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Characterization of components of two amino acid transport systems of Pseudomonas aeruginosa Sluggett, Carol Mary 1970

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CHARACTERIZATION OP COMPONENTS OP TWO AMINO ACID TRANSPORT SYSTEMS OF PSBUDOMONAS AERUGINOSA by CAROL MARY SLUGGETT B.Sc. (Microbiology) U n i v e r s i t y of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS FOR THE DEGREE OF MASTER OP SCIENCE . i n the Department of Microbiology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OP BRITISH COLUMBIA October, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Microbiology The University of British Columbia Vancouver 8, Canada Date 15 Ootober 1970 i i ABSTRACT Isolated membranes of Pseudomonas aeruginosa were found to bind radioactive isoleucine and proline, two amino acids for which active transport systems are known. The active transport systems in whole cells of this organism are energy dependentj the binding systems in isolated membrane preparations are not energy dependent, but are inducible, require magnesium ions and are stable to short periods of sonication usually sufficient to destroy whole cel l s . An assay measuring the binding of radioactive amino acid to amino acid binding protein present in isolated membrane preparations of P. aeruginosa was developed and discussed. Cells induced to high levels of amino acid transport produced equivalent levels of binding on isolation of membranes from these c e l l s . Cells repressed for amino acid transport did not lose a corresponding level of binding on isolation of their membranes, suggesting involvement of more than one protein in the active transport system of that particular amino acid. i i i Evidence was found to substantiate olaims that active transport systems are family speoific, however amino acid i t was also determined that the aliphatic .binding system was not stereospecific. Isolated membrane preparations of P. aeruginosa were found to produce adenosine triphosphate, but this energy-rich, phosphate bond compound did not appear to function in binding of radioactive amino acid to membrane preparations. Its possible functions are discussed. Several methods of isolation of proteins with binding properties from isolated membranes and from osmotic shock supernatant fluids were attempted and discussed. There were indications of a proline binding protein present^but no evidence of an isoleuoine binding protein,in the osmotic shock supernatant f l u i d . The isoleucine binding-protein, or proteins, appeared as an integral part of the cytoplasmic membrane. The data were discussed in an attempt to cla r i f y the mechanism of amino acid transport in P. aeruginosa and to define the role of the amino acid binding proteins in the phenomenon of active transport. iv TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 I. The Permease System . . . . . . . 3 II. The Mechanism of Active Transport. . • 6 III. Binding Proteins . . . . . . . . 8 MATERIALS AND METHODS 16 I. Organisms and Media . . 1 6 II. Preparation of Cell Membranes . . . . 16 III. Uptake of Labelled Amino Aoids . . . 18 IV. Membrane Binding Assay 18 V. Cold Osmotic Shock Treatment . . . . 20 VI. Separation of Binding Protein from the Isolated Membrane Fractions . . .21 VII. Lipase Treatment of Isolated Membranes. • . . . • • . . . .22 VIII. Standard Adenosine Triphosphate Assay. . . . . . . . • . . . 2 3 1. Extraction of ATP . . . .23 2. Assay of ATP 23 IX. Electron Microscopy of Isolated Membranes. 25 RESULTS AND DISCUSSION . .26 I. Osmotic Shock Studies . 26 V Table of Contents (Continued) Page II. Separation of Binding Proteins from Isolated Membrane Fractions '. 33 III. The Binding Assay 38 IV. Stability of Membrane Binding After Storage 44 V. Magnesium Requirement for Binding . . .46 VI. Stability of Membrane Binding to Sonication 48 VII. A Comparison Between Induced and Non-induced Membranes 50 VIII. Binding in the Presenoe of Transport Inhibitors 53 IX. Competition of Amino Acid Binding . . . 56 X. Inhibition of Amino Acid Binding . . . 65 XI. ATP Studies with Isolated Membrane Preparations . . . . . . .73 XII. Vesicle Formation by Isolated Membrane Preparations . . . . . . . 8 2 GENERAL DISCUSSION . .. . . 86 BIBLIOGRAPHY - ... 91 v i LIST OP TABLES Page Table 1. Isolated binding proteins. 10 Table 2. The effect of energy sources on the amino acid binding ability of isolated membrane preparations of P. aeruginosa. 42 Table 3. Stability of isoleucine binding a b i l -ity in membrane preparations under various conditions. 4 5 Table A. Isoleucine binding by sonicated membranes. 49 Table 5 « Competition of proline binding in membrane preparations isolated from induced c e l l s . 5 9 Table 6. Competition of isoleucine binding in membrane preparations isolated from induced oells. 61 Table 7. Inhibition of isoleucine binding by amino acid analogues. 62 Table 8. Structure of amino acids and analogues. 64 Table 9. Inhibition of proline binding by metabolic inhibitors. 68 Table 10. ATP production by isolated membranes of P. aeruginosa. 74 Table 11. ATP production by isolated membrane fractions in the presence of sodium azide and ADP. 76 Table 12. ATP production by isolated membrane fractions in the presence of potassium cyanide and dinitrophenol. 78 Table 13. Affects of inhibitors and ADP on binding during formation of ATP in isolated membrane preparations. 80 v i i LIST OF FIGURES 14 Fig. 1. The incorporation of C-proline into c e l l fractions of P. aeruginosa. 27 Fig. 2. Proline binding by concentrated shock supernatant f l u i d . 28 Fig. 3. The incorporation of ^C-isoleucine into c e l l fractions of P. aeruginosa. 30 Fig. 4* Isoleucine binding by concentrated shock supernatant f l u i d . 31 Fig. 5» Isoleucine binding by membrane fractions of _P. aeruginosa. 35 Fig. 6. Elution profile of isoleucine binding protein from the Sephadex G-200 column. 37 Fig. 7. Isoleucine uptake by isolated P. aeruginosa membranes: binding assay of Kaback and Deuel (1969). 40 Fig. 8. Isoleucine uptake by isolated P. aeruginosa membranes: standard binding assay. 41 Fig. 9. Isoleucine binding by membrane fractions with and without magnesium. 47 Fig. 10. Isoleucine binding by membrane fractions grown under 2 conditions. 51 Fig. 11. Uptake of ^ C-isoleucine by induced and non-induced cells of P* aeruginosa. 52 Fig. 12. The incorporation of -proline into c e l l fractions of P. aeruginosa using repressed and induced c e l l s . 55 Fig. 13. Proline binding by membrane fractions of P. aeruginosa. 66 Fig. 14. Electron microscopy of membranes. 84-85 ACKN07/LEDGEMENTS I wish to express my thanks to Dr. A. P. Gronlund, my researoh supervisor, for her help and criticism during the course of my researoh and in the preparation of this thesis, and to Dr. J. J. R. Campbell for his conoern and interest during the course of my research, I would also like to thank Mrs. Janet Halliwell for her assistance in carrying out the ATP assays, Mrs. Teresa Walters for the electron microscopy, and Mr. B i l l Page for the drawing of the figures in this manuscript. Lastly, I wish to thank the faculty, staff, and especially my fellow students for their toleranoe and fellowship throughout the course of this study. INTRODUCTION The transport phenomenon, or the selective perm-eability of the c e l l membrane, has received a great deal of attention in recent years. Kinetic studies undertaken with whole cells have established that bacteria are selectively permeable to such essential metabolites as carbohydrates, amino acids, and ions; however the precise nature of the selection mechanism i s s t i l l in doubt. Recently, successful attempts have made to isolate components of transport systems for several carbohydrates and amino acids. The components isolated so far consist of proteins or a group of proteins; the i n i t i a l function of which appears to be the binding of the substrate to be transported at the c e l l membrane. These binding proteins appear to reside on or very near the surface of the c e l l membrane, but their exact role in the transport process, after the i n i t i a l binding, i s as yet unknown. Previous studies by W. W. Kay (1968) indicated that the amino acid transport systems of Pseudomonas  aeruginosa were inducible, specific and energy depen-dent, but isolation or further characterization of the binding proteins was not carried out. It was the object of this investigation to examine an amino acid binding protein from this organism and to study the require-ments of the binding process. LITERATURE REVIEW I. The Permease System It has been known for several years that the transport of carbohydrates and amino acids by bacteria was probably facilitated by one or more proteins which were generally stereospecific, and which were under genetic and physiological control. The term "permease" has been designated to describe the specific proteins involved and the "permease system" to describe the transport process (Rickenberg, et a l . , 1956$ Cohen and Riokenberg, 1956; Cohen and Monod, 1957). Mutations in a well-circumsoribed locus governing p-galactoside permease activity on the Escherichia ooli genome have resulted in the disappearance of specific permease activity. In the presence of chloramphenicol which inhibits protein synthesis, or growth in the presence of p-fluor©phenylalanine which i s known to be incorporated solely into protein, synthesis of active permeases were effectively inhibited (Kepes and Cohen, 1962). The stereospecificity of the permease systems has also been well demonstrated. The ^-galactoside configuration i s required for induction of synthesis of the appropriate permease in E. coli? sugars devoid of this steric configuration are unable to promote induction. toside permease to the exclusion of any other permease system. In addition, the activity of the permease i s tosides have an affinity for the permease. The transport of any material implies the conveyance of some object by some other medium across, through, or away from the object's i n i t i a l position. Movement, mediated by some carrier, i s implied. As a consequence of transport across a permeability barrier, and as a means of measuring transport ability, one can note an increase in the internal concentration of the transported oompound. Conversely, the absence of a transport system is indicated by the inability of the organism to metabolize certain substrates even though i t possesses the enzyme systems required for catabolism. It i s the role of the carrier or permease to activate catalytically the equilibrium of the concentration of the substrate on either side of the membrane—as illustrated below. B-galaotosides w i l l induce the synthesis of the ^ -galac-very specific as only B^-galactosides and B-thiogalao-A + b Ab b + A outside membrane inside 5 This system appears to follow the kinetics and specificities of an enzyme-substrate reaction. The kinetics of the accumulation of metabolites within the c e l l also appear to indicate that the stereospecifio sites of the carriers act only as intermediates, not as the f i n a l acceptors. Accumulation can be blocked in several ways; for example, by adding a surface active agent, by blocking the conversion of an energy source to utilizable energy, or by blocking the synthesis of cellular protein. Under these conditions, the organism becomes cryptio even though i t possesses competent intracellular enzyme systems for the metabolism of the compound and i s able to metabolize closely related compounds at a high rate. Specific sites on the transport proteins act as catalysts for the entry of utilizable substrates (Cohen and Monod, 1957). The permease system appears to be an excellent control mechanism for the bacterium. The rate of entry of metabolizable growth compounds and consequent sub-strate utilization, enzyme induction or repression and feedback inhibition of biosynthetic enzymes can be effectively regulated. Using the j3-galaotoside permease system in E. c o l i as an example: i f lactose was hydrolyzed faster than was necessary for respiration and growth, glucose and galactose would accumulate and fin a l l y diffuse into the medium providing nutrients for competing bacterial species; therefore permease aotivity and synthesis i s repressed once intracellular levels of galactose and glucose reach certain concentrations. Induoed synthesis of intracellular metabolic enzymes i s also dependent on and parallel to induced synthesis of the permease system. The synthesis of B-galactosidase, an intracellular enzyme of E. c o l i , i s parallel to the synthesis and dependent on the activity of the galactoside permease system. It would appear that permease synthesis could be the rate—limiting step in the metabolism of galactose. The "all-or-none" response of bacterial cells to an induoer probably establishes a balance most favourable for the survival of the species (Kepes and Cohen, 1962). II. The Mechanism of Active Transport Models have been devised to explain the entry of amino acids and carbohydrates into bacterial c e l l s . The model of Britten and MoClure (1962) for amino acids i s perhaps the best known and consists of the following aspects. The model includes diffusion of the external 7 amino acid into the c e l l , subsequent collision of the amino acid with an unoccupied carrier molecule and the formation of a complex with the stereospecific carrier molecule. This complex diffuses through the c e l l until i t collides with an unoccupied site. In a reaction requiring energy, the amino acid i s transferred from the carrier molecule to the active site in the pool. As the pool level rises, free carrier becomes lower in con-centration and the pool level approaches equilibrium. The rate of formation of the carrier complex with free amino acid f a l l s until i t equals the rate required for protein synthesis. A l l such models assume an i n i t i a l substrate-binding step to a specific active site on the outer surface of the c e l l membrane. This step would not logically be the energy-requiring step in transport, as i t i s d i f f i c u l t to imagine the translocation or the production of energy at the outer' surface of the bacterial membrane. The second step is one about which very l i t t l e i s yet known: the translocation of the substrate or substrate complex across the membrane. It might be possible to follow this process using the technique of fluorescence spec-troscopy. With this method, one i s able to establish the degree of polarity of a particular region of a protein, to measure distances between groups in a protein, to determine the extent of f l e x i b i l i t y of a particular protein, and to measure the rate of very rapid conforma-tional transitions (Stryer, 1968). By following the change of the fluorescent portion of the translocation protein or i t s substrate, i t might be possible to define this l i t t l e known prooess. A variation of the single transporter theory has been suggested by Pardee (1968), and i s as follows: on binding with a substrate molecule the transport protein changes i t s conformation, opening a passageway through the membrane and thereby allowing facilitated diffusion through the hole. This conoept could also be tested by fluorescence spectroscopy. The f i n a l step in a l l membrane transport models involves the release of substrate into the cellular cytoplasm and the return of the active site, or carrier, to i t s i n i t i a l form, with the probable utilization of energy at this f i n a l step. The active transport systems require energy at one or more of these steps allowing the inflow reaction to be more effective than the outflow process (Pardee, I968). III. Binding Proteins The main difficulty confronting researchers 9 attempting to study transport phenomena has "been the isolation of the component protein or proteins oonoerned. Nossal and Heppel (1966), using cold osmotic shock treat-ment of E_. c o l i , were able to demonstrate non-dialyzable membrane fractions and non-dialyzable factors released into the shock flu i d which functioned in the active transport of inorganic ions, certain sugars and amino acids. The released factors were protein in nature and several have been isolated and characterized as having substrate-binding a b i l i t i e s . Using Heppel's technique, Pardee (1966) isolated a sulfate ion binding protein from Salmonella typhimuriumt a B-galactoside transport protein has been isolated by Fox and Kennedy (1965)5 and a leucine, isoleucine, and valine binding protein was isolated by Anraku (1968). A l i s t of isolated transport binding proteins i s shown in Table 1. The exact location of these "shocked-off" proteins i s questionable. Heppel (I967) has suggested that this group of proteins i s found on or near the membrane surface, perhaps in the periplasmic space between the c e l l wall and c e l l membrane. Theoretically then, these proteins, while not mediating the entire transport 10 Table 1. Isolated binding proteins BINDING SUBSTRATE ORGANISM SOURCE REFERENCES Leuoine E. c o l i shook fl u i d Piperno & Oxender, 1966 Ibid., 1968 Leuoine E. c o l i shock f l u i d Anraku, 1967 Ibid., 1968 Arginine E. c o l i W shock f l u i d Wilson & Holden, 1969 Histidine S. typhi- shock f l u i d Ames & Roth, 1968 murium Ames & Lever, 1970 Sulfate £>. typhi- shock f l u i d Pardee, 1966 ion murium Ibid., 1967 Phosphate E. co l i shock f l u i d Medveczky & Rosenberg, ion 1969 Galactose Lactose Sugars-PEP-P-transferase E. c o l i shock f l u i d Anraku, 1967 Ibid., 1968 E. c o l i membrane Fox & Kennedy, 19^ 5 E. c o l i membrane Kundig, Ghosh & Rose-man, I964 PEP system S. typhi- membrane murium PEP system S. aureus membrane Kundig, Ghosh & Rose-man, 1964 Hengstenberg, Egan, Morse, 1968 PEP system A. aerogenes membrane Sapico, Hansen, Walter, Anderson, 1968 aPEP-P-transferase phosphoenol pyruvate-phospho-transferase 11 process, may be the i n i t i a l recognition site for specific transportable substrates. Since this group of proteins composes approximately 5 per cent of the total cellular protein (Nossal and Heppel, 1$66) and also includes nuoleases and phosphatases, i t i s inconceivable that every potentially metabolizable substrate w i l l have a binder protein in this group. That i s , there probably i s not a single transport mechanism applicable to each transportable substrate. Underlying a l l of the reports of the isolation of these proteins (Table l ) i s the following question: are the isolated proteins actually involved in transport? Most of the evidence linking these proteins to the transport phenomenon i s indirect. Transport-negative mutants lacking a specific binding protein or proteins are unable to transport certain substrates. In some cases where more than one protein i s required for transport, a mutation affecting one of these proteins w i l l render the entire system inoperable. Induction of protein synthesis by the specific substrate i s often the only means by which a transport protein may be present in a c e l l . Reversible inhibitors such as substrate analogues, which may induce synthesis of the transport protein, but 12 w i l l not "be transported by i t , and also protein reactive reagents w i l l block the binding of the substrate to the appropriate protein and hence interfere with transport of the substrate into the cells. However, these are a l l indirect pieces of evidence and i t i s s t i l l not known i f transport actually results from the activity of binding proteins. The demonstration of the role of a protein or group of proteins in transport could be carried out by recon-stituting the system; that i s , by adding the necessary component parts to a transport-negative system. Most applications of this approach have had only qualified success. Anraku (1968) has found that the restoration of galactose transport to osmotically shocked E. c o l i does not depend entirely on the readdition of binding protein alone; another non-dialyzable, non-binding fraction of the shock f l u i d must also be added to reconstitute complete transport activity. Pardee (1968) reported only partial success in restoring sulfate transport to shocked S. typhimurium, but Kaback (1968) has been able to fully reconstitute the sugar transport system in E_. c o l i . It i s interesting to note Kaback's success— the phosphotransferase system i s not a single protein, 13 whereas the systems of Anraku and Pardee presumably are. Further attempts to isolate the transport systems in bacteria may yield several proteins which constitute several steps in the transport process. A binding protein may be required to attach the substrate close to the c e l l membrane, with a second, energy-requiring, enzyme-catalysed step required to translocate the substrate across the membrane to the c e l l interior. Kaback (1970) has noted that surface phenomenon, i.e. bacterial chemo-taxis, which i s not directly related to the transport process, but does involve the same degree of speci-f i c i t y and i s subjected to the same type of genetic control as the binding proteins, may also function in the transport process. Knowledge of the exact role of these proteins in the transport process should come as a result of more detailed studies of the properties of the proteins involved. Most of the proteins isolated have not yet been studied closely. The binding proteins which are not affected by inhibitors of energy-linked processes may serve merely as attachment points on or near the membrane surface prior to actual transport; the energy requiring steps to follow. Pardee's sulfate-binding 14 protein has not shown enzyme-like activities, and in particular, energy-linked reactions were not required to bind sulfate ion to the protein. In addition, the binding protein was not soluble in l i p i d solvents and therefore i t probably does not funotion by diffusing across the membrane with i t s bound substrate. Conformational changes in the sulfate-binding protein did not occur when sulfate ion was added to isolated preparations. A complex mechanism of sulfate transport i s anticipated from these results (Pardee, 1968). In order to relate struoture to funotion, amino acid sequence studies to determine primary struoture should be carried out on isolated binding proteins. Secondary structure bonding and tertiary and quaternary structure folding might then be correlated to the primary structure to produce an overall illustration of the protein. Studies to determine the reacting groups at the active site are important to the understanding of the protein's function. How does i t recognize other molecules and select the correct one as i t s substrate? Wherever a protein can be crystallized from i t s environment, X-ray crystallography can be used to determine folding and shape of the moleculej diffusion, sedimentation and 1 5 viscosity measurements w i l l determine shape and size, and gel f i l t r a t i o n w i l l determine size. Its function might be related to structure more concretely by the use of fluorescence spectroscopy. The i n i t i a l problem, however, i s the isolation of the protein from i t s cellular environment. More complete descriptions of the transport pheno-menon and of isolated transport proteins of various metabolites from microorganisms are very adequately presented in several review articles (Britten and McClure, 1962; Kay, 1968; Kaback, 1970). 