MEMBRANE PROPERTIES OF THERMOPLASMA. ACIDOPHILA Dissertation for the Degree of Ph. D. MiCHIGAN STATE UNIVERSITY MARY J. RUWART 1974 A LIBRARY 3. f‘.’ii(f}.;ff'fifl Sta-t3 : ~ in ‘ oh-“‘~*-~H'§mz’ This is to certify that the thesis entitled MEMBRANE PROPERTilES OF THERMOPLASMA ACIDOPHILA presented by Mary J. Ruwart has been accepted towards fulfillment of the requirements for Ph.D. degree in Biophysics 04ka Major professor Dec. 21, I973 Date 0.7 639 5’ e. BINDING av ‘ HUM? & SUNS' M BINDERV lllt. FiaSfia amcoplagr CEHS in :3 “9 "embrar 3" a disc? EJEIECTFOr “Warrice brake COuh ABSTRACT MEMBRANE PROPERTIES OF THERMOPLASMA ACIDOPHILA By Mary J. Ruwart Plasma membranes were isolated from Thermoplasma acidophila, a mycoplasma-like organism which grows Optimally at pH 2 and 590 C. Cells in concentrated suspensions were lysed by titrating to pH 9.3. The membranes were purified by washing at pH I0.0 and centrifuging on a discontinuous sucrose gradient. Membrane purity was assessed by electron microscopy, deoxyribonucleic acid content, and poly- acrylamide gel electrophoretic behavior. Small quantities of mem- brane could be prepared by a combination of osmotic shock and soni- cation. These membranes were indistinguishable from those prepared xi§_high pH lysis as judged by the above criteria. Gel electrophor- etic patterns and amino acid composition of cells and membranes were found to differ significantly. The lipids contained small amounts of fatty acid esters and larger amounts of branched long—chain alkyl ethers. The fluidity and structure of Thermoplasma acidophila membranes were investigated with electron paramagnetic resonance techniques. K,\ (1" \) (‘\ . Cue to the re! :e:e~.dent lio' than labellim o In the grow» ;ces relative frat Thermal “:95 kl‘Oi", F i‘CSltion of “D'd. HDOQ ml (:9 W C-, this TiC 0t Sirgr WOW cho atcear To U Mary J. Ruwart Ga 7‘s": Due to the relative homogeneity of the lipid matrix, sharp temperature- dependent lipid transitions were detectable with stearate spin labels. When labelling was performed at pH 2, transitions were observed at ISO and at 600 C., although Thermoplasma acidophila was grown at 560 C. With- in the growth range of this organism (45° to 600 C.), the fluidity under- goes relatively little change. Order and rotational parameters indicate that Thermoplasma acidophila has one of the most rigid biological membra- nes known. Partial interaction between radicals located on the sixteen position of the stearic acid were observed at low molar ratios of label to lipid. Upon denaturation of the membranes at temperatures greater than 65° C., this type of spectrum is gradually converted to one characteris- tic of strong interaction. Extensive interactions were also noted when nitroxy cholestane was incorporated into the native membrane. Our results appear to be consistent with a model wherein at least some of the lipids are arranged in a cluster rather than the conventional bilayer. MEMBRANE PROPERTIES OF THERMOPLASMA ACIDOPHILA By Mary J. Ruwart A DISSERTATION Submitted to tichigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics I974 This d] to w parent ‘IEBFS . DEDICATION This dissertation is dedicated to my loving husband and especially to my parents whose lifelong encouragement has sustained me these many years. The a ter hustan ard advice tfianks are C. C. Ewefl analysis, tethnical is gratefi H539. ”*0! author 6|. ihese Iriv This CO"Tract FUDIIC he lnSIITUTe The COODE East Lang ACKNOWLEDGEMENTS The author wishes to acknowledge the support and encouragement of her husband, relatives, friends, and co-workers. The helpful comments and advice of the committee members were very much appreciated. Special thanks are extended to Dr. D. T. A. Lamport, Dr. G. G. Hooper, and Dr. C. C. Sweeley, as well as their colleagues, for assistance in amino acid analysis, electron microscopy, and mass spectrometry respectively. The technical assistance of Mr. G. Smith, Mr. D. Jaquet, and Ms. N. Brittain is gratefully acknowledged. Very Special commendation is made to Dr. A. Haug, whose guidance and encouragement made this project possible. The author also wishes to thank Dr. A. Lang for enabling her to carry out these investigations in the MSU/AEC Plant Research Laboratory. This research was supported by the U.S. Atomic Energy Commission Contract No. AT-(ll-l)—l338. The author was also supported by U. S. Public Health Service Training Grant No. GM-Ol422 from the U.S. National Institutes of Health and by the College of Osteopathic Medicine, with the cooperation of the Biophysics Department, Michigan State University, East Lansing, Michigan. water I L‘tas‘er I I TABLE OF CONTENTS Chapter I GENERAL INTRODUCTION . Chapter II ISOLATION AND CHARACTERIZATION OF THE PLASMA MEMBRANE . Introduction . Materials and Methods . . . . . . . . . . . . . Growth of Cells . . . . . . . Collection of Cells . . . . . . . . . Preparation of Membranes . . . . . . . . . . . I). Cell rupture . . . . . . . . . . . . a). b). c). d). e). Osmotic shock . . . . Denaturants and solubilizing agents Freezing . . . . Mechanical lysis . . . . . . . . . Lysis by high pH . . . 2). Membrane Purification . a). Membranes from high pH lysis . . . Lipid Extraction . . Fatty Acid Ester and Alkyl Ether Derivatives . Gas Chromatography . . . . . . . . . . . . . Thin Layer Chromatography . . . . . . . Electron Micnoscopy . . . . . . . . . . . . Mass Spectrometry . . . . . . . . . . . . . Analytical Methods . . . . . . . . Gel Electrophoresis . Amino Acid Analysis . . . . . . . . . . . . Page 33?*er I I ChaDTer IV Chapter III Chapter IV Preparation of Flagella-llke Filaments Results . . . . . . . . . . . . . . . . . . Growth Characterization . . . . Membrane Preparation . Precipitation of Soluble Cytoplasm . Membrane Purity. . . . . . . Membrane Characterization . . . . Protein Composition . . . . . . . . . . Lipid Composition . . . . . . Flagella-like Filaments . . . . . . Discussion . . . . . . . . . . . . . . . . ELECTRON PARAMAGNETIC RESONANCE STUDIES ON THERMOPLASMA ACIDOPHILA MEMBRANES Introduction Materials and Methods . . . . . . . . . . . Results . . . . . . . . . . Discussion . . . . . . . . . BIBLIOGRAPHY . . 26 . 3| . 3| . 36 ..44 . 49 . 52 . 53 . 54 . 56 . 68 . 80 ll... ' P.~ V: a. T . . T .I .C A. . 7d LIST OF TABLES Amino Acid Composition of Cells and Membranes. . . . Lipid Distribution in Thermoplasma acidophila. . . . Fatty Acid Ester Determination in Thermoplasma acidophila . . . . . . . . . . . . . . . . . . . . . Percentage Fatty Acid Composition in Thermgplasma aCidOphila Lipids. O O O O O O O O O O O C I O O 0 O Distribution of Alkyl Ethers in Thermoplasma acidophila Lipids O O O O O O O O O O O O O O O O O I O O I O O O 0 Spectral Parameters of Fatty Acid Spin Labels Incorporated into Thermoplasma acidophila Membranes . . . . . . . . . . vi Page 34 35 40 4| 43 6O - I - Figure LIST OF FIGURES Page Growth curve of Thermoplasma acidophila at pH 2 and 56°C. Cultures were inoculated with a IO% (v7v) aliquot taken from a 22 hr. culture. Aeration was continuous throughout the growth period. . . . . . . . . . . . . . . . . . . . . . I6 Scanning electron micrograph of critically-point dried late log phase I, acidophila cells shadowed with gold- palladium. C O O O O O O O O O O O O O O O O O O O O O O O 0 '8 Transmission electron micrograph from thin sections of I} acidophila cells prepared as described in Methods. . . . 20 Transmission electron micrograph of thin sections of I, acidophila stained as described in Methods. Cells were suspended in water and sonicated for one hour as described In Methods. Sedimentable material was washed ten times with O.I M phosphate buffer, pH 7.4. . . . . . . . . . 23 Transmission electron micrograph of negatively stained "amorphous" material collected after cytoplasm, solubilized by high pH, was reprecipitated by titrating to acidic conditions (pH less than 5.5). . . . . . . . . . . . . . . . 25 Effect of pH on cell lysis. O———O pH change in an aqueous cell suspension (6 mg/ml) upon dropwise addition of 0.04 N_NaOH. X———X Release of protein observed when an aqueous suspension of cells (6 mg/ml) is diluted ten-fold by buffers of varying pH. All buffers used were I M. Buffers at pH 9, IO, and II were glycine-sodium chloride; at pH 8 and pH 4, citrate-NaOH; at pH 7, 6, and 5, phosphate-NaOH. . . . . . . . . . . . . . 28 Transmission electron micrographs of thin sections of I: acidophila membranes prepared by high pH lysis, washed once at pH IO, purified by density gradient centrifugation, and washed two times with distilled water. The membranes were stained as described in Methods. . . . . . . . . . . . 30 vii ‘73-"! Sam 3; at A.“"; dC-E‘IC a; s.:ge:.e: 35 393: TI Gels sf ' phila. I 117731.; av tilde: Figure 8. Page Sodium dodecyl sulfate polyacrylamide gel of proteins precipitated from sonication fluid with 5% trichloro- acetic acid in boiling water (IO mins.). The gel was subjected to electrophoresis and stained with Coomassie blue as described in Methods. . . . . . . . . . . . . . . . . . . 33 Schematic profiles of sodium dodecyl sulfate polyacrylamide gels of cell and membrane preparations from Thermoplasma acido- phila. Absorbance was measured at 550 nm. GeTs were sdbjected TE—ETectrophoresis and stained as described in Methods.. . . 38 Transmission electron micrograph of a I, acidophila cell. This specimen was critically-point dried and shadowed with gOId-‘paIIadium. o o o o o o o o o o o o o o o o o o o o o o 46 Transmission electron micrograph of flagella—like filaments collected from I, acidophila. Filaments were negatively stained with phosphotungstic acid. . . . . . . . . . . . . 48 Electron paramagnetic resonance spectra of fatty acid spin labels in I, acidophila membranes at pH 2 and 56°C. . . . . 