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STRUCTURE OF A GLYCEROL ETHER LIPID AND THE PURIFICATION AND PARTIAL CHARACTERIZATION OF A GLYCOPROTEIN FROM THE PLASMA MEMBRANE OF THERADPLASMA ACIDOPHILUM By Li Lillian Yang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements fbr the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1978 ABSTRACT STRUCTURE OF A GLYCEROL ETHER LIPID AND THE PURIFICATION AND PARTIAL CHARACTERIZATION OF A GLYCOPROTEIN FROM THE PLASMA MEMBRANE OF THERMOPLASIM ACIDOPHILUM By Li Lillian Yang Thermoplasma acidbphilum, a mycoplasma-like organism, grows Opti- mally at pH 2 and 56°C. Since this organism does not have a cell wall, the plasma membrane directly interfaces with the harsh environment. The molecular mechanisms underlying temperature adaptation of micro- organisms are not well understood although much information is available on adaptational changes in lipid side chain structures. With respect to physico-chemical parameters, such as membrane lipid fluidity and lipid phase transition temperature, the growth temperature range of a microorganism seems to depend on the ability to regulate its membrane lipid fluidity within a certain range. The first part of this disser- tation is focused on answering the question of what are the key para- meters of the structural and functional relationships of the lipids in the T. acidbphilum membrane that will allow adaptation to growth at high temperature and low pH. Five spectroscopic techniques: infrared, proton magnetic resonance, gas chromatography-mass spectrometry, elec- tron impact ionization-mass Spectrometry, and carbon 13-nuclear mag- netic resonance, were used to elucidate the lipid side chain structure. Li Lillian Yang The results supported the structure of two repetitively methyl branched C40 side chains that were ether-linked to two glycerol molecules. 80% of the lipid analyzed had the cyclopentane cyclization structure in their side chains. The branched alkyl side chains and their ether linkages with the glycerol backbone have survival value for this ther- mophilic and acidophilic organism. To further understand the tempera- ture effects of T. acidbphilum, cells were adapted to growth at 37°C. Cells grown at 37°C contained lipids with 42% more cyclopentane cycli- zation than the 56°C-grown cells. In 37°C—grown cells, phospholipid and serine content decreased by about 10%, carbohydrate content in- creased by 5?. Electron paramagnetic resonance studies demonstrated an increase in membrane lipid fluidity of 37°C-grown cells with an upper transition temperature at 35°C which was shifted down by 10°C compared with cells grown at 56°C. Membrane-bound ATPase activities also indi- cated similar changes upon adaptation. There is a close correlation between membrane fluidity and physiological functioning of this membrane— bound enzyme. The second part of this dissertation is dedicated to the partial characterization of a procaryotic glchprotein found in the T. acido- philum membrane. The purified membrane glycoprotein from T. acidbphi- Zum had an apparent molecular weight of 152,000 daltons with less than 10% carbohydrate content (w/w). The carbohydrate moiety consisted mainly of mannose residues with branched a 1+2 linkages at the non- reducing ends of the glycopeptide. The reducing end was an N-glycosi— dic linkage between the amino acid asparagine and N-acetylglucosamine. The glycoprotein accounted for 40% (w/w) of the total membrane proteins. Li Lillian Yang The nonreducing ends of the glycopeptide showed a highly branched pattern which extended like a protective coat over the entire cell surface. The hydrophobic interaction between carbohydrates and protein prevents the membrane proteins from thermal inactivation. The stereochemistry and the confbrmation of the carbohydrate chains in conjunction with water turgor may contribute to the rigidity of the membrane and the cation binding. dedicated to all woman warriors ii ACKNOWLEDGEMENTS I wish to thank my major professor, Dr. Alfred Haug, for his guidance, encouragement and friendship during these four years of graduate study. I wish to thank my guidance committee members, especially Dr. Derek T. A. Lamport for numerous discussions and support. Special thanks are extended to Dr. Charles C. Sweeley. Dr. Sun-sang J. Sung, and their colleagues for assistance in mass spectrometry. I wish to thank Chuck Caldwell for his understanding, support and doing some of the figures. This research was supported by the Department of Energy Contract EY—76-C-OZ-1338. iii TABLE OF CONTENTS LIST OF TABLES ....................... ........................ LIST OF FIGURES..... ...... ..................... ............ .. LIST OF ABBREVIATIONS................ ..... ...... ......... .... CHAPTER I GENERAL INTRODUCTION ............ . .................. CHAPTER 2 MATERIALS AND METHODS........ ...... . ........ . ...... MATERIALS. ......... . ..... ........... ..... ............... METHODS. 0000000000 000.00..O...OOOOOOOOOOOOOOOOOOOOOOI er.th Of CE‘iSo 00.0.00...OOOOOOOOOOOOOIOO ..... 0000 Membrane Preparation. ............................. Extraction of Lipids............................... Silicic Acid Column Chromatography................. Preparative Thin-Layer Chromatography ...... . ....... Transmethylation of Lipids. ....................... Methanolysis. ... .............. ...... ...... . ...... Degradation of Glycerol Ethers (i) Alkyl Chloride Derivatives....... ......... (ii) Alkyl Alcohol Derivatives..... ...... ..... Determination of C-methyl Groups................... Gas-Liquid Chromatography (6C) for Membrane Lipid Derivatives............................. Combined Gas Chromatography-Mass Spectrometry...... Electron Impact Ionization-Mass Spectrometry....... Infrared Spectroscopy........................ ...... Nuclear Magnetic Resonance Spectroscopy i) Proton NMR................................ ii) Carbon IS-NMR.......... ..... . ..... ....... ATPase Assay....................................... Buffer Systems for $05 Gel Electrophoresis and Sample Preparation............ ......... ....... Electron Paramagnetic Resonance (EPR).............. Isolation and Purification of Membrane Glycoprotein (i) by preparative slab gel electrophoresis. (ii) by phenol extraction. .................... (iii) by Sepharose 48 Column Chromatography... (iv) by Con A- Sepharose Column Chromatography. Methanolysis of Carbohydrates..................... . Permethylation for Linkage Studies................. iv TABLE OF CONTENTS (cont'd) Anhydrous Hydrogen Fluoride Deglycosylation .......... Amino Acid Analysis....................... ......... .. Treatments of Glyc0protein with Glycosidases (i) a-mannosidase digestion. .................... (ii) B-glucosidase digestion.................... (iii) 6- -galactosidase digestion. ................ (iv) endo-glycosidase H digestion. ...... ....... . Analytical Methods. . . .................... ......... CHAPTER 3 STRUCTURE OF LONG-CHAIN GLYCEROL ETHERS IN PLASMA MEMBRANE FROM THEMPLASIM ACIDOPHILUM GROWN AT 56°Cooooooo OOOOOOOOOOOO 00 00000000 Introduction .............................................. Results. . . .... .......... .... Infrared Spectroscopic Studies of Compound (I)....... Proton NMR Studies of Compound (I). ... ............. 6C Studies of Alkyl Chloride and Alkyl Alcohol Deriva- tives of Side Chains ..... . .................. GC- MS Studies of the Alkyl Chloride Derivatives. ..... GC-MS Studies of Alkyl Alcohol Derivatives. .......... EI- MS Studies of Compound (I). .......... ..... .. ...... Carbon 13- NMR Studies of Compound (I). .......... Discussion....... ....... ........................... ....... CHAPTER 4 CHANGES OF MEMBRANE PROPERTIES IN mamamsm acmoparwu UPON LOH TEMPERATURE (37°C) ADAPTATION ....... ... .............. .. ........ Introduction ...... . ............... .. ...................... Resu‘ts. . 0000000 .... ......... ......000.. 000000 .... ........ Lipids.... ................................. .... Membrane- Bound Adenosine Triphosphotase... ...... ..... EPR Stadies0..0.0 ........ 0.0 000000000000 0 000000000000 D‘scuSSTon 00000000 .0... 0000000000 0000......0. 000000000 .000 CHAPTER 5 PARTIAL CHARACTERIZATION OF A MEMBRANE GLYCOPROTEIN FROM THEWPWM ACIwPHILWO000.000.00.00.0000 IntrOduction.00 ..... ...... ....... 00........00.0..00. ..... 0 Resu‘ts 00000000 00....0000 0.0.0.... ......... Isolation and Purification of the Glycoprotein....... Pronase Digestion of the Glycoprotein....... . ....... . PAGE 22 22 22 23 23 23 23 24 24 30 35 39 52 60 60 TABLE OF CONTENTS (cont'd) PAGE Determination of Carbohydrate Composition by Methanolysis ..... .............. ...... ..... ......... 83 Amino Acid Analysis of the Glycoprotein........... ...... 83 Glycosidic Linkage Studies of the Glycoprotein..... ..... 83 Digestions of the Glycopeptide by Exo- and Endo- glycosidases........ .................. . .......... 87 Permethylation Studies of the Glycopeptide.. ... 93 Effect of Bacitracin on Cell Growth and Glycoprotein Structure. ........................ ................ 95 Discussion ....... . ..... ...... .................. . ............. 108 References .................................. . ..................... 112 vi LIST OF TABLES TABLE PAGE 1. Proton NMR Chemical Shifts 6 (in ppm) of Glycerol Ether Downfield from THS.... ............................ 31 2. Carbon 13-NMR Chemical Shifts 6 (in ppm) of Glycerol Ether. .0000000000000 ......00000.0000 00000000 . 00000000 57 3. Side Chain Comparison of 56°C- and 37°C-grown Cells as Determined by GC and GC-MS .............. ...... .......... 62 4. Amino Acid Composition of the Purified Membrane Glyco- protein from T. acidbphilum... ...... ...... ......... ..... 86 5. Molar Ratios of Carbohydrate Residues from Purified Glycoprotein and after Treatments with Glycosidases ..... 92 6. Molar Ratios of Partially Methylated Alditol Acetates of Mannose, Glucose, Galactose and Glucosamine Obtained by Permethylation of the Purified Glycopeptide .......... 94 vii LIST OF FIGURES FIGURE PAGE l. Chemical Degradation Scheme of the Glycerol Ether Lipids from the Membrane of Thermoplaama acidophilwn.. ...... 27 2. Infrared Spectrum of Compound (I) (Figure 1) ..... .. ....... 28 3. Proton Magnetic Resonance Spectrum of Compound (I) (Figure1).................... ooooo oooooo ooooooo ea... 29 4. Gas Chromatogram of Alkyl Chloride Derivative of Glycerol Ether Lipids from T. acidophilic» Menbrane............ 32 5. Gas Chromatogram of Alkyl Alcohol Derivative of Glycerol Ether Lipids from T. aaidbphilum Membrane............ 33 6. Mass Spectra of the High-Mass Region of 60 Component 4,5 and 6 of Alkyl Chloride Derivatives........ ....... ... 36 7. Mass Spectrum of the First Component of the GC Trace of the Alkyl Alcohol Derivative of T. acidbphilum Lipid Side Chains0.00000.......0.0..0..000.........0. 4o 8. Assignments of the Ion Fragments of Mass Spectrum Figure 70..0.....00.0.0000000.00.000.00.0.00000.0.... 42 9. Mass Spectrum of the Second Component of the GC Trace of the Alkyl Alcohol Derivatives of T. acidbphilum Lipid Side Chains. .. .............. ....... ...... 44 10. Assignments of the Ion Fragments of Mass Spectrum F‘gure 9......00....000.000.00.000000.000.0.00.0.0... 46 11. Mass Spectrum of the Third GC Component of the Alkyl Alcohol Derivatives of the T; acidophilum Lipid Side Chains...000......0000..00000000 .......... 48 12. Assignments of the Ion Fragments of Mass Spectrum Figure 11.... ..... ......................... .......... 50 13. Carbon 13-NMR Spectrum and the Assignment of the Glycerol Ether Molecule from T. acidbphilum "embrane Lip‘d00000.......0...00...0........000...... 55 viii FIGURE 14. Arrhenius Plot of Membrane-Bound ATPase Activity vs 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Temperature for Both 56°C-Grown (-o-) and 37°C- Grown (-o-) T. acidbphilum...... ........ .... ........... Hyperfine Splitting Parameter 2Th as a Function of Temperature for 56°C-Grown (-o-) and 37°C-Grown (-e-) T. acidbphilum Membrane Labelled with 5N5 ........ Scans of SDS Gel Electrophoresis of the Membrane PmtETns fromr. acidophilm...00.0000.0.000.000..0.... Elution Pattern of Membrane Glycoprotein from Con A- Sepharose Column............................. .......... Scan of SDS Gel ElectrOphoresis of the Purified Glyco- protein from Con A-Sepharose Column and the Molecular Weight Determination................... ...... Scans of SDS Gel ElectrOphoresis of the Purified Glyco- protein in 9%, 7% and SS Polyacrylamide................ Sephadex G-100 Column Chromatography of Glycopeptides after Pronase Digestion................................ Sephadex G-75 Elution Profile of Fraction #9 from Figure 20.............................................. Gas-Liquid Chromatography of Trimethylsilylated Methyl Glycosides of the Purified Glycopeptide ..... ........... Gas-Liquid Chromatography of Trimethylsilylated Methyl Glycosides of the Glycopeptide after a-mannosidase 01geSt10n000.0000.0..0........00.00.00..0..00.....0000. Gas-Liquid Chromatography of Trimethylsilylated Methyl Mass 26. Mass 27. Mass Glycosides of the Glycopeptide after a-mannosidase and B-glucosidase Digestions............... ............ Spectrum of Partially Methylated Alditol Acetate Identified as I,S-di-O-acetyl-Z,3,4,6-tetra-0-methyl- mannitol from the Purified Glycopeptide................ Spectrum of Partially Methylated Alditol Acetate Identified as 1,2,S-tri-0-acetyl—3,4,6-tri-0-methyl- mannitol from the Purified Glycopeptide................ Spectrum of Partially Methylated Alditol Acetate Identified as I,3,5,6-tetra-0-acetyl-2,4-di-0-methyl- mannitol from the Purified Glycopeptide................ ix PAGE 63 65 72 74 76 78 81 82 84 88 90 96 97 98 FIGURE 28. 29. 30. 31. 32. 33. 34. Mass Mass Mass Mass Mass PAGE Spectrum of Partially Methylated Alditol Acetate Identified as 1,3,5-tri-0-acetyl-2,4,6-tri-0-methyl- mannitol from the Purified Glycopeptide.. ..... . ....... 99 Spectrum of Partially Methylated Alditol Acetate Identified as 1,5,6-tri-0-acetyl-2,3,4-tri-0-methyl- mannitol from the Purified Glycopeptide....... ........ 100 Spectrum of Partially Methylated Alditol Acetate Identified as 1,4,5-tri-0-acetyl-2,3,6-tri-0-methyl- glucitol from the Purified Glycopeptide............... 101 Spectrum of Partially Methylated Alditol Acetate Identified as 1,3,5-tri-0-acetyl-2,4,6-tri-0-methyl- galactitol from the Purified Glycopeptide... ....... ... 102 Spectrum of Partially Methylated Alditol Acetate Identified as N-acetyl-N-methyl-1,4,5-tri-0-acetyl- 3,6-di-0-methylglucosaminitol from the Purified Glycopeptide...................... ..... ... ............ 103 Proposed Structure of the Thermoplasma addephilwn Membrane Glycoprotein..... ........ ......... ........... 104 T. acidbphilum Cell Growth Ratios in the Presence and Absence of 8acitracin......... ........................ 107 IR NMR PMR 13C-NMR EPR GC MS GC-MS EI-MS TLC Con A $05 PAS BSA TI EDTA Man Glc Gal GlcNAc 5N5 TCA LIST OF ABBREVIATIONS Infrared Nuclear Magnetic Resonance Proton Magnetic Resonance Carbon 13-Nuclear Magnetic Resonance Electron Paramagnetic Resonance Gas-Liquid Chromatography Mass Spectrometry Gas-Liquid Chromatography-Mass Spectrometry Electron Impact Ionization-Mass Spectrometry Thin-Layer Chromatography Concanavalin A Sodium Dodecyl Sulfate Periodic Acid-Schiff Bovine Serum Albumin Trypsin Inhibitor Ethylenediaminetetraacetic acid Mannose Glucose Galactose N-acetylglucosamine S-nitroxylstearate Trichloroacetic Acid xi CHAPTER I GENERAL INTRODUCTION The mycoplasmas are the smallest and simplest self-replicating procaryotes. The mycoplasma cell contains only the minimum set of organelles essential for cell growth and replication: a plasma membrane to separate the cytoplasm from the external environment, ribosomes to assemble the cell proteins, and a double stranded DNA molecule to provide the information for protein synthesis. Unlike all other procaryotes, mycoplasmas have no cell wall. The cell biology of these organisms is interesting not only to mycoplasmologists but also to the many workers who use mycoplasmas as simple model systems for studying general biological problems, particularly those concerning membrane structure and function. The information explosion has been quite pronounced in mycoplas- mology, as the discoveries of insect and plant mycoplasmas and of myco- plasma viruses in the early l9705 have attracted new workers from differ- ent disciplines. Very different and phylogenetically quite remote from all the parasitic mycoplasmas are the wall-less procaryotEs isolated by Darland et at. (l) from self-heated coal refuse piles. These organisms have adapted to a unique ecological niche--high temperature (56°C optimum) and extremely low pH (2.0 optimum)--hence the name Thermoplaema acidophilum. Though their lack of cell walls justifies their inclusion in the class Mycoplasma, their peculiar DNA and RNA, mobility by flagella, and minimal nutritional requirements, set them apart from all other members of the Myc0plasma. Moreover, they are the only nonparasitic members in this class. The existence of obligate, thermoacidophilic microorganisms is a relatively recent discovery. Four different types of organisms have been isolated, namely, Thermoplasma acidbphilum, and three bacteria, SquoZobus acidocaldarius (2), Bacillus acidooaldarius (3) and an obligately autotrophic iron and sulfur oxidizing bacterium (4) closely related to SuLfoZobue. Since these organisms demand the extremes of a hot acid environment for growth, there is considerable interest in defining their physiological characteristics. An understanding of the anticipated unique chemical structures and biochemical properties of these organisms could find application to a better comprehension of evolutionary processes, microbial modifications of extreme environments and the mechanism of resistance of specific cells exposed to a very acid environment. The known thermoacidophilies possess three distinct types of surface structures. The sporeforming Bacillus acidbcaldarius has a morphologically typical