16 MATERIALS AND METHODS I. Organisms and Media Pseudomonas aeruginosa (ATCC 9027) was used through-out this study. Cells required for experimental pro-cedures were grown in Roux flasks for 12 hours at 30 C in 100 ml of minimal medium containing 0.3$ NH^ HgPO^ , 0.2$ KgHPO^ and 0.5 ppm iron as PeS0^.7H20. The medium was adjusted to pH 7.3 with 20$ KOH. Glucose and MgSO^^RgO were added separately after sterilization from 40$ and 10$ stock solutions respectively, to give fin a l concentrations of 0.4$ and 0.1$. The flasks were inoculated with 1 ml of a 10 hour culture previously grown in the same medium. When high levels of amino acid transport activity were required, the minimal medium was supplemented with 0.075$ of the appropriate amino acid. The culture was routinely checked for purity and the production of pyocyanin by streaking cells onto King's medium (King, Ward and Raney, 1954). II. Preparation of Cell Membranes Cells were harvested from the logarithmic phase 17 of growth (12 hours) by centrifugation for 15 minutes at 7,000 x g at room temperature and were washed once with minimal salts medium without added glucose or magnesium. The concentration of cells was determined by measuring the optical density at 660 nm with a model B spectro-photometer (Beckman Instruments Inc., Fullerton, C a l i f . ) . Cells were resuspended to an optical density of approx-imately 50 . ^  [approximately 20 mg dry weight (of cells) per mlj in 0.5 M potassium phosphate buffer (pH 6.6). Deoxyribonuclease (final concentration approximately 60 ug per ml) was added to the c e l l suspensions prior to passage through a French pressure c e l l (Aminco, Silver Spring, Md.). Whole cells were removed from the extract by centrifugation at 6,000 x g for 15 minutes at 4 C. Cell membranes were separated from the cytoplasmic constituents by centrifugation at 22,000 x g for 40 minutes at 0 C. Membranes were resuspended and washed in 0.5 M potassium phosphate buffer (pH 6.6), reprecipitated by centrifugation at 22,000 x g for 40 minutes at 0 C, and stored as a pellet at 4 C until required. III, Uptake of Labelled Amino Aoida 14 The incorporation of C-amino acids into whole c e l l s 9 protein and pools was determined by the f i l t r a t i o n pro-cedure of Britten and McClure (1962). Cells were filtered, on a 0.45 um pore size f i l t e r (Millipore Corp., Bedford, Mass.) in a Tracerlab E8B precipitation apparatus (Tracerlab, Waltham, Mass.) and immediately washed with 2 ml of minimal medium. This procedure did not remove pool amino acids (Kay, 1968). Dried f i l t e r s were placed in vials containing 5 or 10 ml of scintillation f l u i d (Liquifluor, New England Nuclear Corp., Boston, Mass.) and the v i a l oontents were assayed for radioactivity in a model 725 liquid scintillation spectrometer (Nuclear Chicago Corp., Des. Plaines, 111.). IV. Membrane Binding Assay The binding assay was similar to that described by Kaback and Deuel (1969). Membranes, prepared as above, were resuspended in 0.5 M potassium phosphate buffer (pH 6.6) containing 0.01 M MgSO^  |^ 2-10 ml, approximately 10 mg per ml ( dry weight of membranes )J. One to 3 ml samples were incubated at 30 C in 5 ml beakers and mixing was aohieved with an underwater stirrer (Bronwill Industries, 19 Rochester, N. Y.). L-lroline-U- 4C (265 mCi/mmole) or L-isoleucine-U- 1 4C (262 mCi/mmole) was then added at 6.53 x 10"~?M (specific aotivity 2.0-2.5 uc per ml) and the incubation oontinued for 1-2 hours. Magnesium was adjusted to a fi n a l concentration of 8 x 10""^ M. After incubation, samples, usually 0.5 ml, were pipetted into an equal volume of ice cold 10$ trichloroacetic acid or the reaction mixtures were immediately layered onto 1.2 x 15 cm columns containing Bio-Gel P-10 (Bio-Rad Labora-tories, Richmond, Calif.) which had been previously equilibrated with 0.5 M potassium phosphate buffer (pH 6.6) containing 0.01 M MgS0^.7HgO. The columns were eluted with the same potassium phosphate buffer. Eluates were collected in 1 ml amounts and assayed for radioactivity and protein. The columns were packed such that void and excluded volumes were approximately 10-12 ml. Before reuse, the columns were dismantled and the beads washed and decanted at least 3 times in d i s t i l l e d water and once in 0.5 M potassium phosphate buffer (pH 6.6) containing 0.01 M MgSO^ .THgO before resuspension and repacking of the columns in the above buffer. Por measurement of radioactivity, 0.1 ml of each column eluate was counted in 10 ml scintillation f l u i d or 20 alternatively 20 ul duplicate samples were plated at infinite thinness onto stainless steel planchets and counted with a thin end-window Geiger tube attached to a model 181A scaler equipped with an automatic gas-flow counter (Nuclear Chicago Corp., Des Plaines, 111.). Protein concentrations were determined by the method of Lowry et al.(1951). V. Cold Osmotic Shock Treatment The method was adapted from the cold osmotic shock technique outlined by Heppel (1967). Washed cells (approx-imately 1 gram wet weight) were resuspended in 40 ml of 0.033 M sodium phosphate buffer (pH 7.2) to which was added an equal volume of sucrose in 0.033 M sodium phosphate buffer (pH 7.2), to bring the f i n a l suorose concentration to 20$. To this mixture was added 0 .1 M disodiumethylenedeaminetetraacetic acid to the desired fi n a l concentration (usually 0.1-5.0 umoles per ml). The mixture was incubated on a rotary waterbath shaker (New Brunswick Scientific Co. Inc., New Brunswick, N. J.) at 30 C for 10 minutes, then centrifuged at 13,000 x g for 15 minutes at 4 C. The supernatant f l u i d was separated and the well-drained pellet rapidly dispersed 21 in 80 ml ice cold 5 x 10 M MgClg. The suspension was gently stirred on ice for 10 minutes then centrifuged at 13,000 x g for 15 minutes at 4 C, The supernatant fl u i d was referred to as the "shock supernatant" and the pellet as the "shocked c e l l s " . VI. Separation of Binder Protein from the Isolated  Membrane Fractions Membrane preparations, subjected to the membrane binding assay and removed from the phosphate buffer by centrifugation at 22,000 x g at 0 C for 40 minutes, were resuspended in d i s t i l l e d water with sodium lauryl sulfate added to give a fi n a l concentration of 1 per cent. This mixture, while being continously stirred, was incubated at 30 C for 1 hour, centrifuged at 100,000 x g for 1 hour at 0 C, and the supernatant f l u i d and pellet assayed for radioactivity and protein. Sephadex G—200 columns (Pharmacia, Uppsala, Sweden) were prepared and eluted with a 0.5 per cent sodium lauryl sulfate solution i s d i s t i l l e d water, and calibrated with a dye solution in 0.5$ sodium lauryl sulfate con-taining blue dextran (molecular weight 2 x 10^), yellow dextran (molecular weight 4 x 10^), and cytochrome C 22 (molecular weight 1.2 x 10 ). A 1.0 ml sample of the above 100,000 x g supernatant fl u i d together with 0.5 ml of the dye solution was eluted from the column; 1.3 ml eluates were collected on an automatic fraction collector (LKB-Produkter AB, Stockholm-Brommal, Sweden) and assayed for protein and radioactivity. VII. Lipase Treatment of Isolated Membranes Isolated membrane fractions were treated with lipase in an effort to release amino acid binding proteins. Lipase, isolated from Geotrichum candidum, glycerol ester hydrolase (3.1.1.3.), was purchased from Miles Laboratories, Elkhart, Ind. The pH optimum of the enzyme was reported to be 5.6 in 0.05 M sodium acetate buffer and one unit of enzyme activity corres-ponded to 0.05 meq fatty acid released from olive o i l after 2.5 hours at 30 C. The enzyme was added at a f i n a l concentration of 1 mg per ml to membrane fractions resuspended in the appropriate buffer, the magnesium concentration ad-justed to 8 x 10"^ M and the "^C-amino acid to a f i n a l concentration of 6.53 x 10~^ M. Incubation was carried out as previously outlined, after whioh the reaction 23 mixtures were passed through Bio—Gel P-10 columns previously equilibrated with the reaction buffer. VIII. Standard Adenosine Triphosphate Assay 1. Extraction of Adenosine Triphosphate (ATP) ATP was extracted from membrane fractions by aspirating a 0 .5 ml sample into boiling 0.02 pi Tris-HCl buffer (pH 7.8) and boiling 10 min. The mixture was cooled on ice, centrifuged at 8,000 x g for 15 min. at 4 C> and the supernatant fluids were stored at - 2 0 C unti l the ATP assays were carried out. According to Holm-Hansen and Booth (1966), there i s no breakdown of ATP in extraction periods of up to 30 minutes. 2 . Assay of ATP The luciferin—luciferase mixture was prepared by grinding 10 mg of dessicated f i r e f l y t a i l s (Sigma Chemical Co., St. Louis, Mo.) with 1 .0 ml 0 .1 M arsenate buffer (pH 7.4) (Sigma Chemical Co.) for 1 hour on ice. The mixture was centrifuged at 15,000 x g for 30 minutes at 4 C and the clear yellow supernatant stored at 4 C for at least 14 hours prior to use (but not longer than 48 hours). 24 ATP was assayed by a modification (J.J.R. Campbell and J. Halliwell, unpublished data) of the method of Stanley and Williams (19^9)• A 1:1:1 mixture of sodium arsenate buffer [o,l M arsenate, 0.04 M magnesium sulfate (pH 7.4)] * phosphate buffer £o.01 M phosphate, 0.04 M magnesium sulfate (pH 7.4)]s d i s t i l l e d water, was prepared and dispensed in 3 ml aliquots in scin-t i l l a t i o n vials to be used in the assay. The vials were equilibrated at 4 C for 1 hour. A sample of a freshly-prepared standard ATP (approximately 2 x 10 M) or 25 ul of the membrane extract or diluted extract was added to each v i a l . The scintillation spectrometer (UNILUX II, Nuclear Chicago Corp., Des Plaines, 111.) was set for counting tritium with a preset time of 0.100 min. The counter was switched out of coincidenoe and used on the "fast print setting". A 50 ul sample of the enzyme preparation was added to the v i a l at zero time, the v i a l was shaken and placed in the sample well. After 15 seconds, counting commenced. The f i r s t 6 second count was discarded, the background subtracted from the second and third counts, and their average taken as the reading for that sample. A linear c a l i -bration with good reproduceability was obtained by this method. IX. Electron Microscopy of Isolated Membranes Membrane suspensions were fixed and stained by several different methods. One set was fixed in 2.5 per cent glutaraldehyde in cacodylate buffer for 1 hour, then post-fixed in 1 per cent osmium tetraoxide for 15 minutes (Pease, 1964). A second set of membranes was fixed in unbuffered potassium permanganate for 15 minutes (Pease, 1964), and a third set was fixed simultaneously with 2.5 per cent glutaraldehyde and 1 per cent osmium tetraoxide (Hirsch and Fedorko, 1968). After embedding, sectioning and deposition on copper grids, sections were stained for 1 minute with 5 per cent uranyl acetate in d i s t i l l e d water diluted 1:1 with 95 P Q r cent ethanol, followed by lead citrate for 4 minutes (Fahmey, 1967). After drying, the preparations were examined in a Phillips 300 electron microsoope at 60 KV. 26 RESULTS AND DISCUSSION Attempts were made to isolate an amino acid binding protein from shock flu i d aocording to the method of Heppel (1967) previously outlined and also fr6m the isolated membrane fraction of cells grown in the presence of the particular amino acid. I. Osmotic Shook Studies rate of The initial^incorporation of radioactive proline into whole cells and the incorporation after cold osmotio shock treatment are. compared in Fig. 1. The rate of transport decreased 2 fold (from 8.12 x 10"^. : to 3.51 1 10 ^  moles per min.