58 Temperature-dependence of 2T I, acidophila membranes. II or T in spin-labeled a. 2T ' as a function of temperature for |2NS labeled I, acIdophila membranes. O-——O pH 6; x-——x pH 2. b. 2T as a function of temperature for 5N8 labeled I, acndophila membranes. O———O pH 6; x———x pH 2. c. T as a function of reciprocal absolute temperature for l6NS labeled I, acidophila membranes. O-—-O pH 6. . . . 62 EPR spectra of l6NS under various conditions. A. l6NS in water dispersion at 20°C. 8. l6NS bound to four-day-old I, acidophila membranes at 20°C, pH 6. C. l6NS bound to one-day-old I, acidophila membranes at 20°C, pH 6. D. |6NS bound to one-day-old I, acidgphila membranes subjected to 80°C for one hour and cooled to 20°C. The pH is 6. O O O O O 0 O O O 0 O O O O O O O O O O O O 65 viii F. I «a ..l mi I... a: -rwi A3 + .I W: n r) n! r {J 2. r. .I C F a at. T. r1. Tl E. x .. r . rU YI DC 6 I I mu. r1 CJ 1 r. 4 .I I .C . ,r .I I e a I C C e r. Apr . ”d J. I - rJ n e I» u e P o I- . .I .I I. o - .rI O I a F“. a. o o 7i . I . x : g r: .I _ . C C EL I I U, A 8 ”fl.“ D hie] Figure Page l5. Electron micrographs of I, acidophila membranes. A. Negatively-stained native membranes. 8. Negatively-stained membranes after one hour at 80°C. 0 O O O I O O O O O I O O 0 I O O O O O O O O O O 67 I6. Concentration dependence of EPR spectra of l6NS labeled I, acidophila membranes. ———-One-day—old membranes at 20°C, pH 6. The label: lipid ratio is 4 x l0”3. --- The above preparation with the addition of l6NS label giving a flnal label: lipid ratio of 8 x l0‘3. 0—0 The above preparation with the addition of l6NS label giving a final label: lipid ratio of 24 x l0’3. . . . . . . 70 I7. Effect of cholesterol addition on l6NS EPR spectra of labeled I, acidophila membranes. -——-One-day-old membranes labeled with l6NS at pH 6 and 30°C. --- The above preparation after the addition of non-labeled cholesterol in amounts equal to five times the spin label concentrafion (W/W).. O O O O O O O O O O O O O O O I O O O 72 l8. Electron paramagnetic resonance spectra of cholestane spin label in one-day—old I, acidophila membranes at pH 6, 20°C. The molar label: lipid ratio is 4 x l0'3. . . . . . . . . . 76 l9. Membrane models. A. Schematic drawing of a bilayer membrane illustrating the distance between evenly distributed spin labels. 8. Schematic drawing showing a lipid cluster model membrane illustrating the proximity of evenly distributed spin labels. The number of lipid molecules in each model is the same.. . 78 CHAPTER I GENERAL INTRODUCTION I; Biologica recent years I cesses of the 7‘9 CYTOD lasm ‘ransaort and ”It Surfac ic Femgnitic Trbrane 51p“ Eril'enes OI E ”any moc SfCaHY Can t arranqements. C"scribes 1h. “filing the info semenTS Vile lipids 5' Me QIODUIeS subject and ‘ Probably AC' Flo] 99' 38mg wall, making INTRODUCTION Biological membranes have been the subject of much study in recent years because they are intimately involved in the life pro- cesses of the cell. Once considered a mere enveIOpe for containing the cytoplasmic matrix, the membrane is now known to actively control transport and to be the site of macromolecular synthesis and function (70). Surface structures generated by these membranes are immunogen- Ic recognition sites (7|). Furthermore, it appears that the plasma membrane structure may be one of the limiting factors determining the extremes of environment under which an organism may live (72). Many models have been pr0posed for membrane structure, which ba- sically can be classified into two major groups based on their lipid arrangements. The oldest and probably the most substantiated model describes the lipid as a bilayer structure with protein coating or pen- etrating the lipid matrix. The bilayer can be continuous or broken Into segments by the protein (73-76). The second model proposes that the lipids are in clusters, with protein interspersed between or into the globules (77.78). Many excellent reviews are available on this subject and therefore will not further be discussed herein (79). Probably the most widely studied bacterial membrane is that of Acholeplasma laidlawii. Because it is a mycoplasma, it has no cell wall, making membrane isolation and purification relatively easy (80). 4“?! Lipid CONDOSI ‘ vi‘n fatty ac rave indicate- been well stu resonance are In I967, fuse heap anc renditions we and other my. 0i its grout! ”5 probable be obtained Sit'llcture an 9i than 62° emDirical Iy Covered vhic ISm 'IlICh FE at 70° C. 1 “9mm l i( 95 info dire IUnCIIOnal < 'hICh I 93. Inltia long‘Chain ; paEEd 1,0 Ha 3 Lipid composition can be manipulated by supplementing the growth media with fatty acids and cholesterol. Physical studies on this membrane have indicated that its lipids are in a bilayer form. A, Iaidlawii has been well studied In many respects, including electron paramagnetic resonance analysis of its fluidity and thermal transitions (66). In I967, a mycoplasma-like organism was isolated from a coal re— fuse heap and named Thermoplasma acidophila since its optimal growth conditions were pH 2 and 590 C. (I). It differed from A, laidlawii and other myc0plasma in that it did not require lipid supplementation of Its growth media. Because of its lack of a cell wall, however, and its probable relation to a well-studied organism, information could be obtained as to how the hot, acidic environment influenced membrane structure and function. I, acidophila cannot grow at temperatures high- er than 620 C. and this lives very near the high temperature limit found empirically for acidophiles (36). Thermophilic bacteria have been dis- covered which can grow in near boiling water at neutral pH. No organ- Ism which requires a pH less than 3 has been found that can also grow at 700 C. Thus, I, acidophila represents the edge of the acidOphllic, thermophilic frontier at which life may exist. Since the membrane com- es into direct contact with this harsh environment, its structural and functional characteristics may determine the environmental limits at which I. acidOphila may grow. Initial studies revealed that I, acidophila contained branched, long-chain alkyl ethers (2). In this respect, this organism was com- pared to Halobacterium cutirubrum which had been found to contain gtr‘anvl ether: 15 solubi I I zed woentratlons trations. The woes of envil can evoke sim phytanyl ethers. H, cutirubrum grows at high NaCl concentrations and is solubilized in low saline (8|); I, aclggphila requires high proton concentrations for growth, and is solubilized at low hydrogen ion con- trations. These findings lend support to the hypothesis that certain types of environmental stress, regardless of their specific nature, can evoke similar reSponses in different organisms. CHAPTER II ISOLATION AND CHARACTERIZATION OF THE PLASMA MEMBRANE Adv 4 9, II. up he J .3 yo . e C. a- .2 a x: r- . .— . . . we 2... w: difirhmr 6 INTRODUCTION Thermoplasma acidophila, a mycoplasma-like organism with no cell wall, grows optimally at pH2 and 59°C. (I). Investigations of the lipids of this organism revealed the presence of large quantities of long—chain alkyl glyceryl ethers but no fatty acid esters (2). Membrane preparations have been attempted (3), but commonly used Iytic procedures have not been successful (4). Herein, we describe properties of purified I, acidophila membranes obtained in high yield by rupturing the cells at alkaline pH. MATERIALS AND METHODS Growth of Cells. Thermoplasma acidophila was kindly provided by the American Type Culture Collection. The organism was grown at pH 2, 56°C (I). Yeast extract used in this medium was obtained from a single lot (Difco Control #583l97). Several experiments were performed with yeast extract which had been lipid extracted (5). Cultures were inoculated with a l0% (v/v) aliquot taken from a 22 hour culture. Each culture was continuously aerated with sterile, filtered air. Late log phase cells were harvested after 22 hours of growth and treated as described below. Collection of Cells. Intact cells were obtained by centrifugation at 4200 X g for 5 min. at 25°C. Medium components were removed by gently resuspending cells in T-buffer (0.02% (NH ) SO 4 2 4, 0.025% 0.05% M9304, CaCl2.2H20, and 0.3% KH bach shaker at low speed. 2P04, adjusted to pH 2) by shaking on an Eber- Preparation of Membranes. I). Cell Rupture. Osmotic shock: ThermopIasma acidophila cells were exposed to osmotic shock both at pH 2 and pH 7.4. Lysis was attempted with the following four methods: a). Collected, washed cells were diluted l000-fold with deionized, distilled water and stirred for 2 hours. b). Cells were resuSpended in glycerol (20% or 50%), sucrose (25%, 50%, or 70%), 50% glucose, or NaCl (IM or 3M) and stirred for | hour prior to dilution as in (a). Methods (a) and (b) were performed at 4°, 23°, or 55°C in order to determine if temperature affected cell rupture. c). Cells were stirred for l hour in T-buffer, centrifuged at 34,000 X g for 30 min., and then resuspended in 75% T-buffer. These cells were stirred for l hour and the entire procedure 8 was repeated with 50% and 25% T-buffer, respectively. d). Gradual osmotic lysis as in (c) was also attempted with beta-buffer (6). Procedures (c) and (d) were performed in the presence or absence of 0.0125 M Na-EDTA. Denaturants and Solubilizing Agents:’ Cells were resuspended in l M or 5 M urea; 5% or 50% dimethylsulfoxide; 0.I% or |.0% Triton-XIOO; or 0.I% sodium lauryl sulfate. Freezing: Cells suspended in distilled water were frozen in liquid nitrogen and thawed. This procedure was repeated five times. Mechanical Lysis: Cells were suspended in distilled water or O.I M phosphate buffer, pH 7.4, and were treated for l hour with the following: a). Branson automatic cleaner sonicator Model D-50, or b). Raytheon Sonic Oscillator, Model DF-l0l, 0.92 amperes; or 30 minutes with: c). Omnimixer, Ivan Sorvall Omnimixer lnc.,; or d). Sorvall Ribi Cell Fractionator Model RF-l. or e). Biosonik sonicator, 80% full power. Procedures (b) and (c) were also performed in the presence of ll0 micron glass beads (Super Bright Size Il0). Lysis by High pH: This method was most successful for rupturing cells. Cells were collected and washed with T—buffer (l X) and/or water (l-5 X, depending on the experiment). The washed cells were then resuspended in distilled water at a protein concentration greater than l0 mg/ml. The pH was adjusted to 9.3 by adding 2.0 N NaOH dropwise with stirring. The pH was determined with a Beckman Zeromatic ll pH Meter. 