gram-positive cell wall. Protection from H+ apparently involves the cell wall and, possibly, surface sulfonolipids, but the underlying mechanism is unknown. ShZbeobua and its autotrophic iron and sulfur oxidizing relative (4) do not contain a typical bacterial wall. Rather, their cytoplasmic membranes are enveloped by closely packed polygonal subunits composed primarily of protein enriched with charged amino acids,such as aspartate, glutamate and lysine. These subunits apparently are not covalently bonded to the membrane or to one another but retain their integrity through hydrogen bonding. In contrast to the peptidoglycan containing 3. acidbcaldarius, which has a typical bacillary morphology, Sulfblobue possesses a peculiar lobe-shaped morphology. A role for this proteinaceous coat is unknown. Thermoplas- ma acidophilum is the representive of the third type of surface struc- ture. This organism is completely devoid of any wall, being contained by a morphologically typical unit membrane (5). Themaplaema begins to leak protein and nucleic acid at pH about 4.5, while increasing pH on three other thermophilic acidophilic organisms has no effect at all. Hence the integrity of the cell may be maintained by properties of the plasma membrane. Structural and functional membrane parameters may determine the environmental limit within which T. acidbphilun is capable to survive. Elucidation of key parameters is the primary concern of this dissertation. Cell protein profiles indicated a high degree of homogeneity among strains of T. acidbphilum collected at different locations, even though serological tests suggested the presence of at least five different serological groups (5.6). Thermoplasma appears to be coccoidal in shape and cell diameters vary between 0.5 to l.0 micron. Some cells have one or two filamentous projections. T. acidbphilum has a more electron dense cytoplasm than AchoZepZasma Zaidlawii although the membranes have similar thickness, 80-l00 A (l,l3). All the T. acidbphilum isolates can be grown in a simple (compared to other mycoplasmas) medium consisting of inorganic ions, glucose and low concentrations of yeast extract (l). Studies of the T. acidbphilum genome are important because of the questionable relationship between this wall-less,free-living procaryote and the parasitic mycoplasmas. The early reports (l,5) had shown that the DNA of T. aoidbphilum resembles that of the parasitic mycoplasmas in having a G+C content as low as 25%. Later this was proven to be incor- rect. The actual G+C value is about 47% (7,8), closely resembling that of the parasitic Acholeplasma and Spiroplasma species and repre- senting the smallest genome recorded for any nonparasitic procaryote. Another interesting feature of the T. acidbphilum genome is its association with a histone-like protein (9). Histones, basic proteins associated with the nuclear DNA of eucaryotes, have not been found in procaryotes. The histone-like protein of T. acidbphilum resembles eu- caryotic histones in having a high basic amino acid content, but it differs in being unusually rich in amides of acidic amino acids. Recent data (l0) suggest that the histone-like protein condenses the DNA into subunits that are 5 to 6 nm in diameter, each consisting of approximate- ly 40 base pairs of DNA looped around 4 or 6 of protein molecules. Thus the nucleoprotein of T. acidbphilum has a subunit structure similar to that of eucaryotic chromatin but of a simpler nature, as the eucaryotic subunit are larger and contain 8 histone molecules plus l30 to l40 base pairs of DNA each. By association the histone-like protein stabilizes T. acidophilum DNA against thermal denaturation (l0). Furthermore, this association may also protect the DNA from depurination in the hot and highly acidic environment of T. acidbphilum. Even though thermo— plasmas are capable of maintaining a relatively high intracellular pH, estimated at 5.6 (ll) or 6.4-6.9 (l2), the rate of depurination of unprotected DNA at these pH values may be high enough to impose a severe mutational load. Searcy (9) suggested that histones may have evolved independently in eucaryotes and T. acidophilum. However, it is also possible that an organism related to T. acidbphilum was the ancestor of eucaryotic cells, and in this case, histones may have first evolved to 5 protect its DNA from thermal denaturation or depurination. Once evolved, the histones condensed the DNA and thus were a preadaptation for the accumulation of more DNA in the eucaryotic cells. The similar properties of the T. acidbphilum histone and histones of primitive eucaryotes, such as Neurospora and dinoflagellates, are consistent with this hypothesis (9). Thermoplaema acidbphilum, lacking a cell wall and intracytoplasmic membranes, has only one type of membrane, the plasma membrane. Isolation of T. acidbphilum membranes by methods usually employed for myc0plasma have been unsuccessful (l3). Low yields of lysed Themoplaema acido- phiZum are obtained by exposing cells to osmotic shock at pH 2 or pH 7.4, or sonicating them for 30 minutes (l3,l4). Denaturants and detergents at high concentration (5 M urea, l% Triton X—l00, or 0.l% SDS) caused partial solubilization of cellular material, leaving intact cells or amorphous protein aggregates as determined by electron microscopy (l3). High pH (9.3-l0.0) has been applied to induce lysis of the osmotically resistant T. acidbphilum (l3) followed by purification via sucrose den- sity gradient centrifugation. Dne criterion for purity of isolated membranes prepared by high pH lysis is the absence of cytoplasmic components. Transmission electron microscopy has shown the presence of membrane vesicles which are approximately the size of intact cells. The interior of the vesicles is free from electron dense cytoplasm(l3). In contrast to membranes from pH lysis, sonically prepared membranes contain usually smaller vesicles and amorphous debris(l4). The major drawback of the sonication procedure is the cytoplasmic precipitation which occurs at low pH and probably accounts for the amorphous material reported by Smith et a1. (l4). Failure to remove this cytoplasmic material is probably due to the acidic conditions chosen (pH 5). A second criterion of membrane purity prepared by high pH lysis is the failure of contaminating cytoplasmic protein to penetrate low percen- tage polyacrylamide gels, even when gels were loaded with 200 micrograms of protein (l3). In contrast, sonically prepared membrane, which con- tained amorphous material, easily penetrated native polyacrylamide gels (l3). Similar gel patterns are obtained on l0% SDS polyacrylamide gels of cytoplasmic components present in the supernatant of cells sonicated at pH 6.5 (l3). Third, the membrane prepared from high pH lysis is essentially devoid of DNA. The membranes contained less than 0.l% (w/w) DNA. However, membrane from high pH lysis may suffer from the deficien- cy that some membrane proteins or even the membranes themselves, may be solubilized at pH values higher than l0.0 (l3). The isolated Thermoplasma membranes resemble plasma membranes of other mycoplasma and procaryotes in gross chemical composition, being composed mainly of proteins and lipids. The protein comprises roughly three quarters of the mass of the membrane, the balance being primarily lipid. The amino acid composition of the total membrane from T. acido- phiZum has revealed no significant difference from the amino acid composition of mycoplasmal membrane proteins in general (l3,l4). How- ever, the number of chargeable groups is barely half of the number found in mesophilic mycoplasmal membranes, even though the ratio of free -CO0H to free -NH2 (4:l) is identical. The major controversy in T. acidbphilum research lies in the lipid structure of the organism. Most researchers agree on the percentages of the lipid components to be neutral lipid: glycolipid: phospholipid - l:l:3 (13,16). The exact chemical structures of these complex lipids await resolution. In 1972, Langworthy et a1. (16) reported that more than 70% of the total lipid from whole cell (virtually all myCOplasma lipids are located in the cell membrane)is a l,2-subsituted long-chain diether of glycerol. Mass spectrometry data indicate the molecular ions of the long chains are 562 and 560, accounted fbr by the formulas C40H82 and C40H80. Solely from the incorporation of radioactive meva- lonate, they concluded that the long chains have 8 repeating units of isoprenoid. Ester linkages were not detected. After the membrane pre- paration by high pH lysis, membrane lipids were analyzed by Ruwart at a1. (13). Their results agree with most of the lipid structure reported by Langworthy et a1. (16), but disagree on the neutral lipid fraction. Ruwart et a2. (13) found no detectable amount of cholesterol as reported by Langworthy et aZ.