-mg dry weight of c e l l s ) , and the cells were s t i l l viable when plated on King's agar after the shook treatment. The shock f l u i d , after concentration with polyethylene glycol (exclusion molecular weight 20,000; dialysis tubing exclusion molecular weight 12,000), contained 5*85 mg protein per ml and was subjected to the binding assay and sub-sequent gel f i l t r a t i o n . From data presented in Fig.2, and data obtained from trichloroacetic acid preci-pitation of concentrated shook fl u i d , there appeared to be a proline binding protein present with a Figure 1. The incorporation of C-proline into c e l l fractions of P. aeruginosa. Whole cells were preincubated 15 minutes at 30 C in 20 ml gluoose minimal,, medium, after which proline was added to a fin a l concentration of 5 x 10~ M, specific aotivity 0.0825 uc/reaction ml. One-half ml samples were immediately filtered through a 0.45 u pore size Millipore f i l t e r on a Tracerlab precipitation apparatus, washed with 2 ml of glucose minimal media and assayed for radioactivity. At the same time intervals, one-half ml samples were precipitated with an equal volume of cold 10$ trichloro-acetic acid, filtered, washed and assayed for radioactivity. A, before and B, after cold osmotio shock treatment. Symbols: incorporation of radioactivity into O——O, whole cells; D — O , protein; £ & cold t r i -chloroacetic acid soluble pool. Figure 2. Proline binding by concentrated shook - supernatant f l u i d . Shock f l u i d , concentrated with polyethylene glycol, was incubated for 1 hour at 30 C in the standard binding assay, then filtered through A, Bio-Gel P-60, exclusion limit 60,000 and B, Bio-Gel P-10, exolusion limit 10,000. Symbols: o O radioactivity} D---Q , protein. i — T — i — i — i — i — r 1 — i — i — i — i — i — r i Cn i i i i I 1 I I fl I I I I L_ 2 4 6 8 10 12 2 4 6 8 10 12 COLUMN ELUATE (ML) 29 molecular weight greater than 60,000, the exclusion value for Bio-Gel P-60. Repeated attempts to purify the protein further or to reproduce comparable results met with failure. In comparison, data for the attempted isolation of isoleucine binding protein from concentrated shock flui d are presented in Fig. 3 and 4. The rate of iso-leucine transport was reduced by a factor of 16 after cold osmotic shock treatment (Fig. 3). Transport rates were determined after 1 minute incubation with the particular amino acid. No evidence of an aliphatic binding protein was found when the binding assay was carried out on the concentrated shock flu i d (Fig. 4). The cold osmotic shock treatment, the concentration of shock f l u i d , and the binding assay with concentrated shock f l u i d were repeated many times; the concentrations of EDTA in the osmotic shock procedure were varied, times and temperatures of incubation with EDTA were also varied, several methods of concentration of the shock f l u i d were attempted, including DiafloJ) ultra-f i l t r a t i o n and the addition of Sephadex powder, but a l l with negative results. There would appear to be, from the data obtained, 30 1 1 : — i 1 — r M I N U T E S Figure 3. The incorporation of "^C-isoleucine into c e l l fractions of P. aeruginosa. See Fig. 1 for details of technique. A, before and B, after cold osmotic shock treatment. Symbols: incorporation of radioactivity into O——O , whole cell's; • •, protein; & 6 cold trichloroacetic acid soluble pool. Figure 4. Isoleucine binding by concentrated shock supernatant f l u i d . Shock f l u i d , concentrated with polyethylene glycol, was incubated for 1 hour at 30 C in the standard binding assay, then filtered through A, Bio—Gel P-60, exclusion limit 60,000 and B, Bio-Gel P-10, exclusion limit 10,000. Symbols: O——o, radioactivity; •—-O , protein. 32 two quite different meohanisms for the transport of proline and the aliphatic amino acid, isoleucine. The data indicate the possible presence of a proline binding protein in the shock supernatant fluid; no evidence was found of an isoleucine binding protein in shock f l u i d . The proline transport system in P. aeruginosa may consist of a single "recognition" protein on or near the outer cytoplasmic membrane surface, whereas the isoleucine transport system may be more intimately associated with the membrane i t s e l f as a single protein or group of proteins or possibly as a protein-lipid complex. It i s also possible to suggest that the isoleucine binding protein i s present in the shook fl u i d , but i s less stable once separated from i t s cellular milieu or that for binding to be expressed, a component from the shock fluid as well as one from the membrane fraction, must be present. Data from Kay and Gronluhd (196%) tend to support the theory that isoleucine binding protein may. be unstable in the shook flu i d after cold osmotic shock treatment. These authors have reported that of a l l the transport systems of P. aeruginosa, the aliphatic permease system i s the least stable to carbon or 33 nitrogen starvation. The rate of transport of valine and leucine decreased when the organism was starved for glucose and ammonium ions. These authors, however, stated that the changes were not due to destruction of the transport systems, but were due to transient alterations in the cellular environment, perhaps a metabolite in the intracellular pool. A l l other transport systems were extremely stable to nutrient deprivation (proline transport decreasing by only 10 per cent after 10 hours starvation) leading these authors to conclude that the activity of the various systems was not subject to identical regulation mechanisms. II. Separation of Binding Proteins from Isolated  Membrane Fractions Two general methods were used in an attempt to solubilize l i p i d from the lipoprotein mosaic comprising the c e l l membrane. An enzyme from-the fungi, Geo-trichum candidum, glycerol ester hydrolase (3.1.1.3«)» was used in i n i t i a l studies. The enzyme was reportedly active against long chain fatty acid, but inactive against the glycerides of lower fatty acids or lower fatty acid esters. Against membranes suspended in 0.5 M 34 potassium phosphate buffer (pH 6.6), with 0.01 M MgSO^ , the enzyme was completely inactive. It also appeared to be inactive against membranes in 0.05 M sodium acetate buffer (pH 5«5) until the pH was readjusted to less than 5.6, below which point binding of isoleucine to the membrane fraction was extremely poor (Fig. 5)» Pure preparations of lipases whioh are active above pH 5*5 were not available from commercial sources. Because of the marked decrease in amino acid binding at low pH values, this procedure was not pursued further. The second method involved incubating membranes with a detergent, sodium lauryl sulphate, at a f i n a l con-centration of 1 per cent. Both proline-induced and isoleucine—induced isolated membrane fractions were used; however, the isoleucine—induced fractions provided the more encourgging data. Isolated membranes were treated according to the standard binding assay, including passage through a Bio-Gel P-10 column. This latter step was included to remove unbound amino acid, which amounted to 71 per cent of the radioactivity present in the proline reaction mixture after incubation for 2 hours, and 95 per cent of the radioactivity present in the isc— Figure 5* Isoleucine binding by membrane fractions of P. aeruginosa. A, 0 . 5 M phosphate buffer (pH 6.6); B, 0.05 M acetate buffer (pH 5.5); and C, 0.05 M acetate buffer (pH 5.5). A l l reaction mixtures were subsequently treated with lipase for 1 hour prior to passage through Bio-Gel P-10 oolumns. Symbols: O——O » opm; • • , protein. T — i — i — i — n — r r ~ i — i — i — n — " — i — I — i — i — i — i ~ i — r 2 4 6 8" 10 12 2 4 6 8 10 12 2 4 6 8 10 12 C O L U M N E L U A T E (ML) 36 leucine reaction mixture. After treatment of the eluted protein and the bound amino acid with sodium lauryl sulphate, 68 per cent of the radioactivity was present in the supernatant fluid after centrifugation at 100,000 x g for isoleucine-induced membranes and 61 per cent for proline-induced membranes. This indicated that either the amino acid had been dislodged from the binding protein or that, in fact, the binding protein had been dislodged from the membrane fraction. Radioactive samples from the 100,000 x g supernatant fluids con-taining approximately 10 mg protein per ml were passed through Sephadex G—200 columns, and the results indicated, in the case of the isoleucine system, a binding protein or proteins of molecular weight greater than 40,000, but less than 2 x 106 (Pig. 6). The void volume of the Sephadex G—200 column was 5.0 ml, the yellow dextran peak (M.W. 40,000) was 9.0 ml and the cytochrome C peak (M.W. 1200) was 12 ml. It wi l l be noted in Fig. 6 that radioaotive and protein peaks coincide just after the void volume, but before the yellow dextran peak. This i s possibly due to incomplete solubilization of the membrane fraction by the sodium lauryl sulphate with the result that large 37 Figure 6. Elution profile of isoleucine binding protein from the Sephadex G-200 column: void volume 5 ml. Membranes from cells induced to high levels of isoleucine transport were subjected to the standard binding assay, then were solubilized with 1$ sodium lauryl sulphate. The 100,000 x g super-natant fl u i d , after solubilization, was applied to the column, equilibrated and eluted with 0.5$ sodium lauryl sulphate in d i s t i l l e d water. Symbols: Q Q , radioactivity; • • , protein. i 38 pieces of membrane together with binding protein appeared near the void volume of the column. It is also possible that blue dextran combined with the solubilized binding protein and i t s bound radioactive amino acid, thereby speeding up i t s appearance in the column eluate. This system with proline-induced membrane fractions was repeatedly negative with respect to separation of a binding protein or proteins from the intact membrane fractions. III. The Binding Assay In an effort to characterize the amino acid transport system of the organism more completely, other experiments were carried out. The binding assay, as i t eventually evolved, was one based on an assay devised for proline uptake by disrupted membrane preparations by Kaback and Deuel (l°69). Early attempts with equilibrium dialysis, as demonstrated by Oxender (1968), Anraku (1968), were ambiguous. A positive result could often be obtained after 2-4 days at 4 C, with the risk of contamination or leakage or both. The authors mentioned^ achieved equilibrium within 15—24 hours at 4 C. 39 Kabaok's binding assay oontained magnesium, glucose and radioactive amino acid. The requirement for glucose in the system was surprising, but presumably the membranes could metabolize the glucose and produce energy for the binding of the amino acid. When P. aeruginosa membranes were substituted for E. c o l i membranes, (Fig. 7), binding of isoleucine did not take place; that i s , there was no radioactivity associated with the eluted protein. However, by removing the glucose and substituting adenosine triphosphate (ATP), at a f i n a l concentration of 5 umoles per ml, and increasing the concentration of amino acid, successful binding was achieved (Fig. 8) . Glucose apparently interfered with binding of the amino acid or ATP was required, or alternatively a higher concentration of substrate was required for binding to be expressed. To test these hypotheses, ATP was removed from some reaction mixtures and glucose added to others. The results obtained are presented i n Table 2. It was apparent that binding took place without the assistance of ATP, and that glucose at the concentration used in this experiment certainly did not enhanoe, but appeared to actually inhibit the binding of the three 40 LLJ < Z ) _ J U LJL O 2 1 I Q_ U (D O I i i 1 • -/ / -\ V / K \ / / \ \ \ \ / ° \ / 1 \ \ / \ \ — / 1 X X / p I \ / / / J ^ 1 1 I N n » LU r— < 5 . 0 U LL O 2 . 5 ± UJ H o CL CD 2 2 4 6 8 1 0 C O L U M N E L U A T E ( M L ) Figure 7. Isoleucine uptake by isolated P. aeruginosa membranes: binding assay of Kaback and Deuel (1°69), The binding assay included glucose at 3.5 x 10""3 M and isoleucine at 8.7 x 10-6 M # Filtration was through Bio-Gel P-10 beads. Compare with Fig. 8. Symbols: O——0> radioactivity; and 0--0 » protein. 41 COLUMN ELUATE (ML) Figure 8. Isoleucine uptake by isolated jP. aeruginosa membranes: standard binding assay. The binding conditions included 8 x 10"-^  M magnesium, 6.53 x 10-5 M isoleucine and 5 um/ml ATP (final concentrations). No glucose was added as compared with conditions described in Fig. 7. Filtration was through Bio-Gel P-10 beads. Symbols: O O » radioactivity; and protein. Table 2. The effeot of energy sources on the amino acid binding ability of isolated membrane a preparations of P_. aeruginosa CPM MG MEMBRANE PROTEIN 14C-AMIN0 ACID -ATP +ATP +GLUCOSE Proline 4390 2115 1600 Isoleucine 2352 1927 218 Aspartate 1341 1263 234 Cell membranes were isolated as previously described from cells induced to proline, where proline was to be bound to the membrane preparation, isoleucine or aspartate (0.075$ fin a l concentration). The standard binding assay was used throughout with ATP, when present, at 5 umoles/ml f i n a l concentration and glucose at 3.5 x 10-3 M fin a l concentration. No ATP was present in the glucose reaction mixtures. After incubation ( l hour), reaction mixtures were layered onto Bio-Gel P-10 columns and the results presented in the table calculated from the peak tubes where radioactivity ooincided with the bulk of the protein. 