as some negative criticisms. However, it seems that the lipid-globular protein mosaic model has gained much attention in recent years. Bio- physical, biochemical and cytological studies in a wide variety of biological systems lend their support to this model. A well-organized, thoroughly-discussed review on this model is available (9). Models proposed for one membrane system may not be suitable for other systems. One has to be careful when a given membrane structure is generalized. In this thesis, attention has been focused on a particular membrane system, namely, membranes of Acholeplasma laidlawii. Before going into the main body of the thesis, a brief description about this organism seems necessary. Since recent reviews (10, 11), books (12) are available, individual references will not be cited in the following paragraphs. Acholeplasma laidlawii belongs to the order Myc0plasmatales which are a group of procaryotic micro-organisms earlier called pleuro—pneumonia-like organisms, or PPLO. They are generally charac— terized by a small size (0.2 to few u), devoid of cell wall, bound by the boun ment and cont. and 1 are I nechz the T cove] strar Studi in ce Chara ture to 59 RNA a and g growtl With ; exOgex membr; activi the "unit membrane" structure, and lacking any intracellular membrane- bound organelles. A, laidlawii differs from other mchplasmas by not requiring sterol for growth. Its morphology can be coccus, fila- mentous or amorphous, depending on species, growth medium, culture age and the methods employed to examine it. The genome of the organism is circular, of molecular weight 8-9x108 daltons, and have a low G+C content (30 to 36%). DNA replication appears to be semiconservative, and proceeds unidirectionally from at most a few growing points which are believed to be membrane-associated. The ribosomes and the mechanisms of transcription and translation are similar to those of the procaryotes. Phages infecting A, laidlawii cells have been dis- covered. Most of them are bullet-shaped particles and contain single- stranded DNA. Acholeplasma laidlawii has been the subject of many membrane studies in recent years. One obvious reason is its relative simplicity in cellular structures which greatly eases membrane isolation and characterization. Electron microsc0py reveals "unit membrane" struc— ture both in cells and isolated membranes. The membrane contains 50 to 59% protein, 32 to 40% lipid, 0.5 to 2% carbohydrate, 2 to 5% RNA and about 1% DNA. The lipids are composed mainly of phospholipids and glycolipids, with varying amounts of carotenoids depending on the growth condition. Interestingly, the lipids can be highly enriched with a fatty acyl group whose corresponding fatty acid are supplied exageneously. Polyacrylamide gel electrOphoresis of the solubilized membrane exhibits at least 20 to 30 bands. Membrane-associated enzyme activities have been reported. K+ and Na+ transport are not coupled and are transpor strated. Particle plane of a very s 31% u-he Most 1i} Phase t1 detecte< raction fluid 1: permeab: membran. membran. resumes Correct and are independent of the ATPase activity in the membrane. Active transport of K+ ion, acetate, and perhaps D-glucose have been demon- strated. The generally reported membrane thickness is 75 to 100 A. Particles of 75 to 125 A in diameter have been found in the internal plane of the membrane. The cells have a negative surface charge and a very small surface conductivity. Membrane proteins contain 23 to 31% a-helices, 30 to 57% B-pleated sheets, and 13 to 45% random coils. Most lipids in the membrane appear to be arranged in a bilayer manner. Phase transitions of the hydrocarbon chains in the bilayer have been detected with differential thermal colorimetry, BPR, and X-ray diff— raction techniques. At the growth temperature, the membrane has fluid lipid regions which influence important functions such as permeability and osmotic fragility. It is possible to solubilize the membrane by detergents into small "subunits," and form reaggregated membranes after removing the detergents. Lipid bilayer structure resumes in the reaggregated membrane. However, the proteins are in- correctly reassembled in these membranes. in the 131913 possib tion ( lipids influe Increa Permea aggreg the in Proper‘ be