2). Membrane Purification. Membranes from High EH Lysis: Membranes were collected at 34,800 X g for 4 hours. The membrane pellet was resuSpended in half as much water as was used during lysis and the pH of the suspension was adjusted to pH (Tar .,,. “to. deJ VJ: r rh a r.) a r: . . «,4 [I 3 H4 h. Ow fl: II. P . C. v o r. r a P .C A M nl. .l I A . .1 I «I Q 5 AU t .L Ge . .2 ul Bi. ' .r. s :- 3. R. .7 2. . a . a a o .3 n V. o . P. .P- ."d o 1 . w KL M v ,. E J ,. Fw aria IIOws "33 t alvy I T” A l0 with 2N NaOH. The membranes were then collected as before, layered onto a discontinuous sucrose gradient (25%/55%), pH 7.4 and centrifuged on a Beckman L2-65B Ultracentrifuge for 2 hours at 40K with an Sw4| rotor. The membranes appeared as a single band at the interface of the two suc- rose Iayers and were collected and freed from sucrose by washing with water. Lipjd Extraction. Lipids were extracted from cell or membrane pellets after multiple water washings by homogenizing with chloroform-methanol (2:|, v/v). Debris was removed by filtration through a fat—free filter or by low speed centri- fugation. After evaporation to dryness, non-lipid contaminants were removed by passing the extract over Sephadex (7) and eluting lipids with chloroform— methanol (l9:l) and chlcroform-methanol-acetic acid l9:l:4. Silicic acid chromatography (Anisil, l00—200 mesh; |.5 x l2 cm column) was also em- ployed to separate the lipids into three fractions: I). neutral (50 ml chloroform), 2). glycosyl diglycerides (50 ml acetone), and 3). phosphclipids (50 ml chloroform:methanol, 2:l v/v; 50 ml methanol). All glassware used in lipid analyses was acid-cleaned prior to use. All solvents were redistilled. Fatty Acid Ester and AlkyI Ether Derivatives. Fatty acid methyl esters were prepared by incubation of lipid as follows: I). in 2.5% methanolic H SO for 24 hours at 40°C; or 2). in l0% 2 4 methanolic H2804 for 24 hours at 75°C (8); or 3). in l0% methanolic BF3 and benzene (4:l) for 48 hours at 75°C (9). After methanolysis, water was added and the methyl esters were extracted with hexane and washed with water until neutral. Alkyl ethers were converted to their alkane or acetate derivatives (l0). For quantitation by gas chromatography, C methyl ester 2| or C24 alcohol standards were added prior to derivatization. l L r r- -2C' add " _——-'." F. H;*' ‘ .DF.AI "F .- .— vv ‘11 c _, a an"? ' A , ‘ . J- I " , .5; ' fl - F- ”Cl” 5. hr v-q Is. d J “ .-.- ifs.» ~~ .. :. ,6- \ Elsat \ 10 Gas-Chromatography, A Hewlett Packard Model 402 gas chromatograph with a flame ionization detector and with six foot glass columns was used thrOUghout. Fatty acid methyl esters were determined at l70°C on l5% DEGS(80—l00 mesh, Hewlett Packard). Alkyl ether derivatives were separated on |% SE-30 (80-l00 mesh, Hewlett Packard). The ethers were eluted isothermally at 320°C or programmed at 8°/min. from 200°C. Molar response factors were determined empirically from standards obtained from Applied Science. Areas of peaks were determined with a planimeter or by height and width- at-half-height measurements. Thin Layer Chromatography (TLC). Qualitative TLC of the three lipid fractions was performed on Ana- sil-G (250 micron) plates (Analabs, Conn.) as described by Langworthy et al (2). Electron Microscopy, Negative staining was performed with l.5% or 2.0% phosphotungstic acid at pH 6 and 8, respectively. Samples were fixed in 2% glutaraldehyde, 0.033M cacodylate buffer, pH 6.| for 2 hours at 4°C. The sample was collected X19. centrifugation and washed once in O.I M cacodylate buffer containing 0.25 M sucrose. The sample was again centringed and 2% osmium tetroxide in R-K buffer (II) was carefully added to the pellet without disturbing the surface. After I8 hours at 4°C, the preparation was drained and dehydrated through a graded ethanol series. The sample was embedded in Spurr's epoxy resin (l2) and stained with lead citrate and uranyl acetate (l3). Intact cells were dehydrated through graded ethanol and isoamyl ace- tate, and dried in a Bowmar Critical Point Apparatus. Gold palladium (ISO-200 A) was shadow—casted onto the specimen with a Ladd Vacuum Evap- ab- 1"-” I r 4 J‘ J "7' (‘26 ‘ ‘wn , i»? out add c .;’," t: :e = yr I Cda" ll orator. A Philips 300 Transmission Electron Microscope and a AMR 900 Scanning Electron Microscope were used to view the specimens. MassgSpectrometry. All mass spectra were taken with a LKB 9000 Gas ChromatOgraph-Mass Spectrometer equipped with computerized data acquisition (Dr. C. C. Sweeley, Dept. of Biochemistry, Michigan State University, East Lansing, Mich.). Fatty acid methyl esters were run on a 6 ft. 3% SE—30 column; alkyl ether derivatives were eluted on a l% SE-30 (2ft. or 6 ft.) Analytical Methods. Protein was quantitated by the method of Lowry eI_§I, (I4). PhOSphate was assayed using the Bartlett method (I5). Ester and ether derivatives were hydrOgenated as reported (l6). Acyl esters were measured by the ferric hydroxymate method (l7) and by infrared absorption at 5.85 micron (I8) on a Perkin-Elmer Model # 62l grating IR Spectrophotometer. Deoxyribonucleic acid content was determined vIa_the Burton modification (l9). Assays for the presence of phosphonate (20) and plasmalogens (2l-23) were performed. After removal of non—lipid contaminants, lipids were quantitated by a modification of a previously reported colorimetric method (24). Carbohydrate (hexose) was analyzed colorimetrically (25). Glycerol was liberated by 24 hour incubation of lipid at 25°C in BCIS-chloroform (l:l, v/v) and assayed after periodate oxidation. (26). Gel Electrophoresis. SDS polyacrylamide gel electrophoresis was performed as follows: membranes, cells or cytoplasm (precipitated with hot trichloracetic acid) were suspended in 0.I% mercaptoethanol, 0.0l M phosphate buffer (pH 7.|), and l.0% sodium dodecyl sulfate ($05) and incubated at l00°C for 2 min. Sucrose and bromophenol blue were then added. Samples were layered onto 5% 2 -. "3‘“; w I. I, ”35;. and l. .xar (~H . ‘ara:;rsxir Native 12 or l0% (8 or ID cm) polyacrylamide gels containing O.I M phOSphate buffer (pH 7.|), 0.I% 808, 0.026 ml of N,N,N-tetramethylethylenediamine per l00 ml of gel, 0.2% methylene bisacrylamide, and 0.065% ammonium persulfate. The upper and lower buffer reservoirs contained 0.I% SDS and 0.l M phOSphate buffer (pH 7.0). Gels were electrophoresed for IS mins. at l0 mA per gel for 5 hours. Gels were stained overnight in 0.l5% Coomassie Blue R, 7.5% acetic acid, 5.0% methanol. Gels were shaken in destaining solution (5.0% methanol, 7.5% acetic acid) for 6 hours and electrophoretically destained for approximately 2 hours. Gels were stored in distilled water at 4°C. Native polyacrylamide gel electrophoresis was carried out with 2.2%, 4.5%, and 9.0% acrylamide as described by Davis (27) with the following modifications: sample and spacer gels were omitted, pH was adjusted to 8.6, and l0-20 microliter of sample containing 20-200 microgram protein, l0% glycerol, 5% bromophenol blue, and O.I M buffer (pH 8.6) were layered directly on each gel. Samples were electrophoresed from cathode to anode at constant current of l mA/gel for ID min followed by 2.5 mA/gel/hr. Gels were stained for protein with Xylene Brilliant Cyanin G prepared by a modification of the Malik-Berrie procedure (28) as described by Blakesly (29). Gels were stained for 5-8 hours. Stained gels were rinsed two times with distilled water and stored in distilled water at 4°C in the dark. The absorbance of the stained gels was monitored at 550 nm with a Gilford Model 2400 Spectrophotometer equipped with a Gilford Linear Trans- port. Amino Acid Analysis. An aliquot of cells or membranes was dried under nitrogen. 6 N HCI was added and 2.5 x IO-3 micromoles of phenol were added per ml of suspension. dticrolvsis fatten to 0' analysis HE System nod- :a' State I tied Autoll Fil vast-es of were 001 Is for 2,5 ho 38” debri II IlaSl’linr SIOII for C {EntrifuqE tron The ¢ I3 Hydrolysis was complete after I8 hours at 105°C. The suspension was taken to dryness under nitrogen and resuspended in water. Amino acid analysis was performed on a single column accelerated flow Technicon System modified by Dr. Derek Lamport, Plant Research Laboratory, Michi- gan State University, East Lansing, Mich. Results were computed by a modi- fied Autolab System IV. Preparation of Flagella-like Filaments. Filaments were found in the supernatants from T-buffer and water washes of cell preparations (see section "Lysis by high pH"). Filaments were collected by ultracentrifugation of the above supernatants at 40 K for 2.5 hours in a SW—4I rotor. Small pellets containing filaments and cell debris were obtained. Collection of filaments could be faciliatated by washing cells only once in distilled water and stirring the suspen- sion for one hour. Cells, menbranes, and debris were then removed by centrifugation at 34,800 X g for 30 min. and the filaments were harvested from the clear supernatant by ultracentrifugation. LP 4 a .. n, I a no r 2 f... .—I. A U 9 and . PD,’ (a ”J I P F Ge .0 P .v r. .l .93 II | I. I u v . .r:. r e S P, .r .. S E x. c 9 Ir. Ah PIN lK. .I A. y 3 a .. h L I I ‘ , 1‘ I C l I F3 8 e I..