(16), but large amount (30%) of vitamin K2-7. Also Ruwart et al. reported the presence of fatty acid esters (2.1%) in T. . acidophilum membrane lipids by combined gas chromatography-mass spectro- metry and infrared hydroxamate techniques. In 1976, de Rosa et a1. (17) reported that the lipids are based on sn-2,3-glycerol combined as a cyclic diether with a saturated C40 isoprenoid residue which is either acylic, monocyclic or bicyclic. The residues are fbrmed from two phytanyl chains linked head to head. Recently, Langworthy (18) revised the lipid structures to be neither glycerol diethers containing two C40 hydrocarbon chains, nor cyclic glycerol diethers containing a single C40 hydrocarbon, but are diglycerol tetraethers. Membrane lipid structure is one of the major concerns of this dissertation. Since the membrane comes in direct contact with the harsh environment (pH 2 and 56°C), the exact knowledge of the lipid structure may help to elucidate structural and functional relationships in response to environmental stress. The glycolipids and phospholipids of this organism contain mainly glycerol ether residues rather than fatty acid ester-linked glycerides, apparently the result of adaptation to highly acidic conditions, where ester linkages are unstable. The C40 long chains assume the function of thermal stability. Electron paramagnetic resonance studies by Smith et al. (19) indicated that Thermoplasma mem- brane is the most rigid membrane known. Evidence for the existence of a membrane potential is available for T. acidbphilum. The intracellular pH in this organism is estimated at between pH 5.5 and 6.9, depending on the technique used fbr its measurement (11,12). Since the pH of the growth medium was about 2.0, a pH gradient of 3.5 to 4.9 must exist between the outside and the inside of the cells. The finding that metabolic inhibitors and proton- conducting uncouplers did not affect this gradient led Hsung and Haug (12, 20) to conclude that it is maintained passively by a Donnan potential across the membrane, possible generated by charged intracellular macro- molecules. KSI4CN, known to penetrate biological membranes, accumulated in the T. acidophilum cells, whereas tetraethylammonium bromide, a lipo- philic cation, did not, suggesting that the cells are positive on the inside, whereas their surface has a highly negative charge. Hence, some positively charged ions or macromolecules possibly including the histone- like proteins described by Searcy (9), must be present within the cells to create the Donnan potential (21). At pH 2.0, the measured membrane potential is about 120 mV, positive inside, compensating only partly for the huge pH gradient. The membrane potential decreased linearly on in- creasing the external pH, diminishing to less than 15 mV at pH 6.0 (20). Hsung and Haug considered whether ATP can be generated in T. acidbphilum lO crystalline phase transition to an extent dependent on the cation con- centration (25). The presence of monovalent and divalent cations at the polar lipid groups of T. acidbphilum membranes probably induces alterations of alkyl chain conformation, as indicated by the spin probes (22). Raman spectroscopic experiments on aqueous phosphatidylcholine dispersions have demonstrated that Ca2+ ions decrease the proportion of gauche character in the hydrocarbon chains (26). In contrast, K+ caused no modifications. By using electron paramagnetic resonance spectroscopy, Al3+ was shown to produce a dramatic decrease of membrane lipid fluidity on the microorganism at a pH higher than 2. The ability of Al3+ to alter lipid fluidity was enhanced with increasing pH (from 3 to 5). At pH 4, lD’ZM Al3+ increased the lower lipid phase transition by 39°C, and a de- tectable change was observed with A1C13 concentrations as low as lD’SM. The ability of Al3+ to increase the lower lipid phase transition tem- perature of T. acidbphilum is the largest of any cation/lipid inter- action yet reported (23). Thenmoplasma acidbphilum, a mycoplasma-like organism, grows optimally at pH 2 and 56°C. Since this organism doesn't have a cell wall, the plasma membrane directly interfaces with the harsh environment. The molecular mechanisms underlying temperature adaptation of microorganisms are not well understood although much information is available on adapta- tional changes in lipid side chain structures. With respect to physico- chemical parameters, such as membrane lipid fluidity and lipid phase transition temperature, the growth temperature range of a microorganism seems to depend on the ability to regulate its membrane lipid fluidity within a certain range. As reviewed above, the most controversial area ll of T. acidOphiZum research is concerned with the lipid structure. The first part of this dissertation (Chapters 3 and 4) is focused on answering the question of what are the key parameters of the structural and functional relationships of the lipids in the T. acidbphilum membrane which will allow adaptation to growth at high temperature and low pH. Five spectroscopic techniques: IR, PMR, GC-MS, EI-MS and Carbon 13— NMR, were used to elucidate the lipid side chain structure. The contributions of the head groups of the lipid upon temperature adapta- tion were also investigated. To further understand the temperature effects of T. acidbphilum, cells were adapted to growth at 37°C. Cor- relation between membrane fluidity and membrane-bound enzymes were analysed. The second part of this dissertation (Chapter 5) is dedicated to the partial characterization of a procaryotic glycoprotein fbund in the T. acidaphilum membrane. The possible function of this membrane glycoprotein in protecting the organism from its harsh environment (pH 2 and 56°C) was investigated. CHAPTER 2 MATERIALS AND METHODS MATERIALS The fbllowing solvents were of reagent grade: methanol, chloroform, pyridine, toluene, acetic anhydride, dichloromethane, carbon tetrachlo- ride, benzene, acetic acid, acetone, hexanes (Mallinckrodt, St. Louis, Mo.); dimethylsulfoxide ( Fisher, Fair Lawn, N.J.); acetonitrile (Aldrich, Milwaukee, Nis.). All solvents were redistilled prior to use. Chemicals and their sources are as follows: NaH from Alfa Inorgani- cs (Beverly, Mass.); Sephadex G-10, 8-25, G-75, G-100, G-200, LH-20 and Sepharose 48 from Pharmacia (Piscataway, N.J.); silicic acid from Clarkson (Nilliamsport, Pa.); galactosamine hydrochloride, glucosamine hydrochloride, glucose, galactose, mannose from Pfanstiehl (Waukegan, 111.); silica gel 0 TLC plates from Analtech (Newark, Del.); molecular weight calibration proteins from Boehringer-Mannheim (Indianapolis, Ind.); 3% SE-30 on Supelcoport (80-100 mesh), 3% SP-2100 on Supelcoport (80-100 mesh), 3% 0V-225 on Gas Chrom 0 (80-100 mesh) from Supelco ( Bellefbnte, Pa.); mercaptoethanol from Aldrich (Milwaukee, His.); Dowex l, hexamethyldichlorosilazane, trimethylchlorosilane, glycine, bromophenol blue, fuchsin, Coomassie blue, ATP, glucose from Sigma ( St. Louis, Mo.); dialysis tubings from Thomas (Philadelphia, Pa.); nin— hydrin, ammonium persulfate, orcinol, ammonium molybdate from Fisher ( Fair Lawn, N.J.); acrylamide, and NJNf-methylenebisacrylamide from 12 13 Canalco (Rockville, Md.); N,N,Nj,Nf-tetramethyl-ethylenediamine and Photo-Flow 600 from Eastman Kodak (Rochester, N.Y.); silver carbonate, phenol, EDTA from Mallinckrodt (St. Louis, MO.); ethanol amine HCl, EDTA disodium cupric salt from ICN Pharmaceuticals (Cleveland, Ohio); hydrochloric acid, sulfuric acid and iodine from Baker (Philipsburg, N. 0.); Folin Ciocalteu reagent from Harleco (Phila., Pa.); HCl and BCl3 gas from Matheson Gas (Lyndhurst, N. 0.); 5-NS from Synvar (Palo Alto, Ca.); yeast extract from Difco (Detroit, Mi.); o-mannosidase, B—galacto- sidase, a-glucosidase, B-glucosidase, endo-glycosidase H, and endo-glyco- sidase D from Miles (Elkhart, Ind.); Anhydrous hydrogen fluoride reaction apparatus from Peninsula Laboratories Inc. (San Carlos, 0a.). METHODS Growth of Cells Thermplaema acidophilum was obtained from the American Type Cul- ture Collection (ATCC 25905). The organism was grown in a medium con- taining 1.5 mM (NH4)2S04, 4.2 mM MgSDa-7H20, 1.7 mM CaClz-ZHZD, 0.03% KH2P04, 1% glucose, and 0.1% yeast extract (Difco Control #629756). The pH was adjusted to 2 with concentrated H2504 and the medium then auto- claved. A 10% (v/v) inoculum from a 22 hours old culture into the same medium gave the best growth. Each culture was continuously aerated for 22 hours with filtered sterilized air. Late log phase cells were harvested by centrifugation at 4200 g fbr 5 minutes at 4°C. Medium com- ponents were removed by multiple water washings. Cells grown at 37°C were transferred from the same culture line as 56°C-grown cells. The final inoculum fbr 37°C cultures was prepared from cells which had been grown for about 8 days in a culture, which, in turn, had been pre- inoculated (10%) with cells grown for about the same length of time. 14 Cells were adapted from 56°C to growth at 37°C for at least 30 generations. The 37°C—grown cells were harvested as described above. Membrane Preparation Cells were lysed by l M glycine buffer, pH 9.3, 22°C, at a protein concentration higher than 10 mg/ml. Membranes were collected by centri- fUgation