mos1 the the tYpes c carb on CHAPTER II EFFECT OF FATTY ACYL CHAIN LENGTH ON SOME STRUCTURAL AND FUNCTIONAL PARAMETERS OF ACHOLEPLASMA MEMBRANES Introduction The limiting plasma membrane is the only membranous structure in the organism Acholeplasma laidlawii (previously named Mycoplasma laidlawii)(13). The lack of a cell wall structure (14) and the possibility of greatly varying the membrane fatty acyl group composi- tion (15) make it an appropriate system for studying the role of lipids in the structure and function of biological membranes. Extensive studies have investigated the fatty acyl group influences on the structure and function of Acholeplasma membranes. Increasing the quantity of unsaturation enhances non-electrolyte permeability (16), reduces osmotic fragility (l7), and decreases the aggregation of particles on the membrane fracture face (18). However, the influence of the fatty acyl chain length on these membrane properties is apparently less understood (16). This influence will be most easily detected in membranes whose lipids differ greatly in the chain length of their acyl groups. This Study investigated two types of Acholeplasma membranes highly enriched with either a 20- carbon saturated acyl group or a combination of acyl groups averaging 14-carl physic growtl MlChl; sodiu of bo Chica and t units wast (5 mg ethar DEVGI medit aracI was j were They Cell l4-carbon atoms. A pronounced dependence of physiological and physico-chemical properties upon chain length was found. Materials and Methods Organism and Growth Acholeplasma laidlawii (oral strain) was kindly supplied by Dr. S. Rottem (The Hebrew University, Jerusalem, Israel). The basal growth medium consisted of 20g Bacto-tryptose (Difco, Detroit, Michigan) which had been lipid extracted (19), 5g D-glucose, 5g sodium acetate, and 5g tris(hydroxymethyl)aminomethane per liter. The pH of the medium was 8.2 to 8.4 without adjustment. Four g/l of bovine plasma albumin fraction V (Armour Pharmaceutical Co., Chicago, Illinois) was charcoal treated to remove fatty acids (20) and then sterilized by Millipore filtration together with 500 units/ml of penicillin G (Sigma, St. Louis, Missouri). The filtrate was then added to the basal medium. Either arachidic or lauric acid (5 mg/l) was introduced into the growth medium as a 70% aqueous ethanol solution. The final ethanol concentration in the medium never exceeded 0.5%. The cells were originally grown in oleic acid supplemented medium. They could be adapted to supplementation with either arachidic or lauric acid after 6 - 10 daily transfers. The medium was inoculated (1% V/V) with a 24 hour—old cell culture. The cells were grown statically at 37°C, and harvested after 20-24 hours. They were collected by centrifugation at 9,000 xg for 20 minutes at 4°C. A one-day-old culture has a titre of approximately 109 c.f.u. Cell growth was monitored by either viable cell counting on plates or measur [not lipi Edward's at 600 nn Membrane l lysed osr in a smai suspensir gradient at 39,00( hours at of the CI a Bausch converter assayed Osmoti _____11_ (24) wit once and 0-01 M M t0 eithe 600 nm a; The frac or measuring turbidity. The plates contained either tryptose broth (not lipid extracted) with 1% agar and 5 mg/l oleic acid, or modified Edward's medium (21) with 1% agar. Turbidity measurements were made at 600 nm with a Coleman 44 linear SpectrOphotometer. Membrane Buoyant Density The harvested cells were washed twice in B-buffer (22) and lysed osmotically (23). After washing, the membranes were suSpended in a small volume (about 2 ml) of a 1:20 dilution of B—buffer. This suspension (0.35 ml) was overlayed on 3.8 m1 of a linear sucrose gradient (25-55% W/W in 1:20 B-buffer). Centrifugation was performed at 39,000rpm in a SW-56 rotor of a Beckman L2-B ultracentrifuge for 3 hours at 22°C. Fractions were collected after puncturing the bottom of the centrifuge tube and their refractive indices were measured with a Bausch G Lomb refractometer at 20°C. The refractive indices were converted to densitites from a standard table. Each fraction was also assayed for protein content. Osmotic Fragility. The procedure used was essentially that of Rottem and Panos (24) with minor modifications. After harvest, the cells were washed once and resuspended