43 amino acids under study. Aspartio acid was included in this study to ensure that the phenomenon measured was not solubility of isoleucine, an aliphatic amino acid, i n the l i p i d layer of the membrane; or of proline, a basic amino acid, in the lipoprotein portion. Since similar results were obtained with aspartate, an acidic amino acid, i t was assumed that binding of an amino acid to a binding protein or proteins } and not solubilization, was taking place. It i s possible that ATP did not actually inhibit binding, but, in fact, facilitated dissociation of the amino acid-protein complexes, presumably on the inside of the membrane. As P. aeruginosa membranes contain glucose oxidase (Campbell, Strasdine and Hogg, l°6l), then the same may apply to glucose. That i s , the energy gained from electron transfer may have allowed dissociation. The actual point of energy requirement in the active transport of amino acids i s not known, however from these data in P. aeruginosa, i t would not appear to be associated with binding. These results are opposed to those obtained by Kaback and Deuel (l°69) with E. o o l i . Kabaok and Milner (1970) have found that energy provided by the conversion of D-(-)-lactate to pyruvate by 44 D-lactic dehydrogenase i s energy used for the binding of many amino acids by E. c o l i . Pyruvate, fumarate, ATP, phosphoenolpyruvate or diphosphopyridine nucleotide were ineffective as stimulators of the binding of the amino aoids tested. IV. Stability of Membrane Binding After Storage An interesting observation was made when membrane stability studies were undertaken. In order to examine the ability of the membrane preparations to bind amino acids after storing at various temperatures, 30 per cent glycerol was added as a protecting agent to one set of membrane preparations. It was discovered that glycerol protected binding ability at temperatures of —20 C and below, but actually inhibited or interfered, with binding at temperatures of 4 C and above (Table 3). The problem of interference by glycerol may be due to adsorption at the membrane surface and may account for the reduotion in binding noted at -20 C with glycerol as compared with the preparation bound without the glyoerol. The best results appeared with the membrane preparations l e f t at room temperature for 48 hours without glycerol; however, fear of 45 Table 3. Stability of isoleucine binding ability in membrane preparations under various conditions8". CPM — MG MEMBRANE PROTEIN STORAGE TEMPERATURE TIME WITHOUT GLYCEROL WITH GLYCEROL Room Temperature 0 319O 1244 Room Temperature 48 hours 4062 35 4 C 1 week 3855 428 -20 C 1 week 616 2684 -70 C 1 week 463 1352 Membranes were stored at room temperature, 4 C, -20 C and -70 C with and without 30$ glycerol for periods of time extending to 1 week, then were subjected to the binding assay as outlined in Materials and Methods. 46 contamination and enzymic aotivity over extended periods of time forced storage of membrane preparations as a well-drained pellet at 4 C without added glycerol. These studies were carried out on isoleucine-induced membranes, since previous work indicated the aliphatic system was less stable over a period of time at -70 C than was the proline system. V. Magnesium Requirement for Binding The data presented in Fig. 9 demonstrated an enhancement of isoleucine binding by the presence of magnesium ions. Cells were induced to a high rate of isoleucine transport, then harvested, washed and membranes prepared as outlined previously. One set of membranes was then resuspended in 0.5 M potassium phosphate buffer (pH 6.6) and the binding assay carried out as previously outlined, with the exception that magnesium was omitted. The Bio-Gel P-10 column was equilibrated in 0.5 M potassium phosphate buffer (pH 6.6) without magnesium. The other set of membranes was treated as usual, magnesium being added at each step mentionned. The binding of the two preparations differed as measured by opm per mg protein eluted 47 C O L U M N ELUATE (ML) Figure 9. Isoleucine binding by membrane fractions with and without magnesium. The control membrane preparation was treated as in the standard binding assay. The test membrane preparation was resuspended in 0.5 M potassium phosphate buffer (pH 6.6) without magnesium and the binding assay carried out omitting magnesium. The Bio-Gel P-10 column used for f i l t r a t i o n was prepared, equilibrated and eluted with buffer without magnesium. Symbols: » #, radioactivity; and Wh — • , protein with magnesium. O——O > radio-activity; and D-—O , protein without magnesium. 48 from the column as follows: with magnesium — 1012j without magnesium — 630. VI. Stability of Membrane Binding to Sonication In an effort to perhaps release binding protein from whole membrane preparations and to test the effeot of sonication on the binding ability of the membrane fractions, isoleuoine-induced preparations were sub-jected to 30 second, 1 minute, and 2 minutes soni-cation. The sonication was carried out for the times indioated, then 0.5 ml was withdrawn for the binding assay and 0.5 ml withdrawn for f i l t r a t i o n through a 0. 4fpn Millipore f i l t e r , the f i l t r a t e of which was subsequently assayed for binding activity (Table 4)« While the 2 minute f i l t r a t e appears to have some positive binding ability, the amount of protein present was approximately 0.3 mg, and the count obtained on the gas flow planchet counter was 70 per minute, which indicated that the actual result was quite possibly much lower than 500 opm per mg protein. Otherwise, the results obtained indicated that sonication did not significantly hinder the binding of the amino acid and did not markedly reduce the 4 9 A' Table 4 . Isoleucine binding by sonicated membranes . CPM — MG MEJVLBRANE P R O T E I N SONICATION TIME SONICATED MEMBRANE FILTRATE 30 seconds 60 seconds 120 seconds A 5.0 ml suspension of P. aeruginosa membranes as prepared in Materials and Methods was sonicated in a heavy-wall glass centrifuge tube immersed in an ice bath. The sonication was carried out for the times indicated using a Bronwill Scientific Sonicator, rheostat setting of 35• Sonication was carried out continuously for no longer than 30 seconds. When preparations were subjected to longer periods of sonication, a 30 second cooling period was allowed before proceeding. 1810 — 2273 — 1779 83 1760 500 50 size of the membrane fraction that was effecting the binding. VII. A Comparison Between Induced and Non-induced  Membranes A comparison was made between membrane preparations obtained from isoleucine transport induced and non-induced ce l l s . Induced cells were grown for at least 2 transfers in minimal medium supplemented with 0.075 per cent isoleucine, as a l l induced cells were treated in these experiments, while non-induced cells were grown for at least 2 transfers in minimal medium without isoleucine added. Membranes were prepared as outlined previously and subjected to the binding assay. The binding ability of the isoleucine induced membrane preparation was 2.5 times greater than that of the non-induoed preparation (Fig. 10). Experiments with oells grown in the presence of isoleucine indicated that the ability to transport this amino acid was greatly increased over the rate of transport of the amino acid with cells grown in glucose minimal medium. The rate was approximately 3 times faster in induced cells (Fig. l l ) . Kay and Gronlund (19690) have 51 4 6 8 1 0 12 C O L U M N E L U A T E ( M L ) Figure 10. Isoleucine binding by membrane fractions grown under 2 conditions. Cells were induced to isoleucine (0.075$) over 2 transfers in minimal medium as described in Materials and Methods, or cells were grown for at least 2 transfers in minimal medium without addition of iso-leucine. Membranes were isolated and the binding assay carried out as described. Symbols: Q Q, radioactivity; and • • , protein for induced membranes. • # , radioactivity; and protein for non—induced membranes. 52 z f— 1 r M I N U T E S Figure LI, Uptake of C-isoleucine by induoed and non-induced cells of P. aeruginosa. One set of cells were induced with 0.075$ isoleucine for 2 transfers, harvested, washed and uptake determined on whole cells as described in Fig. 1. The concentration of ^c-isoleucine was 5 x 10~6 u # T n e second set of cells were not induced to isoleucine transport, but were other-wise treated similarly. Symbols: incorporation of radioactivity into whole cells, • • , induced; and O — - O • non-induced. 53 shown that wild type cells of P. aeruginosa grown in th© presence of 0.1 per cent proline, coincidentally transport and oxidize proline 5 times faster than non-induced c e l l s . It would appear that the uptake of the amino acid by the binding protein, that i s , the rate of recognition, i s not the limiting step in the active transport process. This i s compatible with the finding that energy in the form of ATP i s not required for the i n i t i a l binding step. The rate limiting step in the transport process must be the point at which energy i s required. VIII. Binding in the Presence of Transport Inhibitors A similar set of experiments to the one discussed immediately above, involved the use of a competitor of proline transport, 3,4 dehydroproline. This structural analogue of proline has been shown to competitively inhibit proline transport (Kay and Gronlund, 1969a). In addition, at high external concentrations of an amino acid analogue, growth of P_. aeruginosa occurred at a greatly reduoelrate (Kay and Gronlund, 1969a)• For 3,4 dehydroproline, the inhibitory concentration appeared to be 0.5 nig per ml, that i s , no growth occurred in minimal medium after 14 hours of incubation at 37 C, and a concentration of 100 ug per ml of the analogue reduced the growth rate to approximately 6 per cent of the normal. Cells grown in the presence of transport, that i s , synthesis of the proline permease system had been repressed by the presence of 3>4 dehydroproline in the growth medium. These authors reported that both thioproline and dehydroproline were transported by the specific proline permease of the organism. Cells were grown in the presence of 100 ug per ml of 3,4 dehydroproline for 24 hours and used, as an inoculum for subsequent transfer into the same medium. Cells from this second transfer were washed 3 times, membranes isolated, and the binding assay carried out. -5 The uptake of 4 x 10 ^ M proline by repressed cells and 1 x 10"^ M proline by control, proline-induced cells i s shown in Pig. 12. The rate of uptake by the proline-induced cells was approximately 25 times (1969c)i as mentionned previously, have reported a 5 times faster rate of transport for proline-induced cells than for non-induced cells grown on glucose 3,4 dehydroproline had a greatly reduced rate of proline of the repressed c e l l s . Kay and Gronlund 55 Z O M I N U T E S Figure 12. The incorporation of C-proline into c e l l fractions of P. aeruginosa using repressed and induced ce l l s . Repressed cells were grown for at least 2 transfers in 3,4 dehydroproline (100 ug/ml); • induced cells received 0.075$ proline for at least 2 serial transfers. Thrice washed cells were then tested for their ability to take up 14C -proline as described for Fig. 1. Symbols: incorporation of radioactivity into O—-O , whole induced cells; and O—KD , protein in induced c e l l s . Incorporation of radioactivity into > # , whole repressed oells; and • • , protein in repressed c e l l s . 