- pl. u) w. ‘I r .c J I F. ‘ twig“ DI C. l I4 RESULTS Growth Characterization. The growth curve of Thermoplasma acidophila at pH 2 and 56°C was determined in order to insure consistent results and to find optimal conditions for harvesting the cells. (Fig. I). I, acidgphila grew similarly on lipid-extracted medium, a finding that is very un- characteristic of other mycoplasma. As shown in Figure I, log phase terminated after 24 hours of growth at 56°C. Colonies could not be counted because solid media could not be obtained under growth conditions. Therefore, all cells were harvested or used as inoculum after a 22 hour incubation period. The rapid propagation of this organism is facilitated by vigorous aeration, since standing cultures grown in our laboratory have characteristics similar to non-aerated cultures as reported by Darland et al. (I). Shaken cultures (2) appear to grow at rates intermediate between those reported for non- aerated cells (I) and those reported herein for rapidly aerated cells. I, acidOphila appears to be coccoidal in shape and cell diameters vary between 0.5 and l.0 micron. Some cells have one or two filamentous projections (Fig. 2). Membrane Preparation. I, acidophila has a more electron dense cytoplasm than AcholepIasma laidlawii (Fig. 3) although the membranes have similar thickness (l00 - 80 ii) (30). Isolation of I. acidophila membranes by methods usually employed for mycoplasma were unsuccessful since they gave extremely low yields. I, acidgphila is very resistant to osmotic lysis. Nonaerated cultures release no protein when exposed to the osmotic shock procedures described in the Methods section. Cells from aerated cultures appear to be more osmotically fragile; approximately l0% of the total cell protein and a yellow pigment were released by osmotic shock or mechanical treatment such as centrifugation. Additional osmotic shock or mechanical treatments did not 15 0 ~4 Figure l. Growth curve of Thermoplasma acidophila at pH 2 and 56 C. Cultures were inoculated with a l0% (v/v) aliquot taken from a 22 hr. culture. Aeration was continuous throughout the growth period. 00540 05 OJ 0 05 F I6 0.5 0.2 00540 O.I 0.05 j I l l 1 l 0 6 I2 13 24 30 36 TIME (hrs) Figure I é‘s . a It) '1 I\) 17 Scanrirc e.ectrc late lcc prase I, acido :at a:-. . chila cells shadowed with gold- Figure 2 1 I'flfl‘ Figure 3. 19 Transmission electron micrograph from thin sections of I, acidophila cells prepared as described in Methods. fp~ up,“ ’1 ._—'LI-”‘._‘.<.. r 20 Figure 3 a , .p. I I . . . .C . x i U .C I .I 3 e . c E t) a r 1/ r II VI CV I. 1.9 I. I a. . e . g .4 .3 C .Hu 2... II I a: » PI, pH r. n . .«flu :d e P P AJ III . Pub .hII I“ P r: a r: .o ;. r . p u I A y :4 . . n d . 2‘ A: p: I .I I n i ’t .5. . ' 2. U otJ L l r In I . 4 .3 . I .u. .s. I .I «Go V Cr; ' "FBI l( ”Exiting; R i 21 release more proTein. RepeaTed freezing and Thawing of The aeraTed cells caused loss of viabiliTy, buT only minimal loss of proTein. No addiTional proTein was released when cells, TreaTed as described above, were washed in one of sixTeen differenT buffers of varying molariTy (l-5M) or pH (2 -7.8). A small amounT of membrane could be obTained by sonicaTion and osmoTic shock (Fig. 4). Such preparaTions could be purified vi§_sucrose densiTy gradienT cenTrifugaTion To give a pure membrane fracTion, buT The meThod was con- sidered unsaTisfacTory due To The low, irreproducible yield (O-5%). DenaTuranTs and deTergenTs aT high concenTraTions (5 M urea, |% TriTon-XIOO, or 0.I% SDS) caused parTial solubilizaTion of cellular maTerial, leaving inTacT cells or amorphous proTein aggregaTes as deTermined by elecTron microscopy. ATTempTed lysis by raising The pH of diluTe cell suspensions solubilized The cells compleTely in agreemenT wiTh previous reporTs (3). PrecipiTaTion by lowering The pH yielded an amorphous maTerial (Fig. 5) which depended upon The proTein concenTraTion, buT generally began aT pH 6.0 and was essenTially compleTe aT pH 4.5. AT pH values lower Than 4.5, The precipiTaTe Tended To form large whiTe vesicles raTher Than a flocculenT maTerial. CompleTe lysis of cells and selecTive solubilizaTion of cyToplasm was achieved by TiTraTing a cell suspension (noT less Than IO mg proTein/ ml) To pH 9.3 as described in MeThods. Care had To be exercised since membranes also became soluble if high pH was mainTained and The proTein con— cenTraTion was reduced. AfTer mulTiple washings wiTh high pH buffers, membranes from such solubilized preparaTions was noT possible. TiTraTing cell suspensions To pH values lower Than 9.0 resulTed in incompleTe lysis as evidenced by The presence of inTacT cells collecTed afTer cenTrifugaTion for 20 min. aT 34,800 X g. Figure 4. 22 Transmission elecTron micrograph of Thin secTions of I, acidophila sTained as described in Mglhggg, Cells were suspended in waTer and sonicaTed for one hour as described in MeThods. SedimenTable maTerial was washed Ten Times wiTh O.I M phosphaTe fuffer, pH 7.4. Figure 4 ' 'fi 0. A'- #3." ~" 4F 4 .1..- --__ n.-- . N, -~. 1 Figure 5. 24 Transmission elecTron micrograph of negaTively sTained "amorphous" maTerial collecTed afTer cyToplasm, solu- bilized by high pH, was reprecipiTaTed by TiTraTing To acidic condiTions (pH less Than 5.5). 25 Figure 5 26 In more diluTe cell suspensions (less Than IO mg proTein/ml), lysis was compleTed beTween pH 6.5 and pH 7.5 (Fig. 6), indicaTing ThaT cell lysis was concenTraTion dependenT. Membrane solubilizaTion as indicaTed by proTein release aT high pH (greaTer Than pH 9.5), conTinued unTil no sedimenTable maTTer was collecTable (pH l2). Similar curves were obTained (Fig. 6) when The volume of base added was ploTTed versus pH in order To observe The release of buffering componenTs (i.e. proTein) from cells inTo The medium. PrecipiTaTion of Soluble QyToplasm. When supernaTanTs of cells which had been TiTraTed To pH 9.3 were adjusTed To acidic pH, The solubilized cyToplasm precipiTaTed. The onseT of precipiTaTion was dependenT upon The proTein concenTraTion in The supernaTanT fluid. IT appears ThaT This proTein was more soluble aT high pH. From These daTa, one can infer ThaT The inTernal pH of I: acidophila is probably in The neuTral range unless iTs cyToplasm is parTiculaTe. These daTa furTher suggesT ThaT The inside and ouTside of The membrane differ greale, since neuTral exTernal pH causes lysis, whereas The inTracelluIar neuTral pH is physiologically accepTable To This organism. Membrane Puriiys One criTerion for puriTy of membranes prepared by high pH lysis was The absence of cyToplasmic componenTs. ElecTron micro- graphs of Thin secTions (Fig. 7) revealed The presence membrane vesicles which were approximaTely The size of inTacT cells. The inTerior of The vesicles was free from elecTron dense cyToplasm. In conTrasT, sonically prepared membranes (Fig. 4) usually conTained smaller vesicles in agreemenT wiTh earlier observaTions (3). The major drawback To This procedure is The cyToplasmic precipiTaTion which occurs aT low pH and probably accounTs for The amorphous maTerial previously reporTed. Failure To remove This cyTo- A .Q I" ~ A .1 4 L‘THJ‘LY’ 'JLLL ‘ Figure 6. 27 EffecT of pH on cell lysis. O—-—O pH change in an aqueous cell suspension (6 mg/ml) upon dropwise addiTion of 0.04 fl_NaOH. X-——X Release of proTein observed when an aqueous suspen- sion of cells (6 mg/ml) is diluTed Ten-fold by buffers of varying pH. All buffers used were I M. Buffers aT pH 9, l0, and II were glycine-sodium chloride; aT pH 8 and pH 4, ciTraTe-NaOH; aT pH 7, 6, and 5, phosphaTe-NaOH. 3.0 - 28 PROTEIN RELEASE (ug ml) 1 q N 11).- (|I.U) pH Figure 6 23"1 Figure 7. 29 Transmission elecTron micrographs of Thin secTions of I, acidophila membranes prepared by high pH lysis, washed once aT pH l0, purified by densiTy gradienT cenTrifugaTion, and washed Two Times wiTh disTilled waTer. The membranes were sTained as described in MeThods. 30 I I I- k!- I. I ; ~ . I. “a O _\ . «Arr f. o, f .5 , O .. . 7. o.. .o.«1l Ii l i, .‘o \ .u .. 1‘1..qu (I . I . Q E I. . ).. . - .....\ _ m... . or a tin... , 4. .f. . a I u .. « 0.. . .r. , wt :1. f .. . r .5 a , , ,. I. . .. _ nu , .1. f I. . . ~ u 6% a J w 3 . .5 .3 , ' l r . be. . ,. v cw: ; a i . .. . . ‘ E ‘ a. 5 p r . x , .35.. .n. a . ., , . ' u I . l . 9 \ A u . ' .(v p i l . . w . i... , v a... i»! i}. . . . . . l | f r «I. e u l x . I .. {x .v/ A. _ L I \o . - a. ‘r . . ~ v ' I \ n o d ‘s I. S N .. , . I . .I V . . .o . I q. . o H . . \ 1"... up / 4 o .15.. o 0. x .. 9 11 _ r U. 4 y > w \. .. I ... sv 0’ L.-. s I .- , a o \ a . o . J. . . I . v w .. . . . . . . .... . (a... 1 . l f, x \ I.‘ I... .‘ I OJ 4 l f u x . 6 t . \Qv .. 1 . I. i a _ . r . , . . a . .. s s . fl . .3‘ we . . . - . u .. i K fl 4 . i. ID. I. \ o. I. a i at! i o . . w 4, i o. o J g . I a l w I w . .1. 4? . a i .i. 3!. p n ,. u: .o. r: n.. I n L ‘, O I V o ; lo . O (J. . i. v . II L‘ . . .\ . .. v \ a .r. . .. .. . v .1. M . Y H; , .. a.) . x. a ‘1 . .. ... 13 £01 0 .. Is . u , d. . . A 34: . " .\ I .l. x; a 1A .’ i ‘ 1 r . (.1 I: . I. I JQ t .r’ .4 g l ¢J . r /.. .. - 4 _ . . . s . .. . 1/. x . _. . . . . ‘ r . ... . A y * .\ i. . .1 J. ._ . v r s o a I. 1.. \. l s S _. . . a. .. . t, 1 . p f O . Q.. ... t a . . . . a . .I, . . . .1 - e V In. (’1 i ‘ . ‘ .W l w. .‘ . 4 .0 s. p a \1 u l d u. . cu . . . l . . o r . . o 1’ s.‘ t .. .c . . J. A I ‘ J 'I a . a. . . _ i u ‘1 .u w .u. . _ .. . C ... . . . . . ‘U. Pf! . v I‘. .«v . . .. 1.34 7.. . V . l .. . r .i q . . , I . V. v a I. r . . p .3... It .. J K .. 7 ... e r U 9 F I. a 1.. 