in a small volume (about 1 m1) of 0.25 M NaCl and 0.01 M MgCl An aliquot (0.2 ml) of this thick su5pension was added 2. to either 2 ml of 0.25 M NaCl or 2 ml of deionized distilled water, and mixed rapidly. Change in turbidity with time was monitored at 600 nm and room temperature with a Coleman 44 linear Spectrophotometer. The fraction of turbidity left was defined as the ratio: These ra (Fig. 4) Osmotic concentr equilibr of the c Glycerol tion of in 200 n ml of is at the 1 mixed wf were be; (Oh) ; strip cl the rec< which i: then Ca} SIBIEd ( 10 0.0.600 nm of cells in water 0.0.600 nm of cells in 0.25 M NaCl These ratios were normalized with reSpect to the zero time value (Fig. 4). Osmotic Swelling_ The osmotic swelling of cells in sucrose solutions at several concentrations was measured according to de Gier g£_§l, (25). Osmotic equilibrium was achieved within one hour at 22°C and the turbidity of the cell suspension was read at 600 nm. Glycerol Permeability The glycerol tranSport was estimated according to a modifica- tion of the procedure of Bangham g£_gl, (26). Cells were suSpended in 200 mM sucrose, and 0.1 ml of this suspension was injected into 3 ml of isotonic glycerol solution in a round cuvette (diameter 1 cm) at the test temperature. The suspension in the cuvette was rapidly mixed with a Vortex-mixer and the measurements of the optical density were begun less than 3 sec after injection. The optical density (0.0.) at 600 nm as a function of time was traced by a Varian G-14A dA strip chart recorder. The initial 0.0. change, 3?} was measured from - 1 the recorder chart and the initial change in reciprocal 0.0., 23:, t which is proportional to the initial swelling rate of the cell, was then calculated. The test temperature was maintained by a thermo- stated cuvette holder. 11 Electron Microscopy After harvesting, the cells were fixed at room temperature with glutaraldehyde and osmium tetroxide, dehydrated, and embedded in Spur. Thin sections were made and stained with uranylacetate and lead citrate and examined with a Philips EM300 electron microscope. The osmolarity of all fixation solutions was closely matched to that of the culture medium to minimize alteration in apparent cell morphology (27). The membrane thickness was measured as the peak-to-peak distance from a densitometer scan of an enlarged photoprint of the micrograph. Lipid Extraction and Determination of Fatty Agyl Group Composition Plasma membranes were prepared from the cells by osmotic shock in deionized water, collected, and washed according to Razin g£_§l, (l7). Membrane lipids were extracted with chloroform-methanol (2:1, V/V), washed with salt solution, and dried under nitrogen according to Folch EE.§1: (28). Methyl esters of fatty acids were prepared by reacting about 4 mg of lipids in 4 ml of 2% (V/V) con- centrated H2804 in methanol at 40°C for 24 hours. The methyl esters were extracted with hexane and quantitatively analyzed by gas chromatography on a diethylene glycol succinate column at 170°C. A Hewlett-Packard 402 gas chromatograph equipped with a flame ionization detector was used. The esters were identified by comparison with standards obtained from Applied Science Lab, Inc., State College, Pennsylvania. Elect ’— Spect oxazo Appro NaCl : tempe: NaCl 1 sion. was at were I with 3 Other . estima' (30], 1 Folin ] as Sta] Chemicz \ and the esters. ONCE pr 12 Electron Paramagnetic Resonance Spectroscopy (EPR) The spin label, 2-(lO-carboxydecyl)-2-hexyl-4,4-dimethy1-3- oxazolidinyloxyl,(12NS), was synthesized in our laboratory (29). Approximately 2x10”7 moles of 12NS were dispersed in 1 m1 of 0.25 M NaCl solution by Vortex agitation followed by brief sonication at room temperature. A membrane pellet which had been washed with 0.25 M NaCl was suspended in about 0.5 ml of the aqueous Spin label disper- sion. The final concentration in the Spin-labeled membrane suspension was about 10 mg protein/ml and 0.1-0.2u moles lZNS/ml. EPR spectra were recorded with a Varian EPR Spectrometer, model 4205-15, equipped with a variable temperature controller, model V4540. Other Chemical Methods The amount of total lipids in a membrane preparation was estimated colorimetrically according to the method of Saito and Sato (30), using cholesterol as standard. Protein