at 34,800 g for 2 hours, then layered onto a discontinuous sucrose gradient (25%/55%) pH 7.4 and centrifuged on a Beckman L2-658 Ultracentrifuge for 2 hours at 40,000 rpm with an SN 41 rotor. The membranes appeared as a single band at the interface of the two sucrose 1aYers and were collected and freed from sucrose by washing with water. Extraction of Lipids Lipids were extracted from cell membrane with chloroform-methanol (2:1 v/v). Further removal of non-lipid contaminants was done by partitiOning the lipid extract with 0.2 volume Of salt solution (27). Silicic Acid Column Chromatography Silicic acid (100-200 mesh) was employed to separate the lipids. The neutral lipids were eluted with chloroform, glycolipids were obtained by elution with acetone, and phospholipids were recovered from the column by final elution with methanol (28). Preparative Thin-Layer Chromatography (TLC) Individual phospholipids and glycolipids were separated on silica gel G plates. Lipids were streaked onto the TLC plate with a preparative TLC sample applicator system (Applied Science Lab.). Plates were deve- loped at room temperature with the solvent system Of chloroform-methanol- water (65:45:8, v/v) for phospholipids or chloroform-methanol (9:1, v/v) fOr glycolipids. Lipids were visualized by iodine vapor. Spots were Scraped and lipids were extracted by successive elution with chloroform- 15 methanol (2:1, v/v), chlorofbrm-methanol (1:2, v/v), methanol, and finally chlorofbrm-methanol-water (50:50:15). Transmethylation of Lipids Samples of lipids were hydrolyzed in 5 ml Of methanol and 0.1 ml of concentrated H2504 fbr 24 hours at 40°C. Fatty acid methyl esters were extracted three times with equal volumes of hexane. The combined hexane phase was washed with water until neutral. Methanolysis Samples of lipids in 2.5% methanolic-HCl were heated at 100°C far 3 hours. The unsaponifiable material was extracted three times with equal volumes of hexane (29). Degradation of Glygerol Ethers (i) Alkyl Chloride Derivatives The unsaponifiable material from methanolysis was treated with BCl3-CHC13 (1:1 v/v) at room temperature for 24 hours. After evapo- ration of the BCl3-CHC13 mixture, the sample was partitioned between hexane and water. The alkyl chloride derivative of the hexane phase was pooled and dried to constant weight under a stream of nitrogen at room temperature (29). (ii) Alcohol Derivatives Hydrolysis Of glycerol ethers was conducted in 57% hydriodic acid (29). Samples were heated under reflux for 24 hours, cooled, and extracted three times with three volumes of hexane. Alkyl iodides contained in the hexane phase were successively washed once with 10% NaCl, saturated solution of K2C03, and finally with 50% Na25203. Alkyl iodides were converted to the acetates by refluxing with silver acetate in acetic acid fbr 24 hours. Alcohol derivatives were prepared from 16 the acetates by hydrolysis in 0.2 N NaOH at 100°C for 2 hours. Determination of C-methyl Groups The method of Kuhn and Roth (30) was used. Samples were sealed into an ampule with 1:4 v/v of concentrated H2504 to 2 N chromic acid and shaken for 12 hours at 135°C. After oxidation, the contents were washed over into a distillation apparatus. After adding one drop of 1% phenolphthalein solution,the distillate was quickly titrated to the first pink tinge remaining for 5 seconds. Gas-Liquid Chromatography (CC) for Membrane Lipid Derivatives A Hewlett-Packard gas chromatograph, Model 402, equipped with a flame ionization detector and 2 m x 2 mm glass column was used for CC analysis of the lipid derivatives. Fatty acid methyl esters were run at 170°C on 3% SP-2100. Alkyl ether derivatives were eluted isother- mally on 3% SP-2100 at 325°C. Areas Of peaks were determined with a Hewlett-Packard integrator, Model 3380 A. Combined Gas Chromatography-Mass Spectrometry (CC-MS) The LKB 9000 gas chromatograph-mass spectrometer was operated with a trap current of 60 uA, an ion source temperature of 310°C and a mole- cular separator temperature of 310°C. (Dr. C. C. Sweeley, Biochemistry Department, Michigan State University) (31). Electron Impact Ionization-Mass Spectrometry (El-MS) Mass spectra were obtained with a Varian MAT mass spectrometer, Model CH-SDF, with a combined electron impact/field ionization/field desorption source. The mass spectrometer was interfaced to a PDP 11/21 minicomputer (Digital Equipment Corporation) in which data collection and storage programs were functional. By direct memory access, this computer was attached to a PDP ll/40 minicomputer (Digital Equipment 17 Corporation) which operated in a time-shared mode employing the multi- task executive program RSX-llD. The outputs from the mass spectro- metric experiments were received from the PDP 11/40 system via a Tektro- nix data terminal, Model 4010, and a Tektronix hard copy unit, Model 4610 (32). Infrared Spectroscopy Infrared spectra were recorded by a Perkin-Elmer Grating Infrared Spectrophotometer, Model 621. Attenuator speed was set at 1100, slit program at 1000 and source intensity at 0.8 A. CC14 was used as solvent. Nuclear Magnetic Resonance Spectroscopy (i) Proton NMR Proton NMR spectrum was recorded on a Bruker NH-180 spectrometer, at 180 MHz. The sample was dissolved in CDC13 with tetramethylsilane as internal standard. (Chemistry Department, Michigan State University). (11) 13c-NMR The 15.08 MHz, proton decoupled 13C-NMR spectrum was obtained using a Bruker HP-60 spectrometer, equipped with Fourier transfbrm operation. The sample (60 mg) was dissolved in 1.5 ml of CDC13 and 10,000 tranients accumulated at 35°C in a 10 mm tube at a sweep width of 3000 Hz, 66° pulse (12 usec.) and 3 sec. rep. time. ATPase Assay The ATPase assay fbllowed the method of Rathbun et a1. (33). Purified membrane sample was incubated at various temperatures, ions and pH's with 0.1 M buffer and ATP as substrate. The controls had no ATP. The reaction was stopped by adding ice-cold TCA and cooled in an ice bath fOr 5 minutes. After centrifugation for 5 minutes at 10,000 g at 4°C, the supernatant solution is decanted into empty test tubes. An 18 aliquot of this supernatant solution is quickly mixed with 3 M acetate buffer and formaldehyde mixture previously pipetted in the test tube. The molybdenum blue color was developed by addition of 2% ammonium molybdate and stannous chloride. The tube contents were quickly mixed and allowed to stand at room temperature. The absorbance of each sample is measured at 735 nm 15 minutes after addition of the molybdate. The protein concentration was detennined as described by Wang et a1. ( 34). Buffer Systems fOr SDS Gel Electrophoresis and Sample Preparation The discontinuous SDS buffer system of Laemmli (35) was used. Separating gels, containing 8% acrylamide, were prepared from a stock solution of 30 9 acrylamide and 0.8 g of NJNf-bis-methyleneacrylamide in 100 ml of water. The stacking gel which usually contained 5% acrylamide was prepared from the same stock solution. The final concen- tration of SDS was 0.1% in both gels and in the electrode buffer. Sample preparation was performed as described by Laemmli (35) except that samples were boiled for 2 minutes. The final membrane protein con- centration in the sample was 2 mg/ml. After addition of glycerol and bromophenol blue, samples were layered onto the gels (10 cm) and run at 5 mA/tube for 35 hours. Proteins were stained with Coomassie brilli- ant blue (36). Carbohydrates were stained as described by Fairbanks at al. (37). Stained gels were scanned spectrophotometrically with a Gilford linear transport system, Model 2520, at 550 nm for Coomassie blue stain, and at 540 nm for carbohydrate stain. Electron Paramagnetic Resonance (EPA) The nitroxide stearate spin label, 5N5, was used for the EPR studies of membranes. Aliquots from a stock solution of spin label in hexane 19 were measured into small glass test tubes; hexane was removed by evaporation. Membrane vesicles were suspended in water at pH 6. The suspension was added to the spin label. The mixture was then soni- cated for 10 minutes. The membrane protein concentration of the mem- brane vesicles ranged from 20 mg/ml to 30 mg/ml. The spin label concen- tration was approximately 0.1% of the lipid weight of the vesicles. All EPR experiments were carried out with a Varian spectrometer, Model E-112, with an attached Variable Temperature Controller (VTC) unit. Temperature calibrations were obtained by comparing VTC settings and a Bailey thermocouple measuring the temperature Of the sample located in the microwave cavity. Isolation and Purification of Membrane Clycoprotein (i) by preparative slab gel electrophoresis Buffer system of Laemmli (35) was used to run slab gels. The Bio- Rad slab gel apparatus was operated at 30 mA with a slab thickness of 3 mm. After electrophoresis, three strips of gels were cut vertically and stained with PAS and Coomassie blue. The gel was reconstructed after staining and glycoprotein bands were cut, and eluted with buffer. SDS was removed by dialysis. (ii) by phenol extraction Membrane was extracted with 50% phenol solution at 70°C for 15 minutes. Material at the interface was collected and dialysed. (iii) by Sepharose 4B Column Chromatography Membrane was solubilized by 1% SDS and loaded onto a 90 x 1.5 cm ( i.d.) Sepharose 48 column. Fractions of 3 ml were collected and assayed for protein and carbohydrate. 