31 plasmic maTerial was probably due To The acidic condiTions chosen (pH 5). A second criTerion of membrane puriTy was The failure of conTaminaTing cyToplasmic proTein To peneTraTe low percenTage polyacrylamide gels (naTive), even when gels were loaded wiTh 200 micrOgrams of proTein. ln conTrasT, oThers (3) found ThaT Their preparaTions, which conTained "amor- phous" maTerial, easily peneTraTed naTive polyacrylamide gels. Similar gel paTTerns were obTained in our laboraTory on IO% SDS polyacrylamide gels of cyToplasmic componenTs presenT in The supernaTanT of cells sonicaTed aT pH 6.5 (Fig. 8). Thirdly, The membrane preparaTion used herein was essenTially devoid of deoxyribonucleic acid. The membranes conTained less Than 0.I% (w/w) DNA. When comparing our resulTs wiTh Those of oTher laboraTories, cauTion musT be exercised, since differenT sTrains, of I: acidophila may have been selecTed for as previously suggesTed (3|). Membrane CharacTerizaTion. The purified membrane was composed of |9% lipid, 5% carbohydraTe, and 76% proTein. Chloroform-meThanol (2:l) ex- TracTions wiTh or wiThouT acid (O.I NHCI) conTained large amounTs of hydro- phobic proTein which musT be removed by column chromaTography for accuraTe quanTiTaTion of lipids (30). The exacT quanTiTy of proTein presenT in each exTracT varied wiTh The lengTh and Type of homogenizaTion during The ex— TracTion and wiTh The ToTaI volume of organic solvenT used. These exTracTs always conTained a 2 To l5 Times as much proTein as lipid (w/w). These consideraTions may accounT for The high lipid-To-proTein raTio reporTed by workers (3) who reporT The presence of amorphous maTerial in Their preparaTions. ProTeln ComposiTion Membranes solubilized and elecTrophoresed on SDS polyacrylamide gels had a paTTern sTrikingly differenT from solubilized Thermoplasma acidophila 13.. .fl ‘I‘kl‘LW W'ZW’ 'dw' Figure 8. 32 Sodium dodecyl sulfaTe polyacrylamide gel of proTeins precipiTaTed from sonicaTion fluid wiTh 5% Trichloro- aceTic acid in boiling wafer (l0 mins.). The gel was subjecTed To elecTrophoresis and sTained wiTh Coomas- sie blue as described in MeThods. 33 Figure 8 .J. I.u“LI.h...~ 4?: 3.5T... mfi! ' it... F 34 TABLE I: AMINO ACID COMPOSITION OF CELLS AND MEMBRANES FROM 1: ACIDOPHILA Amino Acid Cellsa (mole percenT) Membranesa Lys 7.6 i .00 4.05 :_.35 His |.7 :_.l0 |.l5 :_.25 Arg 5.2 : .IO 3.25 :_.05 Asp ll.4 :_.40 8.40 :_.OI Thr 4.8 :_.IO 6.40 :_.30 Ser 4.9 :_.00 6.35 __.35 Glu 9.6 :_.00 6.0 :_.00 Pro 3.8 :_.IO 5.25 :_.05 Gly 8.9 :_.40 7.75 :_.55 Ala 7.l :_.IO 8.40 :_.30 Cys 0.8 :_.00 5.75 :_.05 Val 8.2 :_ .lO 6.90 :_.30 MeT 2.7 :_.4 2.95 _ .05 lsoleu 7.4 :_.20 7.20 _ .20 Leu 7.6 i .20 9.60 :_.00 Tyr 3.7 :_.IO 4.35 i .00 Phe 4.5 i .40 6.25 :_.05 a average of Three deTerminaTions .P.. 3. CV n a T F 2. .C _. . rl .. .. Denr‘c... in"... \l‘ ~ w a." 35 TABLE 2: LIPID DISTRIBUTION IN THERMOPLASMA ACIDOPHILAa MoieT NeuTraIs GI coli ids Phos holi ids __.l __ x J3 P P (umoles/mg lipid) EsTer 0.I4 0.07 0.07 CarbohydraTe 0.3I |.l5 0.58 Glycerol 0.27 0.75 0.84 ETher 0.09 I.08 I.00 PhosphaTe ---- ---- 0.76 ApproximaTe PercenT of weighT accounTed 67 95 83 for PigmenTaTion yellow yellow- red- brown brown a Error is esTimaTed aT 5 To 7% afTer five deTerminaTions. 'FAP ‘Jv : U CV AH. Ly '3 C61 ,. H ‘v ‘— IL— - I nE’E ‘ 4..., —. and: TENT i” ~q v 36 cells (Fig. 9). Cells had approximaTer 32 proTein bands on 808 gels; molecular weighTs varied from l0,000 To I25,000. Membranes conTained l5 proTein bands and four componenTs accounTed for approximaTer 70% of The membrane proTein. NineTy percenT of The membrane proTeins had molecular weighTs less Than 80,000. These resulTs were virTuaIly idenTicaI To Those obTained wiTh membranes prepared osmoTically. The amino acid composiTion of membranes differed from ThaT of whole cells (Table I). The cells conTained more basic and acidic amino acids, while membranes were enriched in Tyrosine and cysTeine. The amounT of hydro- phobic amino acids was approximaTely The same in boTh membranes and cells. The previously reporTed amino acid composiTion (3) was similar To ThaT of our cells. Lipid ComposiTion. General DisTribuTion. Lipids from I: acidophila were fracTionaTed inTo neuTraIs, glycosyl diglycerides, and phospholipids (I7%, 8%, and 75%, respecTively). Glycolipid and phospholipid Thin layer chromaTography (TLC) paTTerns were similar To Those previously described (2). TLC profiles of The neuTraI fracTion differed from ThaT obTained by LangworThy 3:: El: (2): cholesTerol was undeTecTable and viTamin K2-7 was presenT in large amounTs (33% of neuTraI fracTion). NeuTral lipids were observed which migraTed similarly as componenTs 0, R, S, T, V, and W (2). An addiTionaI componenT appeared halfway beTween V and w wiTh a reTenTion Time approximaTing ThaT of monoeTher sTandards. The reasons for These deviaTions may lie in The growTh condiTions employed. The disTribuTion of lipid moieTies in Thermoplasma acidgphila lipids is IisTed in Table 2. One componenT of The phOSpholipids comprising approximaTely IOZ of The fracTion weighT conTained no phosphaTe (2); iT mosT probably accounTed for The high glycerol To phosphaTe raTio in The fracTion. Carbo- 'nalrgusmriau ." ' Figure 9. 37 SchemaTic profiles of sodium dodecyl sulfaTe polyacryl- amide gels of cell and membnane preparaTions from Thermo- plasma acidophila. Absorbance was measured aT 550 nm. Gels were subjecTed To elecTrophoresis and sTained as de- scribed in MeThods. 38 Molecular Weight T-T-F—l I T I r I 1400 2300 3800 6300 l I 10400 Cells I .I _ 7 IIIIIIIIIII w _A “I! I Membranes W LML Figure 9 pl. :. 5» .r J v. I p . . I v P a .C i . IT: a Fir pv r II. F: P I“ PI .. .3 «Hy v ,T “w” ”Ad ad in.4 3v Q. . . ._ av A H. A» Aid Pl A b 7 S. A a “A" Kw r .p... .3 .5 .5 yr: o .4 M7 5. .3 h I I . Q» ‘ .- .fiu n . w: . 3.3. i 4...“. u p . .I . .7. .u. r J .3 .vl m3 .3 . . . . e... \ . . . . . . . . 3' I . . . I . . .x. .3 . . . w u... u." n:- . u “H” t . ... .- . .. n ..v 3. .o ...v N. . in 92-. K: .6. . u . . .na .n‘ . . TIN 51:7 53rd 39 hydraTe was presenT In all fracTions, as was glycerol. In The glycolipid and phospholipid fracTions more eThers were found concomiTanle wiTh a decrease in faTTy acid esTers. Since eTher bonds are generally more sTable Than esTer bonds, such findings may suggesT ThaT The polar lipid classes are closer To The membrane surface. Only 67% of The neuTraI lipid fracTion was accounTed for by These analyses, buT The remainder consisTed of large amounTs of viTamin K2-7 (30%) and small quanTiTies of non-glyceride componenTs. NineTy-five percenT of The glycolipids and 83% of The phospholipids were accounTed for by weighT. LangworThy e: El: (2) showed ThaT The major componenT of The phospholipid fracTion has Two eTher moieTies, one phosphaTe group, and one hexose uniT per glycerol. Thus, abouT l7% of The glyceryl hydroxyl groups in The phospholipid fracTion were eiTher free (lyso) or were bound To an unidenTified moieTy. However, iT is highly unlikely ThaT Iysophospholipids would be presenT in such large amounTs since They are very disrupTive To membrane sTrucTure. Therefore, IT seems more probable ThaT The unaccounTed for l7% phospholipid weighT (Table 2) conTains groups which have noT yeT been analyzed. FaTTy acid esTers. Three meThods were used To esTablish The presence of The faTTy acid esTers (Table 3), alThough each gave differenT values. SemiquanTiTaTive infrared specTroscopy consisTenle yielded high values, while gas chromaTography-mass specTromeTry gave lower ones. Mass specTromeTry was employed To idenTify all meThyl esTer derivaTives of faTTy acids (Table 4). The esTer disTribuTion is similar in all lipid fracTions excepT for The increase in C faTTy acids and The decrease in C in The l6:|’ 020:0' and C20:l l8:2 phospholipids. Several meThyIaTion Techniques were aTTempTed in order To ensure compleTe derivaTizaTion of faTTy acid esTers. ForTy-eighT hour incubaTion aT 75°C in The presence of meThanolic BF and benzene (see MeThods) gave The 3 " v. DP A6 i~=IOn \ I é-Tral EItlcbl lpid \ 3 All Vali OT ”pl: 5 AVSr‘agE \ \ 40 TABLE 3: FATTY ACID ESTER DETERMINATION IN THERMOPLASMA ACIDOPHILAa FracTion HydroxymaTeb Infra-redb GC-MSb (umoles esTer/mg lipid) NeuTral 3.3 :_0.4 8.2 :_2.0 3.7 :_.8 Glycolipid 7.l :_l.0 l0.4 :_2.0 l.9 :_0.7 Phospholipid 2.4 :_0.2 4.3 :_0.3 l.7 :_0.8 a Alllyalues are expressed in microequivalenTs esTer per milligram o pid. Average of five deTerminaTions. Procedures are described in MeThods. I ”.3.— 41 TABLE 4: PercenTage FaTTy Acid ComposiTion of Thermoplasma acidophila Lipidsa Lipid FracTion FaTTy Acid (mole percenT faTTy acid esTer) Chain LengTh Glycosyl NeuTral diglycerides PhOSpholipids IO 0 0.3 0 9 l l l2 0 l.7 0 8 l 2 l4 0 5.3 5 8 5 I I4 I 2 6 2.4 2 6 l6 0 20.l 2| I I9 4 I6 I I3 7 9.8 l7 3 l8 0 l0.9 ll 5 8 I I8 I 2| 6 20.4 2| 5 l8 2 l7.4 l6 8 2 5 20:0 4.4 7.4 l4.0 20:| 2.0 3.I 7.0 a Average of Three deTerminaTions. VariaTion is beTween 5% and l0% for each faTTy acid IisTed. l ‘;'2' MN" '1‘“! -"‘ A" ZarffiST YIelC "r68, hydrol 2r Q‘Wel’ I‘ml e ‘af‘y acid 83' s‘rafions wer :.17ures. F. iaSTed VITO w :zrbonenTs. lizld exTrac “I presumably T “is respec" acid and/or (5 mg, 2.5 Y blg‘Chaln has analyzg "Bighfs Wer t’anCl‘ies pg- ‘9‘9 Consi; Dre‘I/IOUS T‘“ ChrOmaTogr; (Table 5) phOSphol I; To C29 Bl It, Significa. l NO pL' 42 largesT yields and highly reproducible resulTs. For shorTer incubaTion Times, hydrolysis was incompleTe. The presence of long chain alkyl eThers or oTher moieTies in Thermoplasma acidgphila lipids probably sTabilize The faTTy acid esTers. To insure adequaTe amounTs of maTerials, esTer deTer- minaTions were rouTinely made wiTh lipld exTracTed from ThirTy-six liTre culTures. FurThermore, glassware was acid-cleaned, cells and membranes washed wiTh waTer aT leasT Three Times To prevenT conTaminaTion from media componenTs. OTher workers did noT deTecT faTTy acids in Their I, acidophila lipid exTracTs (2). FaTTy acids were presenT in I: acidophila grown on lipid-free media; presumably The organism is capable of synThesizing iTs own faTTy acids. In This respecT I: acidophila differs from mosT mycoplasmas which require faTTy acid and/or cholesTerol supplemenTaTion for growTh. Oleic acid supplemenTaTion (5 mg, 2.5 mg, or l.25 mg/l) prevenTed growTh of I: acidgphila. Long-Chain Alkyl EThers. The disTribuTion of The alkyl eThers in I: acidophila was analyzed by gas chromaTography—mass SpecTroscopy (Table 5). The molecular weighTs were deTermined wiTh aceTaTe derivaTives which indicaTed mulTiple branches per molecule. The mass specTra of The aceTaTes of Two major eThers were consisTenT wiTh 040:0 and C40:I long-chain compounds in agreemenT wiTh previous reporTs (2). Two smaller componenTs appeared To be C4| and C42 by gas chromaTography reTenTion Time sTudies. The disTribuTion of These eTher moieTies (Table 5) indicaTed ThaT The neuTral lipids conTained predominanle 040:0 while phospholipids and glycolipids conTained an equal amounT of C40:|’ The CI8 To C29 alkanes reporTed previously (2) were noT found To be presenT in SignificanT amounTs. No phosphonaTes or plasmaIOgens were found in The lipids of I, acidophila. 