was determined by the Folin phenol method of Lowry gg_gl, (31), with bovine serum albumin as standard. Chemicals Oleic, arachidic and lauric acids were purchased from Sigma, and their purities were checked by gas chromatography of their methyl esters. All solvents were reagent grade and were distilled at least once prior to use. 13 Results Cell Growth Of the fatty acids with even-numbered chain lengths from C10 to C22, only arachidic and lauric acids gave good growth after less than 14 daily transfers. The adaptation was more facile with arachidic than with lauric acid, with about 5 daily transfers needed for arachidic acid compared to 10 for lauric acid. In addition, the cell titre for lauric acid supplementation was at least a factor of five less than that for arachidic acid at the time of harvesting. The poor growth found for supplementation with medium length fatty acids is interesting but not explored further in this work. Acholeplasma laidlawii (oral strain) is reported to have an absolute requirement for an octadecenoic acid (24). Attempts to grow cells on lipid extracted tryptose medium without any fatty acid supplementation failed. Nevertheless, it is possible that minute quantities of residual unsaturated fatty acids in the tryptose are sufficient to fulfill the growth requirement upon supplementation with arachidic or lauric acid. Cell growth depended upon the batch of Bacto-tryptose. Growth usually reached the late log phase in 20—24 hours (Fig. l) but sometimes took as long as 48 hours. Cell Morphologyfiand Membrane Thickness Cell morpholOgy of strain 8 was reported to depend on the nature of fatty acid supplementation (32). However, cells of the oral strain had similar morphology and size distribution when the organism was grown in either arachidic or lauric acid supplemented media. Cells were all coccoid and 0.1 - 0.7u in diameter as revealed Figure 1. 14 Growth curves of A, laidlawii (oral strain) cells in tryptose broth supplemented with 5 mg/l of arachidic acid (0), or lauric acid (A). TURBIDITY 0.| o.< Ol TURBIDITY 0. so O.IO 0,05 0.0! 15 A / O A / l l l l 1 1 l 8 'I6 24 32 4O 48 56 CULTURE AGE (hrs) Figure l 16 with thin section electron microscopy (Fig. 2). The membrane thickness was 70:14 A for both types of cells. Membrane Lipid Fappy Acyl Composition and‘Lipid7Protein Ratio Table 1 lists the total fatty acyl compositions and the lipid/protein ratios for the membranes. The arachidoyl group was found only with arachidic acid supplementation and usually comprised about 55% of the total acyl groups. In one case it comprised 70%. With lauric acid supplementation, the lauroyl, myristoyl, and palmitoyl group percentages were 2, 8 and 4 times greater than with arachidic acid supplementation and the combined quantities of these three groups constituted about 3/4 of the total. The average chain length of the saturated fatty acyl group is about 18.4 carbon atoms for the arachidoyl enriched membranes compared to 14.4 for those enriched with the shorter acyl groups. For each fatty acid supplied, the amount of the oleoyl group was constant. Furthermore, unsat- urated acyl groups comprised a total of about 1/4 of the total fatty acyl groups for both types of membranes, perhaps indicating that unsaturated acyl groups are required for membrane integrity. Thus, two types of Acholeplasma laidlawii membranes are available which differ greatly in the chain length of their saturated acyl groups. The small quantity of lauroyl groups found with lauric acid supplementation suggests that the membrane Structure requires a considerable quantity of longer chain acyl groups. The necessity for Acholeplasma to elongate the lauric acid supplied may be the reason why lauric acid supplementation gives poorer growth than Figure 2. 17 Electron micrograph of A, laidlawii (oral strain) cells grown in arachidic acid supplemented medium. Bar represents 0.5u, cells grown in lauric acid supplemented medium gave similar morphology, size and membrane thickness. .f ... 