20 (iv) by Con A-Sepharose Column Chromatography Glycoprotein isolated by the above three methods can be further purified by Con A-Sepharose column. Glycoprotein was loaded on the column and eluted first with a solution of 0.5% Triton X-100, 50 mM Tris, pH 8.0, 0.5 M NaCl, and then with 0.5% Triton X-100, 50 mM Tris, pH 8.0, 0.2 M a-methylmannoside. Triton X-100 was removed by dialysis. Methanolysis of Carbohydrates Methanolysis of glycoprotein to study the carbohydrate-portion was performed by the method of Chambers and Clamp (38). Samples were hydrolysed in 1.5 N methanolic-HCl in sealed teflon-lined vials at 95°C for 90 minutes with mannitol as internal standard. Silver carbo- nate was added until the solution is neutral. Acetic anhydride was added to re-Nyacetylate the aminosugars for at least 6 hours at room temperature. The sample was then centrifuged and the supernatant frac- tion removed. The residue was washed fbur times with dry methanol. The pooled supernatant fraction was dried under nitrogen at room temperature and derivatized with pyridine/hexamethyldichlorosilazane/trimethyl- chlorosilane (5:1:1, v/v). Trimethylsilylated methyl glycosides were analysed on a Perkin-Elmer gas-liquid chromatograph, Model 910, with a 12 ft 3% SP-2100 on Supelcoport (80-100 mesh) column with initial hold Of 4 minutes at 120°C, then programmed at 0.5°C/min to 185°C, and held at 185°C for 20 minutes. Permethylation for Linkage Studies Permethylation of carbohydrates was performed by the method of Hakomori (39). All the permethylation Operations were done under dry nitrogen. Hexane was dried by refluxing with Ba0 (20 g/liter) for 2 hours, redistilled and stored over molecular sieves. All the other 21 solvents used were redistilled. A sample of NaH (0.9 g of 57% oil emul- sion) was washed 7 times with 15 ml aliquots of dried redistilled hexane. Dry redistilled dimethylsulfoxide (10 ml) was added and allowed to react at 65-70°C until bubbling of hydrogen ceased (approximately 90 minutes). The dimethylsulfinyl ion solution (0.5 ml) was added to samples (0.5 mg)dissolved in 0.5 m1 dimethylsulfbxide and allowed to react fbr 30 minutes with periodic sonications. Redistilled iodomethane (2 ml) was then slowly added and allowed to stand for 2 hours at room tempera- ture. The solutions were then dissolved in 5 ml of chloroform and washed twice with 5 ml water, once with 5 ml 20% Na25203 and thrice with water. The samples were dried under nitrogen with the aid of absolute alcohol and hydrolysed in 0.5 ml 0.5 N H2504 in 95% acetic acid for 24 hours at 85°C. Water was then added and allowed to react fbr an additional 5 hours at 85°C. A small column with 2 ml of Dowex l x 8, acetate form (50-100 mesh) was used to adsorb the sulfate. The column was washed with 2—3 ml of acetic acid. The hydrolysate was evaporated to dryness under nitrogen and reduced with 0.5 m1 of NaBH4 (10 mg/ml) for 2 hours. Re- duction was terminated with the addition of several drops of glacial acetic acid. The solution was dried under nitrogen. Borate was re- moved as its methyl ester by 1-2 drops Of acetic acid and 2 ml methanol, heating in a boilinglvater bath for 5 minutes, and evaporating under nitrogen. The esterification procedure was repeated three more times. The dried sample was acetylated in 0.5-1.0 ml acetic anhydride for 60- 90 minutes at 100°C. After drying under nitrogen with the aid of toluene, dissolved in 2 ml of CHZClz, washed three times with l-2 ml water and drying under nitrogen again, the partially methylated alditOl acetates were ready for analyses by CC or GC-MS. Column packings used for these 22 analyses were 3% 0V-210 on Gas Chrom 0 (80-100 mesh) or 3% 0V-225 on Gas Chrom 0 (80-100 mesh). The mass spectra were interpreted according to Bjdrndal et a2. (40). Anhydrous Hydrogen Fluoride Deglycosylation Reaction vessel containing freeze-dried sample was cooled in liquid nitrogen bath. 10 ml of HF was distilled over from the reservoir. Allow the reaction vessel to warm up to room temperature. At the end of the reaction, evacuate the reaction vessel via a calcium oxide trap. To ensure complete removal of HF, the line was evacuated fbr an addi- tional hour after removal of visible HF. The sample was then taken up in water and passed through an Amicon ultrafiltration cell to separate the carbohydrate and the protein (106). Amino Acid Analysis 5.7 N HCl was added to the purified glycoprotein. Hydrolysis was complete after 18 hours at 110°C. The suspension was taken to dryness under nitrogen and resuspended in 0.01 N HCl. Amino acid analysis was perfOrmed on a single column accelerated flow Technicon system modified by Dr. D.T.A. Lamport, Plant Research Laboratory, Michigan State Univer- sity. Treatments of Glycoprotein with Glycosidases (i) a-mannosidase digestion Reaction mixtures containing approximately 5 mg of purified glyco- protein in 1 m1 of 0.1 M citrate buffer, pH 4.5, were incubated at 37°C with 2 to 3 units of a-mannosidase. Incubation was continued until no additional release of mannose was Observed. The reaction was terminated by introducing 2 ml of 0.2 M borate buffer, pH 9.8. The reaction mix. tures were then passed through an ultrafiltration cell to separate the 23 free carbohydrates from the rest of the glycoproteins. (ii) B-glucosidase digestion The same procedure fOr a-mannosidase was used fOr s-glucosidase. (iii) B-galactosidase digestion The same procedure for a-mannosidase was used only the buffer was different. B-galactosidase has an optimum pH at 7.0. (iv) endo-glycosidase H digestion Glycopeptide in 1 m1 of 0.1 M acetate buffer, pH 5.0 was incubated with 1 unit of endO-glycosidase H fbr 16 hours at 37°C. The reaction was stopped by heating for 1 minute in a boiling water bath and the reaction mixture was ultrafiltrated. Endo-glycosidase H from Strepto- myces griseus hydrolyzes the B-di-N-acetylchitobiose structure in asparagine-linked sugar chains of glycopeptides. Analytical Methods Phosphorus was determined by the method of Fiske and SubbaRow (41). Total nitrogen content was measured according to the method of Lang (42). Glycerol was determined colorimetrically after periodate oxidation (43). Carbohydrate was estimated by the anthrone procedure (44). Choline content was determined by the method of Wells et a2. (45). Serine and ethanolamine was estimated by the method of Dittmer et al. (46). CHAPTER 3 STRUCTURE OF LONG-CHAIN GLYCEROL ETHERS IN PLASMA MEMBRANE FROM THERMOPLASMA ACIDOPHILUM GROHN AT 56°C Introduction Microorganisms have a great capacity to adapt to a wide range of environmental temperatures. The molecular mechanisms underlying temperature adaptation are not well understood although much information is available on adaptational changes in fatty acid composition (47,48). Since temperature has a crucial influence on the structure and function of biological membranes (49,50), it is a reasonable working hypothesis that physico-biochemical membrane properties are fundamental to the ability of microorganisms to exist in thermal habitats. Generally thermophilic microorganisms contain a higher amount of saturated and branched-chain fatty acids (51,52). With respect to physico-chemical parameters, such as membrane lipid fluidity and lipid phase transition temperatures (53,54), the growth temperature range of a microorganism seems to depend on the ability to regulate its membrane lipid fluidity within a certain range (55,56). An attractive system for investigating mechanisms of temperature adaptation is Thermoplasma acidbphilum which grows optimally at pH 2 and 56°C (1). The temperature extremes at which this cell can grow and reproduce are 37°C and 65°C (57). Since its plasma membrane directly interfaces with the harsh environment, structural and functional membrane 24 25 parameters may determine the environmental limits within which the cell is capable to survive. Electron paramagnetic resonance experiments using spin labels demonstrated that the lipid regions of Thermoplaema acidbphilum may be best described as highly rigid (19), even more rigid than those reported fbr the halophilic Halobacterium cutirubrum (58). To understand the high rigidity of the lipid matrix, the structural determination of membrane lipids is important. After we had embarked in this investiga- tion, four different structures have been proposed (13, 16-18); there- fbre, a further Objective of this study is directed to resolve this discrepancy. Results Lipids from T. acidophilum membranes accounted fbr 25% of the membrane dry weight. The relative quantities of neutral lipid: glyco- lipids: phospholipids were l:l:3 (w/w). 