56 minimal medium. However, under the standard binding assay conditions, the amount of ^C-proline bound to induced membrane fractions was only 1.7 times greater than that bound to proline transport repressed membranes. This data then appears to support the data reported for the isoleucine system; binding was only 2.5 times greater for induced membranes than for non-induced membranes, but transport was increased 3 times. It would appear that the amount of proline (or isoleucine) binding protein was not the limiting factor in the rate of proline (or iso-leucine) transport observed with repressed cells; however, i f this was the recognition protein for the entire proline transport system, one would expect i t to be the limiting factor with the proline transport repressed cells since i t s levels in the membrane would be lower. These data suggest then that the presence of 3,4 dehydroproline in the growth medium must repress some other specific component of the proline aotive transport system. IX. Competition of Amino Aoid Binding Several groups, Cohen and Rickenberg (1956), 57 Britten and McClure (1962), Kay and Gronlund (l969a), have observed that amino aoid pool formation was a competitive process for structurally related a m i n o acids. A l l three groups demonstrated that strong competitive interactions were present among the various aliphatic amino a c i d B for the transport system. It was also demonstrated by Kay (1968) that amino acid specific and family specific permeases functioned at low amino acid concentrations; hence for aliphatic amino acids, the binding assay was quite probably measuring not the presence of one, but possibly two permease systems. Kay (1968) also noted that of t h e aliphatic amino acids, leucine, isoleucine, valine and alanine, leucine was transported at the fastest rate, but i t was not as effective a competitive inhibitor for isoleucine uptake in whole cells as was valine, suggesting 2 aliphatic transport systems. Proline transport, however, was thought to be a specifio process, as was shown in E. c o l i (Britten and McClure, 1962)., In order to gain more information on these processes, competition experiments using membrane preparations were carried out. Competition was effected in every c a s e by adding the competitor at 10 ^ M fi n a l concentration, 58 whereas the radioactive amino acid remained at 10 J M final concentration. A l l other aspects of the binding assay remained constant. It became apparent (Table 5) that arginine did not compete with proline to any great degree; however,3,4 dehydroproline, as expected, and isoleucine competed with proline for the binding site. A certain percentage, possibly 5 per cent of the radioactivity, could be accounted for by non-specific adsorption to membraneous material on trichloroacetic acid precipitation. Iso-leucine would not be expected to compete with proline for i t s binding protein unless isoleucine shared a non-specific general permease with proline—this was not evident from uptake data presented by Kay (1968). It i s interesting to note, however, that very high concentrations of leucine, isoleucine or valine (10~^ M) compete with aromatic transport systems in P. aeruginosa (Kay and Gronlund, unpublished data) and perhaps iso-leucine, at very high concentrations, also oompetes with proline transport. In addition, Kay and Gronlund (1969c) nave reported that the ability of the organism to transport tyrosine or isoleucine was signifioantly reduced when P. aeruginosa was grown in the presence of Table 5« Competition of proline binding in membrane preparations isolated from induced cells . COMPETITOR lO""^ CPM — MG PROTEIN $ INHIBITION Proline control 10155 — — 3,4 dehydroproline 5324 47.6 isoleucine 6536 35.7 arginine 8542 15.9 Membranes were isolated from cells induced to proline (0.075$). The binding assay was carried out as described in Materials and Methods with the competitor added at 100 times the concentration of the proline. After 1 hour incubation, 0.5 ml of the incubation mixture was pipetted into 0.5 ml ice cold 10$ trichloroacetic acid and the resulting precipitate assayed for protein and radioactivity. 60 0.1 per cent proline. It i s possible then that high concentrations of isoleucine interfere with proline transport or that a non-specific general permease i s shared by the two amino acids. Similar experiments were carried out with isoleucine (Tables 6 and 7). Arginine and proline did not compete, while valine and leucine, as expected, interfered with binding. It may be noted that while leucine was not as effective a competitive inhibitor for isoleucine uptake in whole cells as was valine (Kay, 1968), the reverse was true for isolated membrane preparations (Table 6). Leucine was 6 per cent more effective, a slight difference. It could perhaps be argued on the basis of these data that approximately 30 per cent of the total isoleuoine uptake oapacity available to the cells was via an aliphatic family specific permease; while the remaining 70 per cent was mediated by an isoleuoine amino acid specific permease. And whereas isoleucine caused a 36 per cent inhibition of proline binding, proline stimulated isoleucine binding by 14 per cent. Isoleucine may be recognized by the proline binding protein, but the reverse obviously does not occur. The reason for slight stimulation of isoleucine binding by proline and the significant stimulation Table 6. Competition of isoleucine binding in membrane a preparations isolated from induced cells . 10~3M CPM — $ $ COMPETITOR MG PROTEIN INHIBITION STIMULATION L-isoleuoine (control) 6600 L-leuoine 4189 36.6 L-valine 4616 30.1 L-proline 7551 14.4 L-arginine 8728 32.2 P. aeruginosa oells were induced to isoleucine 7b.075$) and isolated membranes prepared. Com-petition was effected by adding isoleucine at 6.53 x 10~5M and the competitors at 6.53 x K P ^ M . After 1 hour incubation, 0.5 ml of the reaction mixture was pipetted into an equal volume of ioe cold trichloroacetic acid (10$), and the resulting precipitate assayed for protein and radioactivity. 62 Table 7. Inhibition of isoleucine binding by amino acid analoguesa. Amino Cpm - Mg $ # Analogue (lOT^M) Acid Protein Inhibition Stimulation L-isoleuoine (control) L-norleucine 1-amino—eyelo-pentane—1—car-boxy l i o acid D-allc—iso-leucine D,L-X-amino-isobutyric acid leucine valine isoleuoine valine N-cyclohexyl- leucine ^-alanine D-leucine L-norvaline isoleucine leuoine valine leuoine 2873 923 3070 1406 3167 688 I684 2293 67.9 51.1 76.1 41.4 20.2 6.9 10.2 Membrane preparations induced to isoleucine were sub-jected to the binding assay and analogue concentrations made up to 100 times the concentration of the iso-leucine added. After 1 hour incubation, 0.5 ml of reaction mixture was pipetted into TCA and the pre-cipitate assayed for protein and radioactivity. 63 effected by arginine i s not apparent. Studies with analogues of isoleucine, leucine and valine were undertaken to determine their effect on the binding of isoleucine to the membrane fractions. The D or L configuration of the amino acid competitor appeared to make l i l t l e difference in the case of D and L leucine (Tables 6 and 7)« Both caused approximately 40 per cent inhibition of isoleucine binding. The recognition or binding therefore cannot be stereospecific. The presence of a long hydrophobic chain or a large bulky group at carbon number 3 appears to be required for good inhibition (Tables 7 and 8). L-norvaline has a straight chain of only 2 carbon atoms in length attached to carbon 3 and causes compara-tively slight inhibition, 20.2 per cent. On the other hand, the other analogues have large, bulky groups or long chain hydrophobic groups on carbon 3 which the isoleucine binding protein appears to recognize. Leucine and to a lesser degree, valine, can be included in this group. A l l these analogues are f a i r l y good inhibitors of isoleucine binding. The presence of additional substituents at carbon 2 negate inhibitory activity: for example, D,L-0^ -amino-64 Table 8. Structure of amino acids and analogues . AMINO ACID (or ANALOGUE) STRUCTURE L-isoleucine L-leucine CH,-CHp-CH-CH^ COOH 3 * 6 H 3 NH CH,-CH-CHp-CH=COOH -* CH^ * L-valine NHp CH,-CH-CH-CCOH 3 C H 3 D, L-^-aminoisobutyrio acid NH CH^ -C-COOH 3 CH-> 1-amino-cyclopentane-1-carboxylio acid CH 0—0H o COOH i 2 2;< CH 2—CH 2 ^ N-cyclohexyl-$-alanine L-norleucine L-norvaline ^CHp—CH NHp CH2 ^ CH-NH-CH9-CH SCH 2—CH 2 ^ COOH f a CH3-CH2-CH2-CH2-CH-COOH NH9 l * CH^ -CHg-CHg-CH-COOH D-allo-isoleucine contains the same number of carbon atoms as L-isoleucine, rearranged spatially. 65 isobutyric aoid and 1-amino-cyolopentane-l-carboxylie acid. This may be a result of steric hindrance and suggests that the amino group i s important in attachment to the binding protein. The latter two analogues appear to stimulate binding slightly, X, Inhibition of Amino Acid Binding Studies with inhibitors of active transport in whole cells were undertaken to determine their effect on binding of amino acids to membrane preparations. Sodium azide and iodoaoetamide were i n i t i a l l y used as controls, sine i t had previously been determined that energy in the form produced from glucose, or as supplied by ATP, was not required for the binding assay. Kay (1968), however, had shown these two inhibitors to inhibit active transport of amino acids into whole c e l l s . It was expected that they should have l i t t l e , i f any, effect on binding since energy was seemingly not required at this step. The results of the ex-periments with either proline or isoleucine-induced membrane preparations and 30 mM sodium azide and 1 mM iodoacetamide appear in Fig, 13. Inhibition was caused by sodium azide, generally known to be an Figure 13. Proline binding by membrane fractions of P_. aeruginosa. A, in the presence of 30 mM NaN3 and 1 mM iodoaoetamide. B, in the presenoe of 5 urn per ml ATP. Symbols: 0 0, control cpm and • o, control protein; A, • • , 30 urn NaN^  or 1 mM iodo-acetamide per ml, cpm and* • , protein; B, • • , 5 um ATP per ml cpm and* • , protein. C O L U M N ELUATE (ML) 67 uncoupler of oxidative phosphorylation and an inhibitor of electron transport; hence i t i s associated with energy—yielding or energy-dependent mechanisms of the organism. It w i l l be noted, however, that the presence of ATP did not enhance binding above the control level. Azide was possibly interfering with some membrane function which yields energy in the form used in binding. With the data obtained above, i t appeared advisable to investigate the effect of various other types of inhibitors on isolated membrane systems. Accordingly, a number of protein synthesis inhibitors, oxidative phosphorylation uncouplers and respiratory poisons were chosen (Table 9)• Membrane preparations were re-suspended and treated as for the standard binding assay; hov/ever, prior to addition of the radioactive amino acid, the mixtures were preincubated with the inhibitor at the desired f i n a l concentration. The final incubation was stopped by adding a sample of the mixture to an equal volume of 10 per cent trichloro-acetic aoid on ice. Trichloroacetic acid precipitation was used as evidence of binding activity in preference to the Millipore f i l t r a t i o n prooedure of Kabaok (1969), since precipitation was faster and appeared relatively 68 Table 9. Inhibition of proline binding by metabolic inhibitors 8 -. Cpm - Mg $ $ Inhibitor Concentration Protein Inhibition Stimulation Control 1 4325 puromycin 1.54 X 10" •ht 3830 11.5 — — oycloheximide 1.54 X 10" •\ 5265 21.7 amytal 1.54 X 10" •h 6670 54.2 dinitrophenol 1.54 X 10" •h 5370 24.2 KCN 8.30 X. 10* •** 4095 5.4 hydroxylamine 3.84 X 10" ** 8255 90.8 chloramphenicol 100 ug/ml 484O — — — 11.9 Control 12146 . NaN3 2.50 X 10" " 3 M 4130 66.0 NaN^ 2.30 X 10" 2849 76.6 iodoacetamide 1.54 X 10" " 3M 7804 35.8 p-chloro- 1.54 X 10' " 3M 4320 64.5 mercuribenzoate a Membrane preparations;;induced to proline (0.075$)>were subjected to the binding assay in the presence of the inhibitors listed. Present in each mixture was approx. 30 mg protein/ml. After 1 hour incubation samples were precipitated in 5$ TCA and assayed for protein and radio-activity. 