5. . u u. 3.. , A. .. .3. a. r 3L? 31..., . A. A.» I. Figure 2 19 .ucomoua mm: oumoao chuoe xflco ooumofioafi mnoumo axnpos pace xuumm Heuou on“ we mflmxawca oflmoom -ouuuomm oonmumcw cu .uo>ozo: .maoEOmfi afinuoeoom nmflamcwpmwo uoc oasou cowufiocou menace unou .mocon oHnaov mo noses: on muomou unwwu on» 0» Hanan: “macaw :onnmu mo hopes: on whammy :oHoo can we umoH any on Honaszn .moonuo: one mamfinouaz oom mHHeuoo new .voumufiuaw pace xuuem one 00 00000000 0 0005 00 000000000 0000 .000050000X0 000000000 0 5000 0500 00000000 .000000>00 00000000 0 0005 00 0000000x0 0000 .00005000000 000000000 0 5000 0500 0000000 0 .n0\mamv 0000 000000000 0003 000005000000 500005 00 0300» 0003 000000 o~.oumm.0 No.00mv.o No.0000.o 0m.o000.0 no.000m.o moo.000N.o Uowm UoNN Uom0 moo.00mn0.0 moo.o0000.0 086000“ gflvom 0000000000 500000 00000: 0000000000 00000000 0000000m 0 00000000 00 0000 00000030 0000000 nfluoow 00 05\mu 0000000 0000000 00\000 0000000u00 500002 m .000000502 003000000 na 00 00000000005000 00000000 0>00000m 000 000000000 0000030 «0 00005 47 solution was proportional to the glycerol permeability. Cells grown in pr0pionate were significantly more permeable to glycerol than cells grown in acetate at all three temperatures tested (Table 4). In both cases, the initial swelling rate increased with temperature. At temperatures higher than 45°C, the cells swelled so rapidly that the initial swelling rate became difficult to measure. When a non- electrolyte molecule such as glycerol permeates a lipid barrier, the rate of penetration depends on the packing of lipids. Therefore, the slower permeation in the carotenoid-rich cells suggested a more viscous lipid region in the membrane. This conclusion is consistent with results of the spin-labeling experiments. Osmotic Fragility of Cells The resistance of the cell to the osmotic lysis was measured kinetically. The results showed that prOpionate-grown cells were more resistant to osmotic lysis than those grown in acetate (Fig. 9). After one hour at room temperature the propionate-grown cells had about 10% more turbidity left than the acetate-grown ones. Thus, cells having more fluid membranes are tougher to lyse. This results agrees with the hypothesis that lipid fluidity enhances the membrane tensile strength against osmotic shock (17,56). Discussion The results presented here demonstrate that the hydrophobic regions of the carotenoid-rich membrane are less fluid than the cor- responding regions of the carotenoid-poor ones. Consequently, carotenoid-rich membranes are characterized by higher osmotic 48 Figure 9. Osmotic fragility of A: laidlawii cells. Cells were grown in an arachidic acid supplemented medium containing sodium propionate or acetate (5 g/l). PERCENT TURBIDITY LEFT IOO 90 80 70 60 50 49 , . Sodium Propionote Sodium Acetate l l 1 l 1 l 0 IO 20 3O 4O 50 60 TIME ( min.) Figure 9 SO fragility, lower glycerol permeability, and higher buoyant density. However, no significant change in membrane osmotic fragility was observed when the carotenoid content of strain 8 cells is increased ten-fold (17). Also, protoplasts of Sarcina lutgg_prepared from a colorless mutant or from a wild type strain grown in diphenylamine do not exhibit different membrane osmotic fragility as compared to those from wild type cells (57). Both reports did not present any data about the membrane lipid fluidity and the fatty acyl composition. Nevertheless, it is possible that the fatty acyl composition is modified upon a drastic change in carotenoid content such that the cell can maintain a proper membrane fluidity necessary for normal growth. Consequently, one expects no significant change in osmotic fragility as long as the membrane lipid fluidity stays essentially constant. This argument is further supported by preliminary results from this laboratory that A: laidlawii cells indeed modify their membrane fatty acyl composition and maintain the lipid fluidity within a narrow range when the carotenoid content is ziltered by about 60-fold. Numerous reports demonstrated that cholesterol and a number of its derivatives condense the packing of phospholipids in various biological membranes (58,59), and in model lipid membranes (51,52,25). Our experiments showed that carotenoid molecules may have similar functions. These findings are consistent with the hypothesis that carotenoids and sterols play a similar role in the membranes of Myc0plasmas. However, it is presently unclear how these two types of lipid perform a similar function with entirely different chemical 51 structures. There are at least four