43 TABLE 5: DISTRIBUTION OF ALKYL ETHERS IN THERMOPLASMA ACIDOPHILA LIPIDSa CARBON NUMBER 40:0 40:| 4I:O 42:0 NEUTRALS I.00 :0.|0 0.06 10.00 0.03 10.00 0.00 10.00 GLYCOLIPIDS |.OO 10.04 I.08 10.02 0.22 _+_0.04 0.00 _+_0.00 PHOSPHOLIPIDS I.00:0.0l 0.93:0.0l 0.3l :0.|O 0.06 _+_0.02 a Average of Two deTerminaTions. C 40:0' All quanTiTies normalized To ‘- I I. (I‘ll. ‘I-l-l- 44 Flagella-like FilamenTs. Thermoplasma acidophila appears To be coccoidal in shape and cell diameTers vary beTween 0.5 and 1.0 micron. Some cells have one or Two fiamenTous projechons ( Fig. IO). When cells were viewed individually (Fig. IO), iT became apparenT ThaT each cell had one or Two filamenTs which were ofTen losT from The organism during preparaTion for elecTron microscopy. Washing T. acidophila especially in disTilled waTer, removed These filamenTs from The cells. Flagella-like filamenTs collecTed from These washes as described in The MeThods secTion are shown in Fig. II. Each filamenT is approximaTely 100 R in diameTer and as much as 30,000 R in lengTh. The presence of These filamenTs suggesTs ThaT Thermoplasma acidophila may be an L-form raTher Than a True myc0plasma. Figure IO. 45 Transmission elecTron micrograph of a I, acidoghila cell. This specimen was criTicallv-poinT dried and shadowed wiTh gold-palladium. 46 Figure I0 47 Figure ll. Transmission elecTron micrograph of flagella-like filamenTs collecTed from I, acidophila. FilamenTs were negaTively sTained wiTh phosphoTungsTic acid. 48 Figure II 49 DISCUSSION These sTudies have been underTaken in order To deTermine which characTerisTics of I, acldophila may accounT for iTs Thermophily, acid- ophily, and iTs abiliTy To wiThsTand osmoTic shock despiTe iTs lack of a cell wall. The resulTs indicaTe ThaT These prOperTies may be reIaTed. The lack of cell wall indicaTes ThaT This organism is probably eiTher a myCOpIasma or an L-form. AlThough The classificaTion is sTill unresolv- ed, The exisTing evidence appears more consisTenT wiTh The organism being an L-form. The reasons for The original classificaTion of I. acidophila as a mycoplasma were The following: I). iTs size (0.3-2.0 micron), 2). iTs lack of a cell wall, 3). iTs sensiTiviTy To novobiocin, and 4). iTs low Guanidine plus CyTosine conTenT (l). However, The organism has such a lar- ge range of size, ThaT classificaTion according To size is meaningless. The lack of cell wall is also characTerisTic of L-forms. In addiTion, The pre- sence of flagella-like filamenTs has never been demonsTraTed in mycoplasmas, buT flagella can be reTained in L-forms of flagellaTed bacTeria. Novobio- cin inhibiTs growTh of mycoplasma and I, acldophila, buT The anTibioTic's acTion may be due To iTs specificiTy for cells lacking a cell wall raTher . Than To a specificiTy for mycoplasmas. OTher workers (32) have noT found l.(l), possibly because The in- The low G+C conTenT reporTed by Darland g1_ , iTial isolaTe was noT a pure culTure. FurThermore, Thermoplasma acidophila does noT require faTTy acids or cholesTerol for growTh and is, in facT, in- hibiTed by oleic acid. 50 In spiTe of iTs lack of cell wall, I: acidophila is unusually resisTanT To osmoTic and mechanical sTress. This phenomenon is uncharacTerisTic of mycoplasma. The sTabiliTy of The I: acidophila membrane may be relaTed To iTs exTreme rigidiTy as evidenced by elecTron paramagneTic resonance (EPR) specTra (35). IT is also conceivable ThaT The four major polypepTides (Fig. 9) represenT "sTrucTuraI" elemenT in The proTein-rich (76%) membrane. Brock and Devaux have noTed ThaT acidophily and Thermophily are probably incompaTible, since no organism has been found ThaT grew aT pH 3 or lower and simulTaneously aT TemperaTures higher Than 70°, while bacTeria growing aT alkaline pH can live in boiling or near-boiling waTer. Since I, acidophila grows aT The upper TemperaTure limiT for acidophiles, iT is of inTeresT To invesTigaTe which membrane properTies are mainly deTermined by The low pH and which properTies are relaTed To The high TemperaTure of iTs envrionmenT. Lipids in Thermophiles generally have higher melTing poinTs Than Those of mesophiles; This condiTion is promoTed by increase in IengTh, saTuraTion and branching of The lipids. The lipids of I: acidophila are very long, branched, and 50% saTuraTed and Thus probably have The high melTing poinT characTerisTic of Thermophiles. FurThermore, The high eTher and The low esTer conTenT confer chemical sTabiliTy To The membrane lipid maTrix. Moreover, The high esTer conTenT of The neuTral fracTion would be in accordance wiTh a membrane model wherein The mosT non-polar lipids are relaTively shielded from The harsh environmenT. Enzymes from Thermophiles are generally more resisTanT To heaT denaTuraTion Than Their mesophilic counTerparTs. The reasons for This are noT clear, al- Though hydrophobic forces are almosT always invoked as one of The major 5| sTabilizaTion mechanisms (37). Hydrophobic inTeracTions have maximum sTabiliTy aT 60° (83), which, coincidenTally, is very near To The upper TemperaTure limiT aT which acidophiles have been found To grow. AlThOUgh flagella from Thermophilic organisms have been found To have few charged groups (40), There is a growing body of evidence ThaT ThermosTable enzymes require more acidic amino acids Than Their mesophilic counTerparT (39,44). The solubilizaTion of I: acidophila aT high pH is probably relaTed To The charge disTribuTion of The amino acids (3). AT high pH (greaTer Than 4) carboxyl groups are ionized, resulTing in charge repulsion, desTabilizaTion, and solubilizaTion of The membrane. However, in I: acidophila, These effecTs are minimized, since The organism has only half as many carboxyl and amide groups as mesophilic mycoplasma (3). Moreover, The I: acidophila membrane has fewer -COOH and -NH2 groups Than The ToTal cellular maTerial (Table I). Thus, aT high pH, The cyToplasm of I: acidophila will be solubilized before The membrane proTein, as is indeed found. WiTh These observaTions in mind, iT is possible To SpeculaTe as To whv no acidophiles have been found To grow aT TemperaTures above 70°. As Thermophiles, These organisms may require a high acidic amino acid conTenT; as acidophiles, They require a minimum of ionizable grOUps. Apparenle The highesT TemperaTure aT which boTh requiremenTs are meT is, To a firsT approximaTion, The same one aT which hydrophobic bonds begin To weaken (i.e. 60°). CHAPTER III ELECTRON PARAMAGNETIC RESONANCE STUDIES ON THERMOPLASMA ACIDOPHILA 52 53 INTRODUCTION Thermoplasma acidophila grows opTimaIly aT pH 2 and 590 C (I). This organism has been classified as a mycoplasma, since if lacks a cell wall. Moreover, I, acidophila has several similariTies wiTh HalobacTerium cuTirubrum. BoTh microbes have a low lipid conTenT (less Than 20% of The membrane weighT) and a high incidence of branch- ed alkyl eThers (45, I0). H, cuTirubrum requires a high salT concen- TraTion (greaTer Than 3 M) for growTh and lyses readily when The salT concenTraTion is lowered (below I M) (82). Similarly, I, acidophila has a high proTon requiremenT (greaTer Than IO.4 M; i.e. less Than pH 4) and lyses when The hydrogen ion concenTraTion is decreased (less Than IO-5 M; i.e. greaTer Than pH 5). The proTeins of The membrane are hy- drOphobic and are apparenle desTabilized aT high pH values due To The ionizTion of carboxyl groups (3). FuncTion and sTrucTure of The mem- branes of H, cuTirubrum and several sTrains of mycoplasma have been in- vesTigaTed wiTh elecTron paramagneTic resonance Techniques (epr). Spin label sTudies wiTh H, cuTirubrum revealed The presence of a highly or- dered membrane (46). Herein, we reporT The resulTs of invesTigaTions upon The membrane properTies of I, acidophila. Comparison of These daTa wiTh Those pre- viously for H, cuTirubrunr(46) should yield insighT inTo The abiliTy of The organisms To wiThsTand Their harsh environmenTs. Since 50% of The I, acidophila lipid by weighT is composed of Two long-chain alkyl eThers, 54 This organism should also be an excellenT Tool wiTh which To sTudy co~ operaTive phase TransiTions in membranes. MATERIALS AND METHODS I, acidophila was grown in aeraTed culTures aT pH 2, 560 C. Mem— branes were purified afTer cell lysis aT pH 9.3 (45), and washed Two Times wiTh 40 volumes of disTilled waTer afTer removal from sucnose densiTy gradienTs. ProTein was deTermined by The meThod of Lowry g:_ 91, (I4). Lipids were exTracTed wiTh chloroform: meThanol (Zzl); non- lipid conTaminanTs were removed by Sephadex and silicic acid chromaTo- graphy (45). Lipids were assayed colorimeTrlcaIly by a modificaTion of The meThod of SaiTo and SaTo (24). Organic solvenTs were redisTil- led prior To use. All glassware employed in lipid analyses was acid- cleaned. FaTTy acid spin labels were purchased from Synvar (Palo AlTo, Calif.) and were of The general formula: CHa-(CHQM — c -(CH,),,_, coow 'fl n-niTroxy-sTearaTe (nNS) 55 I6NS = 2-(l4-carboxyTeTradecyl)-2-eThyl-4,4-dlmeThyl-3- oxazolidinyloxyl IZNS = 2-(I0-carboxyldecyl)-2-hexyl-4,4-dimeThyl-3- oxazolidinyloxyl 5N5 8 2-(3—carboxyldecyl)-4,4-dimeThyl-Z-Tridecyl-S- oxazolidinyloxyl