69 similar on a cpm "basis to the f i l t r a t i o n procedure when the two methods were compared. Millipore f i l t r a t i o n yielded slightly higher cpm - mg membrane protein for any one sample. That binding was unaffeoted by t r i -chloroacetic acid precipitation indicates a strong bond linkage, perhaps covalent, between amino acid and protein. Since the binding protein and the radioactive amino acid precipitated with cold 5 Pe** cent trichloroacetic acid, i t was important to ascertain i f the results obtained were due to incorporation of the radioaotive amino acid into new protein by protein synthesis or whether the results truly indicated binding of the amino acid to the binding protein in the membrane preparation. Hence, studies were undertaken with inhibitors of various stages of protein synthesis. Chloramphenicol, which acts at the attachment point of the 30S and 50S ribosomal units, cycloheximide, which interferes with peptide chain elongation, and puromycin , which acts at the addition of the specific amino acid to the terminal adenosine of transfer ribonucleic aoid, were chosen as representatives of eaoh type of protein synthesis inhibitor. 70 Chloramphenicol and oyoloheximide (Table 9) had no inhibitory effect on the binding of the radioactive amino acid to the binding protein. This would indicate, together with the data obtained from trichloroacetic acid precipitation, that the radioactive amino acid i s not incorporated into new protein, but that actual binding to a component in the membrane i s taking place. Puromycin, on the other hand, inhibited binding by 11.5 P e r cent. This slight degree of interference may indicate a non-specific mechanism of i n h i b i t i o n — possibly steric. The antimetabolite may attach i t s e l f to some position on the membrane close to the aotive site for binding and thereby block the reaction of amino acid with binding protein. In addition, i t w i l l be noted that cycloheximide and chloramphenicol stimulated binding 21.7 and 11.9 per cent respectively. This effect may be the result of removal by these inhibitors of unspecified substances present in the membrane environment, allowing more amino acid to react with the binding protein than is available in the normal binding assay. The reported modes of inhibition for these protein inhibitors were not expeoted to be functional under the reaotion 71 conditions established during the binding assay, since there should be no functioning protein synthesis units present after c e l l fractionation and membrane preparation. Membranes were prepared and were washed without magnesium, therefore neither the intact ribosomal subunits nor the messenger ribonucleic acid necessary for protein synthesis should have been present. Hydroxylamine stimulated binding approximately 2 fold above the control level. It may function in the overall reaction: ATP + amino acid + hydroxylamine •—» aminoacyl-hydroxamate + AMP + PPi and which i s catalyzed by amino acid-activating enzymes normally found in the particle-free supernatant f l u i d of c e l l homogenates (Davie et a l . , 1956)• Hydroxyl-amine also forms the aminoacyl-hydroxymate with activated amino acids attached to transfer ribonucleic acid. That binding was not inhibited by the presence of hydroxylamine, together with the slight inhibition by puromycin, indicates that a transfer ribonucleic acid-amino acid complex, which i s cold trichloroacetic acid precipitable, was not formed. Therefore radioactivity associated with trichloroacetic acid-precipitable material was most probably *4C-amino acid bound to 72 a protein component in the membrane. The stability of the binding suggests a covalent linkage. Hydroxylamine may indeed have stimulated the binding of amino acid to protein by shifting the equilibrium of the binding reaction in favour of the amino acid-protein complex. Amytal and dinitrophenol, two compounds which affect the eleotron transport chain of most organisms, do not inhibit binding of radioactive amino acid to the membrane fragments. This was not unexpected since one would not expect a functioning electron transport or respiratory chain to be present after the drastic treatment of the membranes during preparation, although functioning cytochromes have been reported present in such membrane preparations (Kaback and Miner, 1970). That there was slight inhibition of binding by potassium cyanide was not surprising, since cyanide w i l l remove a l l available phosphorus and destroy the activity of the oxidative enzymes of the organism (Strasdine, I96I). The inhibition by azide and iodoaoetamide has been mentionned previously. It has also been suggested that together with i t s effect on substrate level phosphoryl-ation, iodoaoetamide may be interacting with sulfhydral groups present at the active site for binding on the 73 protein. For this reason, p-chloromercuribenzoate, a sulfhydral group inhibitor, was included. It was observed to have a relatively effective role in inhibition, perhaps suggesting that sulfhydral groups may be present at the active site on the binding protein. XI. ATP Studies with Isolated Membrane Preparations. Another interesting aspect of the membranes was discovered in the course of measuring adenosine t r i -phosphate levels in fractions before and after incubation in the standard binding assay. Adenosine diphosphate (ADP) was added to one reaction mixture as a control. The standard ATP assay prooedure, as outlined in Materials and Methods, was followed throughout these studies. At the outset of incubation, of the order of 4.56 x 10~^ moles ATP per mg protein were present in the membrane preparations (Table 10)• Over the incub-ation interval of 1-2 hours, this level decreased by two thirds to I . 6 5 x lO -"^ moles ATP per mg protein, indicating that possibly ATP was being utilized for binding, or more likely, that i t was being degraded by other components in the membrane, suoh as adenosine Table 10. ATP production by isolated membranes of P. aeruginosa . INCUBATION ADP MOLES ATP/MG PROTEIN TIME 5 um/ml 0 4.56 x 10" 1 1 1 hour 1.65 x 10"11 -8 1 hour + 1.39 x 10 During the standard assay for amino acid binding, samples were withdrawn for measurement of ATP levels in the reaction mixture. No 14C-amino acids were added to these reaction mixtures since they would interfere with the luciferin-suciferase assay method. Samples withdrawn were treated as outlined in Materials and Methods. ATP levels decreased from the 0 time control level in a l l cases when ADP was not added. When ADP was included in the incubation mixture, ATP levels consistently increased. Present in each incubation mixture was 27 mg protein/ml. A background level of 3.71 x 10~° moles ATP/ml present as a contaminant in the ADP used in each reaction mixture, was subtracted from the results obtained when ADP was added. 75 triphosphatase. V/hen ADP was added to the incubation mixture, however, ATP was unexpectedly produced. The background level of ATP present as a contaminant in the ADP added to each reaction mixture was subtracted from the results obtained when ADP was added. Experiments were undertaken with inhibitors of the energy production processes of the o e l l . Sodium azide did not inhibit ATP formation, in fact the ATP con-centration was 2.5 times greater than the control at 1 hour and 3 times greater at 2 hours, however the ATP level decreased slightly at 2 hours from the level at 1 hour. This decrease was no doubt due to ATP degrad-ation taking place in the membrane fraction and i t was also evident in the control without azide. Again ADP stimulated the production of ATP to levels comparable to those in Table 10. Under these conditions, ATP degradation on prolonged incubation was most marked as a 19 fold decrease occurring between 1 and 2 hours (Table 11). Sodium azide enhanced the stimulation of ATP production by ADP; that i s , there was approximately 4 times more ATP after 1 hour incubation than found with ADP alone. The results obtained after 2 hours 76 Table 11. ATP production by isolated membrane fractions in the presence of sodium azide and ADPa. INCUBATION NaN, ADP ATP PRESENT —2 TIME (hr) 1.3 x 10 M 5 um/ml moles/mg protein 0 — — 1.83 x 10" 1 — — 1.98 x 10" 2 — — 1.35 * 10" 1 + —• 5.10 x 10" 2 + — 3.98 x 10" 1 — + 1.90 x 10~: 2 — + 0.10 x 10"' 1 + + 7.90 x 10~! 2 + + 12.40 x 10": Samples (0.5 ml) were withdrawn at the times noted and treated as in the standard ATP assay described in Materials and Methods. Membranes were resuspended in 0.5 M potassium phosphate buffer (pH 6.6) with magnesium. Present in each reaction mixture were g 28 mg protein/ml. A background level of 3.71 x 10~ moles ATP/ml present in the ADP as a contaminant was subtracted from the result obtained when ADP was added. 77 incubation suggest that sodium azide functioned by decreasing the rate of ATP degradation. In another experiment, KCN, which acts on electron transport by inhibiting cytochrome oxidase, and dinitro-phenol, an uncoupler of oxidative phosphorylation, were used to determine their effect on ATP formation by the membrane preparations (Table 12). Cyanide and dinitro-phenol alone did not stimulate ATP production to the same extent as did sodium azide and also neither compound inhibited ATP degradation. When ADP was added to the reaction mixtures there was an increase of 160 times the control level after 30 minutes with potassium cyanide and 319 times the control level after 30 minutes with dinitrophenol. At 60 minutes, the amount of ATP added in the ADP overshadowed that amount produced by the membrane fractions. The increases at 30 minutes were probably degraded by the components, such as ATPase, in the membrane fractions or by exohange reactions. ATP degradation was particularly active in this membrane preparation. It i s also evident from this Table 12 when compared to Table 11, that ATP production reaches i t s highest levels at 30 minutes or before, after which time ATP degradation increases. 78 Table 12. ATP production by isolated membrane fractions 3* in the presence of potassium cyanide and dinitrophenol . INCUBATION ADP ATP PRESENT TIME (min) ™ I T O R CONCENTRATION m o l e s / m g p r o t Q i n 0 5.10 X l O " 1 1 30 — — — 6.45 X 10- 1 1 60 — 1.11 X l O " 1 1 1.33 -8 30 + X 10 60 + __b 30 KCN 5.9 X 10_3M — 8.58 X l O " 1 1 60 KCN 5.9 X 10-^ M — 0.87 X IC" 1 1 30 DNP 5.9 X 10_4M 6.81 X l O " 1 1 60 DNP 5.9 X 10""4!! . — O.84 X 10- 1 1 30 KCN 5.9 X 10~3M + 1.03 X 10-8 60 KCN 5.9 X 10"3M + b 30 DNP 5.9 X + 2.56 X lO" 8 60 DNP 5.9 X + _ b a Samples were treated in the manner described in Table 11. b —8 3.71 ^ 10"~ moles ATP/ml was degraded as well as that synthesized at 30 minutes leaving a net negative amount of ATP. 79 It was interesting to note that concurrent with the rise in ATP levels in reaction mixtures supplied with ADP, there was also a rise in binding of radio-active amino acid to membrane fractions, except in reaction mixtures containing cyanide or sodium azide (Table 13). The mechanism of ATP synthesis in the membrane fractions i s a matter of conjecture. Attempts to measure adenylate kinase in the preparations were unsuccessful. It has been reported that P. aeruginosa has cytochromes a, ag, b, c, o^, and cytochrome oxidase and this combination can synthesize ATP (Azoulay, 1964). T n e only requirement apparent i s for a group of pigments with enough difference in redox potential to furnish energy to synthesize ATP from ADP (Smith, 1961). It i s possible that such a system is in operation here. Barnes and Kaback (1970) have reported that energy formed by the conversion of D-(-)-lactate to pyruvate mediated by membrane bound D-(-)-lactic dehydrogenase is the energy source for the uptake of proline as well as other amino acids by isolated membranes of E. c o l i . Kaback also reported that the effect of D—(-)-lactate on amino acid transport is apparently not exerted through the production of 80 Table 13. Affeots of inhibitors and ADP on binding during formation of ATP in isolated membrane preparations . CPM — $ $ ADDITIONS CONCENTRATION MG PROTEIN INHIBITION INCREASE Control — 10,015 — — ADP 5 um/ml 13,625 — 3.6 NaN, 1.3 x 10_2M 4,245 57.5 ~ Control — 34,050 — — KCN 5.9 x 10~3M 13,287 61.0 — DNP 5.9 x 10-4M 34,693 — 2.0 In parallel with incubation mixtures prepared for the measurement of ATP levels, membrane fractions were resuspended and treated as outlined for the standard binding assay. Radioactive amino acid was added to these reaction mixtures, whereas i t was not added to mixtures intended for the lucerferin— luciferase assay. Present in each incubation mixture was 26 mg protein/ml. After 1 hour incubation, 0.5 ml was pipetted into 0.5 ml of 10$ ice cold t r i -chloroacetic acid and the resulting precipitate assayed for protein and radioactivity. Differences in the control cpm-mg protein values are due to the age of the membrane preparations. Proline was the amino acid bound. 