0n TLC plates the glycolipids could be separated into 8 bands, where 70% (w/w) were monoglycosylated. The carbohydrate content was 88% glucose and 11.5% mannose. 0n the other hand, phOSpholipids could be separated into 9 bands where one band accounted for 60% (w/w) of the total membrane lipids. Previously reported experiments showed that these two lipid classes comprise 83% (w/w) of the total lipids (13). After recovery from the TLC plates, material from each individual glyco- or phOSpholipids band was trans- methylated. Approximately 0.3% (by weight) of fatty acids were fOund in either type Of lipid class, which agreed with previously reported results (13). As determined by gas-liquid chromatography, the chain lengths of the fatty acids varied from C15 to C20. Since ester-linked fatty acids are acid-labile, this finding is consistent with the 26 ability of growth at pH 2 of T; acidbphilum; The unsaponifiable material from transmethylation was treated with 2.5% methanolic-HCl at 100°C for 3 hours (glycolipids) or 5 hours (phos- pholipids). Methanolysis reaction will cleave the head group of the lipids, and leave the glycerol backbone intact (Figure 1). The unsaponi- fiable material (I) (Figure I) was extracted three times with equal volumes of hexane. The hexane phase was pooled and dried under nitrogen. Thin-layer Chromatogram of compound (I) developed in the solvent system chlorofbrm/diethyl ether 9/1 (v/v) showed a single band regardless of the origin of the lipid class or band. The dried material (I) was taken up in CCl4 for infrared measurements, in C0613 for NMR studies, or in hexane fer direct probe mass spectrometry studies. Infrared Spectroscopic Studies of Compound (1) Infrared spectrum of compound (I) (Figure 1) is illustrated in Fi- gure 2. The unsaponifiable material (I) gave 0H absorption at 3590-3620 cm'l, alkyl stretching at 2350-2950 cm'l, C-H bending at 1455 and 1370 cm’l, ether C-0-C stretching at 1110 cm", and primary hydroxyl C-0 at 1040 cm". The absorptions of C'C, CeC-H, c-o and CO0H were absent. These absorp- tions indicated that the side chains were linked to the glycerol backbone by ether linkages, and there were no ester or double bonds in compound ( 1), hence the name glycerol ether. Bands at 840-890 cm"1 provided evi- dence for the possible existence of cyclo-alkane ring. This was further supported by the presence of a -CH2- scissoring vibration at 1455 cm'1 which occurred in cyclopentane and cyclohexane derivatives. Proton-NMR Studies of Compound (I) The proton magnetic resonance spectrum obtained for the glycerol ether is illustrated in Figure 3. The assignment of chemical shifts 27 H2 -O-R| HZC‘O‘RI H -O-R2 2.15%melhdnnlla—l-ICIt H (I-O-Rz + hg HzC-o-hg H2C-OH Ll PlD GLYCEROL ETHER (I) Fifi-(HR. /BC|3/RIC(IJ)R2C|+ glycerol HC-O-Rz I HzOl HZC-OH \RI' + RZI ... §HO| H201 MOAC RI : ROAC NoOH A , ROH (Ill)- hg = head group Figure 1. Chemical Degradation Scheme of the Glycerol Ether Lipids from the Membrane of Themaplasma acidoPhilum. 28 anon on n— N. coo— o— 89 .3 v.53“: A: egg—=8 .3 .5383 v9.22: .. so. a 9222335 _ mod. con 82 80a comm 95m 83 m o n v n 3205...... 502533 .N ee=u_e 8 53 .8 Dream) mumswvu nN 29 .A~ mean—my A~v vcaoaeou eo escuoonm unencumoc u—uucaa: coves; ficaavw N n v m p h p- p- .n wean—u b 30 of proton signals of glycerol ether is summarized in Table 1. The PMR spectrum showed a triplet at 6 0.83, 0.86 and 0.89, assigned to the methyl groups -CH3. The resolved signals, those at 6 1.25 were assigned to the interior methylene groups, CHZ-Cflz-CHZ, those at 5 1.55 were assigned to the ring methylene groups, those at 6 1.72 were assigned to the interior nethine groups, QM, those at 6 2.15 were assigned to the primary hydroxyl, -0H, and those at 53.50, 3.63 were assigned to fl-C-D-Cflz and -Cfl2-0H res- pectively. There were no c-c-g, or -CO0H absorption, which was consistent with the infrared spectrum. Both the PMR and IR spectra supported the structure of the unsaponifiable material (I) to be an ether-linked lipid with methyl branching and the possible existence of cyclo-alkane ring in the side chains. No double bond was detectable in either spectrum. GC Studies of Alkyl Chloride and Alkyl Alcohol Derivatives of Side Chains To further study the detailed structure of the side chains, (I) was either reacted with 8Cl3/CHC13 to their alkyl chloride derivatives (11) (Figure 1), or reduced to their alkyl alcohol derivatives (III)(Figure 1). Both derivatives did not elute on the gas chromatograph, equipped with a 6 ft 3% SP-2100 column, until 325°C. A rough estimate from the retention times indicated that the side chain should consist of more than 35 carbon atoms. Since high molecular standard for GC analyses was unavailable, combined GC-MS was used to determine the molecular weights and the structures of the lipid side chains. The alkyl chloride derivatives gave 7 CC peaks where peaks 43-6 ac- counted fbr 90% of the lipids analyzed (Figure 4). However, the alkyl alcohol derivatives showed 3 GC peaks with the ratio #1/#2/#3 - 20/45/30 (Figure 5). At first these differences were rather confusing. The dis- crepency was resolved when the GC-MS and EI-MS spectra were analysed. 31 Table l. Proton NMR Chemical Shifts 5 (in ppm) of Glycerol Ether Downfield from TMS. 6 (PPM) Assignment 0.83, 0.86, 0.89 -CH3 1.10 -Cfl3 next to ring 1.25 -CH2-CH2-CH2- 1.55 -CH2 in ring 1.72 -CH' 2.15 -0H 3.50 .fl-C-O-Cflz- 3.63 -Cflz-0H Figure 4. 32 I.l.l m Z 0‘ i o. m 32 2 a: l i O U m '7 r“ V D 2 6 10 14 RETENTION TIME (min) Gas Chromatogram of Alkyl Chloride Derivative of Glycerol Ether Lipids from T. aoidbphilwm Membrane. GC analyses were perfbrmed on a 6 ft 3% SP-2100 column, operated isothermally at 325°C, with helium as carrier gas. 33 .muoguoz c— voawcumou mu wouncono mu: gascuouueocgu mom on» .855: 53.388 .e soc» mu.a.4 coguu pocouapu we o>euuspcua poeou~< pzxp< eo Escuoumeocgu new .m mean—d 34 ON AEEV 92:. m. h .m weaned ZO_._.Zm_._.mm o_ _ D BSNOdSBH 80103130 35 Organic halides in general, particularly di-halides, undergo several on- column reactions, including rearrangement, loss of one or more molecules of HCl, or H I Cl exchange with the liquid phase, even on non-polar phases in glass columns. This is particularly true at high temperatures (>250°C). For alkyl derivatives of (I), because of the chain length, column tempe- rature was set at 325°C.Some of the GC peaks were generated by de-chlori- nated products of the alkyl chloride derivatives.F0r alkyl BICODOI» the column temperature was also set at 325°C, however, alkyl alcohol was TMS- derivatized which is not known to undergo any rearrangement or exchange reactions. GC-MS Studies of the Alkyl Chloride Derivatives The GC-MS data of the alkyl chloride derivative will be discussed first. Peak #4, which accounted fOr 28% of the side chains (Figure 4). generated a very similar fragmentation pattern as that of peak #6 (18%). CC component #4 gave a molecular ion at m/e 592 with the molecular formu- la C40H77Cl, whereas peak #6 gave a molecular ion at m/e 628 with the molecular fbrmula of C40H73C12 (Figure 6). Peak #3 apparently correspon- ded to a mixture of C40H73 and C40H79Cl. Peak #5 and peak #7 also gene- rated similar fragmentation patterns. Since alkyl chloride derivatives were thermally unstable as discussed above, peaks 3, 4 and 5, which were identified as monochloride derivatives, might be a result of on-column conversion of dichloride derivatives: peaks 6 and 7. The sum of the peak percentages that contributed to the fbrmulae C40H30Clz, C40H73Clz and C40H75C12 were 18%, 46% and 32% respectively. These values are in good agreement with that of the alkyl alcohol derivatives (Figure 5). Although the alkyl chloride derivatives looked confusing, they provided valuable infbrmation concerning the structures of the side chains. ‘In the mass chromatograms, pronounced peaks appeared in the 36 .co. capauu—oe use mucomucaoc z .muocuu: :. voawcumov no «so vo—csuu no: zuuosocuuoam amus-xgaucmouaeoegu mam vac—nsou .moseua>—coo «o'copzu —»x.< me m use m.¢ neocoaeou ow we covaox mm~:-=a.: use mo acuooam mun: .m unau'd 37 One GRAIOVU 000 «on Den :2 «or .m mcammd O\E 0mm and can Own AIISNSINI BAIIV'ISU 38 low-mass region which were characteristic for hydrocarbon-type ion fragments. The relative intensities of these ions decreased as m/e increased. The base peaks were either at m/e 55 or at m/e 57. Ion fragments which contained chlorine could be identified from the isotopic clusters of the mass spectrum (59), because monochlorinated ions are characterized by relative intensity ratios of pm+l/Rm = 0.5 and Pm+2/ Pm = 0.42, where P is the relative intensity. In the case of dichlori- nated ions, the relative intensity ratio is sz/Pm . 