Spin labelled cholesTane (4',4'-dimeThyl-spiro-(cholesTane-3-2'-(I',3'- oxazolindine-3'-oxyl))) had The following sTrucTure: CK. . a... 7<_O 4, AliquoTs from a sTock soluTion of spin label dissolved in hexane were measured inTo small glass TesT Tubes; hexane was removed by evap- oraTion. Membrane vesicles were suspended eiTher in waTer aT pH 6 or in T-buffer (45) aT pH 2, and added To The spin label. The mixTure was Then sonicaTed for I0 min. on a Branson sonicaTor. The proTein con- cenTraTion of The membrane vesicles was beTween l0 and 20 mg/ml. Un- less oTherwise sTaTed The spin label concenTraTion approximaTed 0.I% of 56 The lipid weighT of The vesicles. Assuming an average molecular weighT of ISOO for The lipid (based on The composiTion of The major dieTher phospholipid)l2) The lipid To sTearaTe spin label molar raTio was abouT 250:l. All elecTron paramagneTic resonance (epr) specTra were deTermined on a Varian V-4502-I5 specTrophoTomeTer equipped wiTh a Varian variable- TemperaTure accessory, V-4540. An aqueous sample cell was used Thro- ughouT. The roTaTional moTion of The spin label was calculaTed from The following equaTion (47): IO r" = 6.5 x Io‘ wo {(ho/h_,)'/2 -l} (I) The order paramefer Sn was calculaTed as previously described (46, 48- 50): Sn = 0.568 (T'l| " T'l)/a' (2) where a = I/3 (T' + 2T'1) ll Samples were prepared for elecTron microscopy wiTh l.5% phosphoTungsTic acid, pH 7.0. Specimens were viewed on a Phiips 300 Transmission Elec- Tron Ml crosoope. RESULTS Typical epr specTra of T. acidophila membranes in disTilled waTer (pH 6.0) aT 560 C are shown in Fig. I2. Since ascorbic acid does noT . §t1 i 11 Figure |2. 57 Elecfron paramagneTic resonance specTra of faTTy acid soin labels in I, acidophila membranes aT pH 2 and 560 C. 2Tu' k l H 06 ~——2T4.’—-' We 2 l..___,, (5;? 7“: [MNS / /I\/ 16NS 59 reduce The signal heighT, if is assumed ThaT The faTTy acid spin label has been incorporaTed info The membrane lipid maTrix. Ascorbic acid would desTroy unincorporaTed spin label or ThaT exposed on The surface (5|). lmmobilizaTion of The spin label is evidenT aT all TemperaTures TesTed, buT is especially pronounced aT low TemperaTures (Table 6). This lmmobilizaTin ls indicaTive of a rigid membrane sTrucTure. OTher invesTigaTors have found ThaT as The niTroxide group is placed closer To The hydrocarbon and of The sTearaTe molecule, The roTaTional free- dom of The group usually increases (5|), since The free radical pene- TraTes deeper inTo The hydrocarbon region of The membrane bilayer. AT TemperaTures lower Than 400 C, 5N5 in I, acidophila membranes reflecTs greaTer or equivalenT membrane rigidiTy as compared To ThaT seen by The IZNS label (Table 6 and Fig. l3). However, above 40° C, The IZNS label apparenle experiences an environmenT wiTh greaTer rigidiTy Than ThaT of The 5N5 label (Table 6 and Fig. l3). The TemperaTure dependence of I, acidophila membrane fluidiTy is il- lusTraTed In Fig. l3. When The membranes were labeled aT pH 6.0 wiTh 5N5, TransiTions were observed aT l5° and 45° C. No differences were observed when membranes labelled aT pH 6.0 were resuspended in pH 2.0 buffer. However, if labelling was performed aT pH 2.0, The 450 C Tran- slTion was abolished and anofher TransiTion appeared aT 60°C. This sug- gesTs ThaT The pH aT The Time of labelling deTermines The membrane do- main To which The 5N8 spin label migraTes. The IZNS spin label consisTenle indicaTed a TransiTion Tempera- Ture aT 60° C, regardless of The pH aT The Time of labelling. A TransiTion 6O TABLE 6. SPECTRAL PARAMETERS OF FATTY ACID SPIN LABELS INCORPORATED INTO THERMOPLASMA ACIDOPHILA MEMBRANESa Lang_ TemperaTure Sn 2} ng|(G) In (nsec 5N5 37° i.5 l5.4 57.2 --- l2NS 37° --- --- 57.2 --- l6NS 37° --- --- 53.8 8.3 5N8 55° 0.7l l4.4 55.5 5.6 IZNS 55° 0.71 l4.5 --- ll.7 I6NS 55° --- --- 45.2 2.2 5N5 80° 0.54 l6.8 47.9 2.9 IZNS 80° 0.54 l4.3 --- 5.6 I6NS 80° --- --- --- 1.4 aMembranes were labelled and specTra were Taken aT pH6. Where no values for a parTicular parameTer are given, The specTral shape was such ThaT measuremenT and calculaTion of ThaT parameTer was noT possible. Error is esTimaTed aT l0% for all parameTers excepT ZTII' where The error is wiThin 3%. 6| Figure l3. TemperaTure-dependence of 2Tll or T in Spin-labeled T. acidOphila membranes. a. 2T" as a funcTion of TemperaTure for IZNS labelled I, acidophila membranes. O-—-O pH 6; X-—-X pH 2. b. 2Tll as a funcTion of TemperaTure for 5N5 labelled I, acidophila membranes. O-——O pH 6; X-—-X pH 2. c. T as a funcTion of reciprocal absoluTe TemperaTure for I6NS labelled I, acidoghila membranes. O--O pH 6. 62a cm 00 0V om m_ 6Lsm_u o om 00 0V on nV on no 00 no :.—.N 62b no:c_+coo m_ 0L:m_u g Aommp: 63 also occurred aT i5° c. The TranslTion aT 600 c was also observed wiTh l6NS; however, The lower TemperaTure TransiTion oould noT be deTecTed since specTra of Those low TemperaTures did noT allow calculafion of T values. The SpecTra of I6NS labelled membranes (Fig. l2) were obTained wiTh membranes which had been sTored aT 4°C for four days or more. Fresh preparaTions were found To produce specTra such as shown in Fig. l4c. This Type of specTrum appears To be oomposiTe of The normal I6NS spec- Trum (Fig. l4b) and one which can be generafed by placing The spin la- bel groups in close pnoximiTy To each oTher (Fig. l4d). Earlier inves- TigaTors (52-54) observed This Type of specTrum only when The membranes were highly doped (greaTer Than l0%) wiTh spin label. The specTrum shown in Fig. l4c was obTained only wiTh fresh membrane preparaTions and was sTable for Two days af 50 C. When The TemperaTure of These la- belled membranes was raised above 650 C, however, The specTrum oonverT- ed To a virTually compleTe spin-spin inTeracTion specTrum (Fig. l4d). This Irreversible conversion occurred wiThin l0 To 60 min., depending upon The TemperaTure and The sample used. The exacT TemperaTure aT which The conversion began and The Time Taken for compleTe conversion varied (15°C, :30 min.) wiTh The preparaTion. This conversion caused major al— TeraTions in The membranes (Fig. IS). The membrane vesicles decreased in size, reTained The phosphoTungsTic acid sTain, and aggregaTed. Since The parTiaI spin-spin inTeracTion SpecTrum indicaTed ThaT The anroxlde radicals were in close proximiTy (Fig. l4c), if was of inTer- esT To deTermine wheTher some of The I6NS spin labels were forming a Figure I4. 64 EPR specTra of I6NS under vari0us condiTions. A. B. I6NS in waTer dispersion aT 20° C. I6NS bound To four-day-old I, acidoghila membranes aT 20° C, pH 6. I6NS bound To one—day-old I, acidophila membranes aT 20° 0, pH 6. I6NS bound To one-dav-old I, acidoohila membranes subjecTed To 800 C for one hour and cooled To 20° C. The pH is 6. “a I I fill: Figure I5. 66 EIecTron micrographs of I, acidophila membranes. A. NegaTively-sTained naTive membranes. 8. NegaTively-sTained membranes afTer one hour aT 80° C. 67 Figure )5 68 clusTer due To low solubiliTy in The membrane lipid. If This were The case, one mighT expecT ThaT furTher addiTion of I6NS label would increase The inTeracTion beTween spin labels. However, This was noT observed. AddiTion of more l6NS resulTed in a specTrum wiTh less in- TeracTion characTerisTics (Fig. l6). Thus, iT appeared ThaT There were a limiTed number of siTes available wherein I6NS free radicals were able To inTeracT wiTh each oTher. Apparenle, These siTes were The firsT ones filled or modified in The presence of excess I6NS. Ad- diTion of unlabelled cholesTerol also reduced The inTeracTion beTween I6NS molecules in The labelled membranes (Fig. l7). T. acidophila membranes labelled wiTh The spin label cholesTane exhibiTed a specTrum indicaTive of close inTeracTion beTween spin la- bels (Fig. l8). LiTTle change was noTed in This specTrum as a func- Tion of TemperaTure from 50-600 C. DISCUSSION Previous invesTigaTions wiTh physical meThods supporT The Theory of a bilayer lipid organizaTion in biological membranes (55-64). Fnom order parameTer sTudies on faTTy acids wiTh Spin labels aT differenT posiTions along The alkyl chains, rigidiTy gradienTs have been deTecT- ed in membrane lipids (62, 65-67). In esTablished bilayer membranes such a gradienT has been demonsTraTed and iTs presence has been used To infer a bilayer sTrucTure in less sTudied sysTems (68). AT Temp- eraTures below 400 C, 5N5 appears To sense a similar environmenT as l2NS, which is locaTed in a more rigid lipid region Than I6NS in ““3 &'\"’ .‘ _n. _gudr-r' Figure I6. 69 ConcenTraTion dependence of EPR specTra of I6NS labelled I, acidophila membranes. ———-0ne-day-old membranes aT 20° 0, pH 6. The label: lipid raTio is 4 X l0-3. --- The above preparaTion wiTh The addiTion of I6NS label giving a final label: lipid rafio of 8 X l0-3. 0-0 The above preparaTion wiTh The addiTion of I6NS label giving a final label: lipid raTio of 24 x l0‘3. 7O o_ opao_d muav— “a 7| Figure l7. EffecT of cholesTerol addiTion on I6NS EPR specTra of labelled I, acidophila membranes. ——-0ne-day-old membranes labelled wiTh I6NS aT pH 6 and 30°C. --- The above preparaTion afTer The addiTion of non-labelled cholesTerol in amounTs equal To five Times The spin label concenTraTion (w/w). 