major types of carotenoid in A: laidlawii membranes (60). Whether one or several of these pig- ments is responsible for the control of membrane fluidity remains to be investigated. The nature of interaction between carotenoids and membrane lipids is unknown. If the forces are mainly hydrophobic, one expects that the interaction would depend on the characteristics of hydrocarbon chains of adjacent lipids, such as the degree of unsaturation, branching, and steric configuration. Experiments elucidating such a5pects may be carried out by enriching membranes with suitable fatty acyl groups other than the arachidoyl one. CHAPTER IV REGULATION OF MEMBRANE LIPID FLUIDITY IN ACHOLEPLASMA LAIDLAWII Introduction The lipid fatty acyl composition of Acholeplasma laidlawii can be drastically changed (61). Normally its membrane does not contain sterols; however, if offered in the growth medium sterols can be incorporated into the membrane up to 3-4% (62). The membrane carotenoid content can also be altered to a large extent by appro- priately feeding the organism (47,48). All these biochemical altera- tions influence the membrane lipid fluidity (39,56,59) which plays a crucial role in many membrane functions, such as permeability (59), membrane-bound enzyme activity (35), tranSport of certain nutrients (63), and osmotic stability (56). Therefore, it is reasonable to raise the question whether and how the membrane fluidity of Acholeplasma cells is regulated in response to certain stresses. For this purpose the cells were grown at a reduced temperature or the plasma membrane was enriched with an extreme amount of carotenoid pigments. 52 53 Methods Organism and Growth Acholeplasma laidlawii (oral strain) was originally from Dr. S. Rottem (Hebrew University, Jerusalem, Israel). The growth medium contained lipid-extracted tryptose brothisupplemented with arachidic acid (Smg/l). The concentration of sodium acetate, however, was raised (20 g/l) to obtain highly pigmented cells, and sodium prOpionate (20 g/l) was substituted for acetate when pigment-depleted cells were needed. The high concentration of acetate or propionate did not change the pH of the strongly buffered medium (pH 8.4). For 37°C-grown cultures, the inoculum (1%,v/v) was a 24-hour old culture which had already been adapted to an arachidic acid and sodium acetate (5 g/l) supplemented medium. The inoculum for 28°C cultures was prepared identically except that it had been adapted to 28°C. Cells were grown statically in the dark and harvested by centrifuga- tion at the late log phase. Preparation of Plasma Membranes The procedures of Razin g£_al: (17) were followed. Analysis of Fatty Acyl Composition and fieterminafion o Membrane Carotenoid Content Lipids were extracted from isolated plasma membranes according . to Folch e£_al: (28). The optical absorption at 450 nm of the "Folch lower phase" was measured with chloroform: methanol CZ:1,v/v) as reference. The membrane carotenoid content was expressed as OD4so/mg membrane protein. The methyl esters of lipid fatty acyl groups were 54 prepared, extracted and analyzed gas-chromatographically as described before. Membrane proteins were assayed by the method of Lowry gt_al, (31). Spin-Labelingof Membranes The general methods to introduce spin-labeled fatty acids into isolated membranes have been described in Chapter III. Two fatty acid spin labels were employed, namely, 2-(lO-carboxydecyl)-2-hexyl- 4,4-dimethyl-3-oxazolidinyloxyl, (12 NS), and 2—(3-carboxypropyl)-4,4- dimethyl-2-tridecyl-3-oxazolidinyloxyl, (5 NS), which were obtained from Synvar, Palo Alto, California. These fatty acid spin labels intercalate into the hydrOphobic region of the membrane (55). The hyperfine Splitting 2T| from the EPR Spectrum was chosen to determine the mobility of the Spin label in the membrane (Fig. 10) (55). A large splitting indicates a high degree of orientation of the Spin label and therefore a rigid micro-environment around the label. At temperatures above 25°C, accurate measurements of 2TI became rather difficult when spectra of lZNS-labeled membrane were recorded. In these cases, the relative rotational correlation time Tc of the spin label was calculated according to formula (1). A large value of Tc results from a slow rotational motion of the spin label, and