0.7. In the absence of Chlorine, the ion fragment has a relative intensity ratio Of sz/Pm = 0.1 (60) (Figure 6). The mass spectra from each GC component revealed the presence Of methyl branches in the alkyl chloride chains. It is possible to deduce the position of methyl branching by searching for relatively high peaks, so-called a-eliminations. In the absence of branches, the relative intensities decrease exponentially as m/e in- creases. As independent evidence, the Kuhn and Roth (30) reaction was used to determine the number of methyl branchings of the side chains. Application of the sulfuric acid-chromic acid mixture cleaved the methyl branches to give acetic acid. After separating acetic acid from sul- furic and chromic acid by distillation, acetic acid was quantitated by titration. The results derived from the Kuhn and Roth reaction showed an average of seven methyl branches in the alkyl chloride side chains. From the GC-MS data of alkyl chloride derivatives of compound (I), it was concluded: (1) each side chain has 40 carbon atoms; (ii) from the molecular ions and the isotope patterns, that the side chains have two functional groups per chains, hence gave the dichlorinated derivatives; (iii) because of the lack of absorption characteristic of double bonds, as evidenced by PMR and IR data, the molecular formulae of C40H78C12 39 and C40H75C12 indicated certain cyclic structures in the chains; (iv) there existed evidence of methyl branchings in the chains; (v) alkyl chloride derivatives were thermally unstable. GC-MS Studies of Alkyl Alcohol Derivatives The detailed GC-MS analysis was carried out on the alkyl alcohol derivatives of compound (I). Alkyl alcohols were derivatized with pyri- dine/hexamethyldichlorosilazane/trimethylchlorosilane 10/5/2. Figure 5 shows the gas Chromatogram of the derivatives. The corresponding mass spectra are shown in Figures 7, 9 and 11. Figure 7 shows the first com- ponent of the GC trace (Figure 5) which accounted fbr 20% of the side chains. The molecular ion was identified at m/e 738. After losing one methyl group from the TMS, ion fragments were seen at m/e 723. Pronounced peaks were seen at m/e 708, 634 and 559 by losing one or both TMS (Figure 8). The next prominent peak was at m/e 354, which was exactly half of m/e 708. This was a strong indication of a symmetric molecule. This was further supported by the fragment m/e 280, which was half of m/e 559+HI. The fragmentations after m/e 280 differed either by 42, 28 or 14 mass units (m/e 253, 239, 225, 211, 169 and 143). This suggested repetitive branch- ing patterns. The major difference between the mass spectra of GC compo- nent 2 (also 3) and GC component 1 was the pronounced peak at m/e 165 and m/e 253. The fragment m/e 165 was completely absent in component 1, and fragment m/e 253 was not an outstanding peak compared to peaks close by. The mass spectra 2 and 3 (Figure 9 and 11) looked rather similar, only spectrum 2 showed mixed character of components 1 and 3. Component 3 which accounted for 32% of the total side chains had a symmetric mass fragmentation pattern (Figure 11). The molecular ion was identified at m/e 734. Further fragments were seen at m/e 719, 705, 646, 631 and 557 4O .muoguoz c. uoa.cumou mu vouacoao no: cuuoeocuuoam mmue-;nocmou~socgo mam ooom ex; on» 55.5 8; Be: 33888 a .6 mos—postoo :53: To. E .5 do 32» on as» ea «559.8 «Cr... 23 do .5333 man: .5 ouamwu 41 .s mean—m o): Own CON Omw 00m 0mm 00m on? 00¢ >PPPPthPPDLer brlePhthLbbeL J (1‘44114 ‘— # one. mom .8 mos nNh mmm row x . rom cnw o—x cog _ z.au>rcoo pogoup< Fxxp< ogu we come» um new we acocoaeou vacuum age do escuuonm mun: .m oc:m.u .m weaned $6 45 omr oor omw oow own oom omq 00¢ hvm mON Nnm wMN_NN mmm mom mx row roq .ow tom 0mm oom 0mm mom mNN mMN mmN ofix QE oom kiwi :. mm. on— N z*2\,4§;¥7\( D ..W e M lei- “ £0 mm. nl _ .. W lg" mw_ 1”” _ a 1. mm. mNN AH" mmm rlj. mmm .. SN mmm a now 08 EN mum Rm mm» "3F. 48 .muoguoz c. won—Lumen mu vouacono no: couosocuuoam unsangaacmouoeoccu men coco ax; as» .225 he; 33.. 53588 .... ...5 to 8:23.50 2:82 32 e5 .3 22858 8 2:: e: .3 538% 3.: .—~ weaned 49 0MP Pblbbbrbbbbb on» .- mean—u Q:_ 00h 0mm com 0mm 00h mi. wow .mw hon 00w omq 00¢ woe one 0x .0N .0¢ :3. .00 0mm g «on . ecu ac.uuom a: cuuarsupou a o.~ o.H a." o.~ uc—somcuapm «one» m.~ a.” m.~ «mouuopuu woes» m.~ m.n m.m uuouapm m.o m.m m.- H.o¢ unoccue «coruuomvv omovvmouuu—amim meowumom*u umovvmouapmuu omavvmouapo-m coyumuu.u umevwmoccce-a umukuoccu513 omuvvmoccus-e cvuuosnouapu sou». Levee guumo monou.mouxpu cue: mucoEuuoep Levee use cvuuocnouspc um.».c=n soc» “mavrmoa uuucuxgongou we amo.uc¢ gape: .m «pan» 93 that the reducing end comprised two glucosamine residues (chitobiose), a-linked, followed by mannose (1-2 residues)(94). The inability of removing the last trace (1-2 residues) of mannose with a-mannosidase suggested that at least one of the mannose residues was B-linked. Permethylation Studies of the Glycopeptide To determine the linkages, the glycopeptides were subjected to per- methylation (39). Partially methylated alditol acetates were analyzed by combined gas chromatography-mass spectroscopy and the data were inter- preted according to Bjdrndal et al. (40). The identification of the partially methylated derivatives of mannose, glucose, galactose and glucosamine was established both by their retention times in the gas- liquid chromatograms and by mass spectrometry by their fragmentation patterns, and were further compared with known reference standards. Primary fragments are formed by fission between carbon atoms in the chain. Fission between a methoxylated and an acetoxylated carbon atom is pre- ferred over fission between two acetoxylated carbon atoms. Hhen a molecule contains two adjacent methoxylated carbon atoms, fission between those two atoms is preferred over fission between one of these and an acetoxylated carbon atom. Both fragments from this fission are detected as positive ions. Secondary fragments are formed from the primary ones by single or consecutive loss of acetic acid (m/e 60), methanol (m/e 32), ketene (m/e 42), and formaldehyde (m/e 30). Permethylation and mass Spec- tral analysis of the glycopeptides after acid hydrolysis, borohydride reduction, and acetylation gave evidence fer the presence of 2,3,4,6-te- tra-O-methylmannitol , 3,4,6-tri-0-methylmannitol, 2,4,6-tri-O-methylman- nitol, 2,3,4-tri-O-methylmannitol, 2,4-di-O-methylmannitol, 2,3,6-tri-O- methylglucitol, 2,4,6-tri-0-methylgalactitol and 3,6-di-O-methylgluco- saminitol (Table 6). The presence of 2,3,4,6-tetra-O-methylmannitol 94 Table 6. Molar Ratios* of Partially Methylated Alditol Acetates of Mannose, Glucose, Galactose and Glucosamine Obtained by Permethylation of the Purified Glycopeptide 2,3,4,6-tetra-0-methylmannitol 4.1 ( 8.2) 3,4,6-tri-O-methylmannitol 18.4 (36.8) 2,4,6-tri-0—methylmannitol 6.1 (12.2) 2,3,4-tri-O-methylmannitol 5.1 (10.2) 2,4-di-0-methylmannitol 3.5 ( 7.0) 2,3,6-tri-O-methylglucitol 3.1 ( 6.2) 2,4,6-tri-O-methylgalactitol 1.5 ( 3.0) 3,6-di-O-methylglucosaminitol 1.0 ( 2.0) *Molar ratios were calculated using 3,6-di-O-methylglucosaminitol as 1.0. The values in parentheses were calculated as mol carbohydrate/glycopro- tein. 95 (Figure 25) as the only tetra-methylated carbohydrate, indicated that mannose residues were the nonreducing ends. The presence of an abundant amount of 3,4,6-tri-O-methylmannitols (Figure 26, 1,2-substitued) was consistent with the ability of the glycoprotein to bind Con A. The presence of 2,4-di-0-methylmannitol (Figure 27, 1,3,6-substituted) was indicative of a branched glycopeptide. From the molar ratios (Table 6), it was obvious that the glycopeptide branched at 7 locations. 2,4,6- tri-O-methylmannitol (Figure 28, 1,3-substituted) and 2,3,4-tri-O-methyl l““* mannitol (Figure 29, 1,6-substituted) seemed to have about equal amounts. After a-mannosidase digestion, the 2,4-di-O-methylmannitol and 3,4,6-tri- O-methylmannitol derivatives were drastically reduced. This indicated “he. that the branching was mainly at the outer mannose residues which amounted to 70% (Table 5) and which were linked predominantly via 6 1+2 linkages. The middle inner core of the glchpeptides consisted of l,4-substituted glucose (Figure 30), l,3-substituted galactose (Figure 31), l,2-, l,3-, and l,6-substituted mannose. The innermost of the glycopeptide was l,4-substituted glucosamine residues (Figure 32). The inability to re- move the last trace of mannose with a-mannosidase (Table 5), suggested fiih» that there probably existed one mannose residue which was B-linked to the distal N-acetylglucosamine residue (Figure 33). Effect of Bacitracin on Cell Growth and Glycoprotein Structure Thermquasma acidbphilum, a myc0plasma-like organism, grows optimally Q; at 56°C and pH 2. The existence of the membrane glycoprotein leads to the fellowing studies on the structural and functional relationship of this procaryotic glycopeptide. The protective and lubricating roles of the glycoproteins from epithelial secretions are well known. 00 the~mem- brane glycoproteins found in Thenmoplaama acidbphilum have the similar 96 82888:”. 8:75.. 2: 5.: 52:35:52-9...53 ... m. .6. 1385-884: 8:353 3302 .362 823.8: 325...: co .568 m 3.: .3 2.3: oxE oom 0mm oom Om: oofi on now m20~IU .muillwmmmuz 3. mON ma; 01V 3.168 ozoumllllum..- I 00 1mm 11111 m ..022 J” mm. t. t. 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