72 Figure 17 73 I, acidophila membrane vesicles (Table 6 and Fig. l3). Similar obser- vaTions aT These TemperaTures have been made wiTh H, cuTirubrum (46). Above 40° C, however, l2NS in I, acidophila membranes was apparenle in a more rigid environmenT Than SNS and I6NS labels. This behavior has noT been observed in esTablished bilyer sysTems. The lipid regions of I, acidophila membranes may besT be describ- ed as "rigid", since for all labels TesTed, boTh The order and roTa- Tional parameTers indicaTe less fluidiTy Than has been reporTed for H, cuTirubrum, The mosT highly ordered membrane known. This rigidiTy appears consisTenT wiTh The high resisTance To osmoTic shock noTed in I, acidgphila (4). No osmoTic procedure has ever been reporTed To lyse more Than 50% of These organisms (3, 4) alThough iT lacks a cell wall. Evidenle, in I, acidophila, The membrane is sTrong enough To proTecT The cells againsT osmoTic shock possibly by mainTaining a greaTer rigi- diTy Than ThaT found in The membranes of oTher organisms sensiTive To osmoTic changes. The presence of high amounTs of branched long-chain alkyl eThers (molecular weighT = 532 To 560) and The large proTein: li- pid raTio (4:l, w/w) probably conTribuTe To The high rigidiTy observ- ed in I, acidophila. Sharp TransiTions were deTecTed in I, acidophila membranes wiTh all sTearaTe spin labels TesTed. This observaTion is consisTenT wiTh The relaTive lipid homogeneiTy (over 50$ of The lipid weighT is com- posed of Two alkyl eThers (45)) and wiTh The assumpTion ThaT The faTTy acid labels are locaTed in The membrane lipids (5i). Fresh I, acidophila membrane preparaTions labelled wiTh I6NS re- sulTed in specTra (Fig. l4c) similar To Those observed in phosphafidyl 74 efhanolamine vesicles highly doped (more Than 35le2 mole sTeroid/ mole lipid) wiTh spin-label cholesTane (52-54). Upon applicaTion of Spin-label To a limiTed region of an arTificial lipid bilayer, laTer- al diffusion was accompanied by a gradual change in specTral Type; namely, from exchange specTra (Fig. l4d) To The usual Three line paT- Tern (Fig. I40) (69). AlThough The molar raTio of spin-label To lipid is only 4 X ID"3 in our experimenTs, inTeracTion is unquesTionably ob— served (Fig. I4c). The decrease in inTeracTion observed upon furTher addiTion of I6NS is inconsisTenT wiTh The concepT of a uniform bilay— er or a spin label pool devoid of membrane lipids, since eiTher hypo— Thesis would predicT sTronger inTeracTion wiTh increased concenTraTion of spin label. These findings could possibly be explained by assum- ing ThaT aT IeasT some of The lipids in I, acidophila are arranged in a clusTer, perhaps as illusTraTed in Fig. l9. AddiTion of excess la- bel or of unlabelled cholesTerol could cause expansion or possibly dis- rupTion of The clusTer, resulTing in less inTeracTion beTween labels (Figs. l6 and I7). AlTernaTely, only a few clusTer siTes may be avail- able where l6NS Spin labels can inTeracT wiTh each oTher. Once These clusTer siTes are filled, spin labels migraTe To oTher membrane regions (9. g. bilayer), Thus producing a specTrum wiTh less inTeracTion char- acTerisTics. lf membranes are labelled only wiTh a spin-label choles- Tane probe, sTrong inTeracTion is observed in I, acidophila membranes. This mighT be expecTed since non-polar molecules would prefer To mi- graTe inTo The cenTer of The lipid clusTer. Hydrophobic proTeins mighT also peneTraTe inTo regions of This clusTer, increasing The rigidiTy of l1 1 "AU . I 1"] 01‘ Figure I8. 75 ElecTnon paramagneTic resonance specTra of cholesTane spin label in one-day-old I, acidophila membranes aT pH 6, 20°C. The molar label: lipid raTio is 4 x l0’3. 76 a. opsa_d mu nvp i.I I... Figure I9. 77 Membrane models. A. SchemaTic drawing of a bilayer membrane illusTraTing The disTance beTween evenly disTribuTed spin labels. 8. SchemaTic drawing showing a lipid clusTer model mem— brane illusTraTing The proximiTy of evenly disTribuTed spin labels. The number of lipid molecules in each model is The same. 79 The inferior of The clusTer, and acoounTlng for The observaTion ThaT regions near The l2NS label are less fluid Than Those of 5NS aT Temp- eraTures above 400 C. Thus, The resulTs presenTed herein indicaTe ThaT I, acidophila membranes are The leasT fluid biological membranes yeT reporTed. Much of The spin label behavior in The lipid maTrix appears To be more con- sisTenT wiTh lipid clusTers Than wiTh a lipid bilayer. CHAPTER IV BIBLIOGRAPHY 80 8| BIBLIOGRAPHY Darland, 6., Brock, T., Samsonoff, W., and ConTi, S. (I970) Science I29, l4l6. LangworThy, T., SmiTh, P., and Mayberry, W. (I972) J. BacTeriol. ll2, ll93. SmiTh, P., LangworThy, T., Mayberry, W., and Hougland, A. (I973) J. BacTeriol. ll6, l0l9. Belly, R., and Brock, T. (I972) J. Gen. Microbiol. 23, 465. Henrikson, C. and Panos, C. (I969) Biochem. 8, 646. Pollack, J., Razin, S., Pollack, M., and Cleverdon, R. (l965) Life Sci. 4, 973. Rouser, G., KriTchevsky, C., and YamamoTo, A. (I967) in "Lipid ChromaTographic Analysis" Vol. I (Ed. G. MarineTTi) Marcel Dekker, lnc., New York. Flexer, S. and Wein, J. (I963) Biochim. Biophys. AcTa IQ, 423. Jardon, T. and Rider, F. (l965) Anal. Biochem. IQ, l93. KaTes, M., Yengovan, L., and SasTry, P. (l965) Biochim. Biophys. AcTa 28, 252. Kellenberger, E., RyTer, A., and Sechaud, J. (I958) J. Biophys. Biochim. CyTol. fl, 67l. Spurr, A. (I969) J. UlTra. Res. 29, 3I. Venable, A. and Coggeshall, G. (l965) J. Cell Biol. 25, 407. Lowry, 0., Rosenbrough, N., Farr, A., and Randall, R. (I95l) J. Biol. Chem. I93, 265. BarTleTT, G. (I959) J. Biol. Chem. 234, 466. AppelqvisT, L. (I972) J. Lipid Res. I3, I46. RapporT, M. and Alonzo, N. (I955) J. Biol. Chem. 2l7, I99. DITTmer, J. and Wells, M. (I969) in "MeThods in Enzymology" Vol. l4 (ed. J. LowensTein) Academic Press, New York. 20. 2|. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3|. 32. 33. 34. 35. 36. 37. 38. 39. 82 BurTon, K. (I956) Biochem J. 22, 3I5. Aalbers, J. and Bieber, L. (I968) Anal. Biochem. 22, 443. Klenk, E. and Friedricks, E. (I952) Z. Physiol. Chem. 290,I69. Skidmore, W. and EnTenmorn, C. (l962) J. Lipid Res. 2, 47I. RieTsema, R. (I954) Anal. Chem. 22, 960. SaiTo, K. and SaTo, K. (I966) J. Biochem. 22, 6l9. Dubois, M., Gilles, K., HamilTon, J., Rebers, P., and SmiTh, F. (I956) Anal. Chem. 22, 350. Wells, M. and DiTTmer, J. (l965) Biochem. 2, 2459. Davis, B. (I964) Proc. NaTl. Acad. Sci. U. S. l l, 404. Malik, N. and Barrie, A. (l972) Anal. Biochem. 49, I73. Blakesly, R. (I974) Thesis, BiochemisTry DeparTmenT, Michigan STaTe UniversiTy., EasT Lansing, Michigan 48823. Nelson, C. (I968) Lipids 2, 267. Razin, 5. (I967) Ann. N. Y. Acad. Sci. l43, ll5. FreunaT, S. (l972) in "PaThogenic Mycoplasma" AssociaTe Science Publishing Co., Ciba FoundaTion, New York. Chan, M., Virmani, Y., Himes, R., and Akagi, J. (I972) Ann. MeeT. Amer. Soc. Microbiol., 72 nd, Philadelphia. RuwarT, M. SmiTh, C., and Haug, A. (I973) in preparaTion. Chan, M., Virmani, Y., Himes, R., and Akagi, J. (l972) Conf. Ex- Treme Environ., Ames Res. CenTer, NASA, MoffeTT Field, Calif., June. Bmck, T. and Darland, G. (T970) Science I69, I3l6. SingleTon, R. and Amelunxem, R. (l973) BacT. Rev. 22, 320. MalleT, G. and Koffler, H. (1957) Arch. Biochem. Biophys. 21, 254. Campbell, L. and Cleveland, P. (I96I) J. Biol. Chem. 236, 2966. ll 40. 4|. 42. 43. 44. 45. 46. 47. 48. 49. 50. 5|. 52. 53. 54. 55. 56. 57. 58. 59. 60. 6|. 83 Campbell, L. and Manning, G. (I96I) J. Biol. Chem. 236, 2962. SingleTon, R., Kimmel, J., and Amelixer, R. (I969) J. Biol. Chem. 244, I623. Tanaka, M., Haniu, M., MaTsueda, C., Yasunobu, K., Himes, R., Akagi, J., Barnes, E., and DevanThan, T. (l97l) J.B.C. 246, 3953. Barnes, L. and STellwagan, E. (l973) Biochem. I2, l559. STellwagen, E., Cronlund, M., and Barnes, L. (l973) Biochem. l2, l552. RuwarT, M. and Haug, A. (l973) in preparaTion. Esser, A. and Lanyi, J. (l973) Biochem. I2, I933. KeiTh, A., Bulfield, G., and Sniper, W. (I970) Biophys. J. I2, 6I8. Seelig, J. (I970) J. Am. Chem. Soc. 22, 388i. Hubbel, W. and McConnell, H. (l97l) J. Am. Chem. Soc. 22, 3l4. McFarland, 8. (I973) in "MeThods in Enzymology? in press. McConnell, H. M. and McFarland, 8. (I970) QuarT. Rev. Biophys. 3, 9|. Trauble, H. and Sackmann, E. (l972) J. Am. Chem. Soc. 24, 4499. Sackmann, E. and Trauble, H. (l972) J. Am. Chem. Soc. 25, 4482. Sackmann, E. and Trauble, H. (l972) J. Am. Chem. Soc. 22, 4492. BuTler, K., Dugas, H., SmiTh, l., and Schneider, H.,Biochem. Bio- phys. Res. Comm. (I970) 22, 770. WorThingTon, C. (l97l) Fed. Proc. 22, 57. WorThingTon, C. and Blaurock, A. (I969) Biochim. Biophys. AcTa I73, 427. Blaurock, A. (l97l) J. Mol. Biol. 29, 35. Engelman, 0. (I970) J. Mol. Biol. 31, HS. Engelman, D. (l97l) J. MOI. Biol. 22, l53. LiberTinI, L., Waggoner, A., JosT, P., and GriffiTh, 0. (I969) Proc. NaT. Acad. Sci. U. S. 21, I3. 62. 63. 64. 65. 66. 67. 68. 69. 70. 7|. 72. 73. 74. 75. 76. 77. 78. 79. 80. 8|. 84 Hubbell, W. and McConnell, H. (I969) Proc. NaT. Acad. Sci. U. S. 95-, 20. Hsia, J., Schneider, H., and SmiTh, I. (I970) Biochim. Biophys. AcTa 202, 399. BuTler, K., SmiTh, l. and Schneider, H. (I970) Biochim. Biophys. AcTa 2l9, 5l4. JosT, P., LiberTini, L., HeberT, V., and GriffiTh, 0. (l97l) J. Mol. Biol. 22, 77. RoTTem, S. Hubbell, W., Hayflick, L., and McConnell, H. (I970) Biochim. Biophys. AcTa 2I9, |04. Simpkins, H., Panko, E., and Tay, S. (l97l) J. Mem. Biol. 2, 334. Landsberger, F., Compans, R., Choppin, T., and Lenard, J. (l973) Biochem. l2, 4498. Devaux, P. and McConnell, H. (I972) J. Am. Chem. Soc. 22, 4475. RoThfield, L. (l97l) "STrucTure and FuncTion of Biological Mem- branes", Academic Press, New York. RapporT, M. and Graf, L. (I969) Progr. Allergy I2, 273. Brock, T. (I967) Science l58, IOI2. Davson, H. and Danielli, J. (I952) "PermeabiliTy of NaTural Mem- branes", 2 nd ed., Cambridge UniversiTy Press, London: RoberTson, J. (I964) in "Cellular Membranes in DevelopmenT", (ed. Locke, J.), Academic Press, New York. Lenard, J. and Singer, S. (I966) Proc. NaT. Acad. Sci. U. S. 22, I828. Deamer, D. W. (I970) Bioengineering I, 237. SjosTrand, F. (I963) J. UlTra. Res. 2, 340. Lucy, J. (l970) NaTure 227, 8l5. Singer, 5. (I97l) in "STrucTure and FuncTion of Biological Mem- branes", (ed. RoThfield, L.), Academic Press, New York. RoTTem, S. and Razin, S. (I966) J. BacTeriol. 22, 7l4. Lanyi, J. (|97I) J. Biol. Chem. 246, 4552. . . . ?.ior 1w“... ~ 85 82. Larsen, H. (I967) Advan. Microbiol. Physiol. I, 97. 83. Scheraga, H. (I963) in "The ProTeins", Vol. I (ed. H. NeuraTh), p. 477, Academic Press, New York.