therefore indicates a viscous micro-environment. Results Carotenoid Content of Membranes When cells were grown in a high-acetate medium (20 g/l), large quantities of yellow pigments accumulated in the membrane. On 55 the other hand, cells looked pale if they were obtained from a high- pr0pionate medium (20 g/l). The carotenoid content in these two types of membrane differed by about S7-fold (Table 5). Recently, we have observed a lO-fold difference in membrane carotenoid content when the growth medium contained a lower concentration of sodium acetate or pr0pionate (5 g/l). If the concentration of acetate was raised to 20 g/l, the membrane pigment content became appreciably higher (OD4SO/mg membrane protein increased from 0.66 to 2.3). Howeven a similar four-fold increase in propionate concentration only yielded a slightly lower carotenoid content (OD4SO/mg membrane protein decreased from 0.06 to 0.04). Therefore, pr0pionate at a concentra- tion of 5 g/l was already sufficient to block virtually all the caro- tenoid pigment synthesis. On the other hand, cells were highly capable of synthesizing and accumulating carotenoid pigments when large quantities of acetate were available in the medium. Fatty Acyl Composition of Total Membrane Lipidsflin Carotenoidfiich and Carotenoid- Poor Cells Although the same amount of arachidic acid was present in the medium, the lipids of carotenoid-poor cells became enriched with the arachidoyl group up to 56%. The carotenoid-rich cells, however, only had 35 % (Table 5). The average chain length of the saturated fatty acyl groups was 18.7 and 17.1 carbon atoms for carotenoid-poor and -rich cells, respectively. Moreover, lipids of acetate-grown cells also contained more unsaturated fatty acyl groups than those of cells cultured in prOpionate. Small amounts (less than 1%) of saturated .na\uamv kuu uavwguawa so“: voucoaoammsm undone a“ crop» one: magnum 5(5 o~.~ n.~ ~.m o.n m.em a.” a.o~ n.m n.m o.v~ - a.m - o.o~ -- m.~ ouuwwwm «o.c o.m n.¢ ~.~ m.mm m.n m.a n.m ~.~ q.o~ o.o Q.” ~.o ~.~ «.0 n.“ unnumwmmua nnwououm nosoa\ofioav . . . . . . unwmwummw mason» ona ~.c~u d.86 o.o~u ~.¢~u H.osu o.m«u a one o one suede o «do o ”Ho o usu cause cacao acouaoo vounHSuanc: a~\uo~u vuocououuu \vouuusuaw . .ueouuouueu Aw ouoav cofiuwnoaaou fixes spasm suave: «.mocuunao: Hazaflkufi a“ mo acoucou vwozououuu vac coauunomaou ~Ao< spasm can“; «each .m vague 57 fatty acyl groups with odd-numbered carbon atoms were found in the lipids of propionate-grown cells. In conclusion, cells seemed to modify their fatty acyl composition towards shorter chains and higher unsaturation in response to a large accumulation of carotenoid pigments. Membrane Lipid Fluidity of Carotenoid- Rich and Carotenoid-Poor Cells The membrane lipid fluidity was measured with the spin- labeling technique. Typical EPR spectra of membranes labeled with 12NS are shown in Fig. 10. Table 6 lists the values of the two motion parameters measured at various temperatures. Both parameters of 12NS in carotenoid-poor membrane were smaller at all temperatures tested, e.g., Tc at 37°C was about 10% lower. Thus the carotenoid-poor membranes were slightly more fluid than the carotenoid-rich ones. This result was unexpected in view of our previous report where a ten-fold alteration in carotenoid content led to 20-60% difference in Tc at 37°C. An interpretation is provided by the findings in the present studies that the pronounced increase in carotenoid content was accompanied by a concomitant modification of the fatty acyl composition. An increase in carotenoid content made membrane lipid regions more rigid. 0n the other hand, shorter acyl chains and more unsaturated acyl groups led to a more fluid membrane. As a net result, the membrane lipid fluidity was maintained within a narrow range. k|l‘._u- v.21— 58 .unuwu on pmoH scum momaouucfl sumcouum ufloflm ofluocmmz .mHoan cflam veuuuomhoucwzs seam mamcmwm oueofiucw mzonu< .Uoom um pouuouou one: muuuomm .flfl\m omv oumcoflaoum summon no opeueoe seamen Henuflo mcflcfimucoo Esfivoe m a“ czonm one: mHHou .wflzmfivwmfi na Eoum monaHnEoe uoumaomfi oucfi vopmuomuoucfi mz- Hosea swam one we «upcomm managemeu ufluocmmemhmm coupooam k a. .La a, is .. :4, I . c. :fiil... Ii. . . .. FEE, .1 .oH ouzmflm 59 I! mmDOO TI 0. ll\\ w._.