81 stable high energy phosphate compounds such as ATP or phosphoenolpyruvate. This i s probably also true of P. aeruginosa as ATP had no stimulatory effect on isoleucine or proline binding. Kaback has stated that concentrative uptake of proline involved electron transport. He has also reported (Barnes and Kaback, 1970) that the |-galac-toside uptake system i s coupled to the membrane-bound D-lactic dehydrogenase via an electron transport chain, but does not involve oxidative phosphorylation. Effective oxidative phosphorylation inhibitors such as dinitrophenol, azide and cyanide inhibited lactose uptake but not as inhibitors of oxidative phosphorylation, but rather as inhibitors of proton conductors. Azide and dinitrophenol, as well as amyta], affect the electron transport chain of most organisms. Barnes and Kaback (1970) state that the inhibition of binding by cyanide and azide i s consistent with the observed anaerobic inhibition of lactose uptake by membrane preparations and indicates the involvement of oxygen as a terminal electron acceptor. The previously observed inhibition of binding by iodoacetamide and p-chloromercuribenzoate (Table 9 ) may 82 have been due to the inactivation of the binding protein rather than inhibition of energy production. Inhibition of binding by sodium azide or KCN may implicate a requirement for energy for binding from electron trans-port, however, the system in P. aeruginosa must differ markedly from that in E. c o l i as neither amytai nor dinitrophenol inhibited binding of proline or isoleucine. XII. Vesicle Formation by Isolated Membrane Preparations. Electron microscopy of membrane preparations was carried out to determine whether P. aeruginosa membranes behaved as E. c o l i membranes after isolation (Kaback and Deuel, I969). It was found by these authors that isolated membranes formed roughly circular vesicles of various sizes. A similar result was obtained with isolated membranes of P_. aeruginosa (Fig. 14). Circular vesicles formed, eaoh having a double membrane boundary. Several of the larger vesicles contained smaller vesicles and several had 2 layered double membranes, but none appeared to contain ribosomes or other intracellular or membrane-associated components. It was f e l t that the vesicles formed spontaneously, since several methods of fixation were employed during the preparation for 83 electron microscopy, and in each case, vesicles resulted. The reasons for this phenomenon are not known. The formation of vesicles by isolated membrane preparations of P. aeruginosa i s a phenomenon which raises another very basio question. How does one know which protein i s being measured-—one normally situated on the inner cytoplasmic surface or one normally on the outer periplasmic surface. Perhaps i f there i s only one protein involved in the actual translocation process, i t i s the one measured since the membrane i s approximately 70 Angstrom units wide, a size not unusual for a protein of this nature (Pardee,1967). Figure 14. E l e c t r o n microscopy of membranes. I s o l a t e d membrane prepara t ions were f i x e d and s t a ined as o u t l i n e d i n M a t e r i a l s and Methods. A . f i x e d w i t h 2 . 5 $ g lutaraldehyde f o r 1 hour, then p o s t - f i x e d i n 1 $ O S O 4 . Note the va r ious s i z e s and the double membrane surrounding the v e s i c l e s , x 1 4 4 , 5 0 0 B . f i x e d w i t h 2 . 5 $ g lutara ldehyde and 1 $ 0 s 0 4 s i m u l -taneously . Double membrane fragments are present which do not form v e s i c u l a r s t r u c t u r e s , x 8 5 , 0 0 0 C. f i x e d as i n A . Note the smal le r v e s i c l e s "budding" from the l a r g e r , 2 l ayered double membrane v e s i c l e , x 5 3 , 0 0 0 D. f i x e d i n unbuffered KUnO^. The va r ious s i z e s of v e s i c l e s can be noted, x 4 2 , 1 ) 0 0 E. f i x e d as i n D. Note the many smal ler v e s i c l e s w i t h i n the l a r g e r ones, x 1 4 4 , 5 0 0 85 86 GENERAL DISCUSSION When attempts to separate binding proteins either from shock f l u i d or from isolated membrane preparations met with limited success, i t became expedient to study the binding process in more detail, using whole membrane preparations rather than the isolated protein. Son-ication studies on isolated membrane fragments together with repeated, unsuccessful attempts to release iso-leucine transport binding protein from whole cells and membrane fragments with detergents or enzyme, have indicated that the aliphatic permease system i s quite possibly an integral part of the organism's membrane, or that i t i s unusually unstable. The proline transport binding protein i s probably less closely associated with the membrane and may, in fact, be released into supernatant fluids under certain shock procedures. The active transport systems of P. aeruginosa require a utilizable energy source in order to accumulate proline or isoleucine within the c e l l . Kay (1968) and others have shown that inhibitors of energy metabolism, such as azide and iodoacetamide, abolish transport of 87 the amino acid. However, Kay stated that the actual transport process was possibly unaffected by the inhibitor, and that i t was the ability to maintain amino acid pools that had been lost. He presented data that indicated the actual uptake of proline was an energy independent process and that another energy dependent function was involved, presumably to keep the amino acid in the cell. More recently, Barnes and Kaback (1970)> using isolated membrane vesicles of E_. coli, have 'suggested that laotose uptake and possibly the uptake of certain amino acids (Kaback and Milner, 1970), is dependent upon the presence of a functioning electron transport chain, proton acceptors and the presence of D-(-)-lactic dehydrogenase in the membrane. The conversion of D-(-)-lactate to pyruvate by this enzyme provides the energy required for binding of lactose or the amino acids. Oxidative phosphorylation or the formation of i high energy-rich phosphate bonds such as those present in ATP are not an integral part of this system. The data presented here indicate that the uptake system for proline and isoleucine is slightly different in P. aeruginosa. Energy in the form of exogenous 88 ATP was not required for binding of proline, isoleuoine or aspartic acid; however, binding was inhibited by sodium azide, iodoacetamide and, to a lesser degree, by potassium cyanide. Kaback has reported inhibition of binding or lactose uptake by these compounds as inter-ference with proton acceptance and hence with the terminal portion of the electron transport chain in E. c o l i . Kay (I968) however, stated that azide inter-fered with intracellular amino acid pool maintenance, and not amino acid uptake. It would appear, therefore, that P. aeruginosa has some form of energy requirement for amino acid binding, but that the system i s different from that proposed for E. c o l l . The systems are similar only in that they both do not require high energy-rich phosphate bonds for binding. The results obtained from competition experiments with proline-induced membranes were unexpected. In uptake studies with whole cells, isoleucine and arginine did not compete with proline; however, using isolated membrane fractions, isoleucine and arginine are effective competitors for proline binding. This suggests either that the i n i t i a l binding may not be as specific a process as was once hypothesized or that high concentrations of 8 9 isoleucine cause interference with the proline uptake system in P. aeruginosa. Valine and leuoine oompet'e with isoleucine for the active transport system of aliphatic amino acids in whole cells as reported by Kay ( 1 9 6 8 ) and also competed for the isoleucine transport binding protein in isolated membrane fractions. Analogues of isoleucine, leucine and, to a lesser degree, valine, also afforded competition for isoleucine indicating the aliphatic amino acid transport system was family specific, although i t was found to be non-stereospecific. The apparent production of ATP by P. aeruginosa membrane fragments is an interesting phenomenon, which possibly has l i t t l e to do with the binding of amino acid to the binding protein present in the membranes. ATP production was unaffected by azide, indicating the possible presence of a functioning electron transport chain, which may be able to furnish ATP to the membrane vesicle. The function of this ATP and the mechanism of its formation is unknown. It is possible that this energy might be utilized for the release of bound amino acid from the membrane to the intracellular pool or for the 90 translocation of membrane transport proteins to their original configuration after transport of their substrates. 91 BIBLIOGRAPHY 1. Ames, G.P., and J.R. Roth. 1968. H i s t i d i n e and aromatic permeases of Salmonella typhimurium. J . B a c t e r i o l . £6: 1742-1749. 2. Ames, G.F., and J . Lever. 1970. Components of h i s t i d i n e transport: h i s t i d i n e - b i n d i n g p r o t e i n s and hisP p r o t e i n . Proc. N a t l . Acad. Science 66: 1096-1103. 3. Anraku, Y. 1967. The reduction and r e s t o r a t i o n of galactose transport i n osmotically shocked c e l l s of E s c h e r i c h i a c o l i . J . B i o l . Chem. 242: 793-800^ 4. Anraku, Y. 1968. Transport of sugars and amino acids i n b a c t e r i a . J . B i o l . Chem. 243* 3116-3135. 5. Azoulay, E. I964. Influence des cond i t i o n s de cu l t u r e sur l a r e s p i r a t i o n de Pseudomonas  aeruginosa. Biochim. Biophys. Acta 92: 458-464. 6. Barnes, E.N., and H.R. Kaback. 1970. B-galactoside transport i n b a c t e r i a l membrane preparations: energy coupling v i a membrane-bound D - l a c t i c dehydrogenase. Proc. N a t l . Acad. Science 66: II9O-II98. 7. B r i t t e n , R.J., and P.T. McClure. 1962. The amino ac i d pool i n E s c h e r i c h i a c o l i . B a c t e r i o l . Rev. 26: 292-335. 8. Campbell, J.J.R., L.A. Hogg, and G.A. Strasdine. 1962, Enzyme d i s t r i b u t i o n i n Pseudomonas aeruginosa. J . B a c t e r i o l . 83j 1155-1160. 9. Cohen, G.N., and H.V. Rickenberg. 1956. Concen-t r a t i o n s p e c i f ique re'versible des amino acides chez E s c h e r i c h i a c o l i . Ann. I n s t . Pasteur 91* 693-720. 10. Cohen, G.N., and J . Monod. 1957. B a c t e r i a l permeases. B a c t e r i o l . Rev. 21: 169-194. 92 11. Davie, E.W., V.V. Koningsberger, and F. Lipmann. 1956. The isolation of a tryptophan-activ-ating enzyme, from pancreas. Arch. Biochem. Biophys. 65_: 21-38. 12. Fahmey, A. 1967. An extratemporaneous lead citrate stain for electron microsoopy. In Arceneaux, C«, ed., Proc. 25th Ann. Meeting Electron Microscopy Soc. Am.: 148-149. 13. Fowden, L., D. Lewis, and H. Tristram. 1967. Toxic amino aoids: their action as anti-metabolites. Adv. in Enzymol. 2£: 89-I63. 14. Fox, C.F., and E.P. Kennedy. 1965. Speoifio labeling and partial purification of the M protein, a component of the B^-galactoside transport system of Escherichia c o l i . Proc. Natl. Acad. Science 5J.: 891-899. 15. Hengstenberg, W., J.B. Egan, and M.L. Morse. 1968, Carbohydrate transport in Staphylococcus  aureus. VI. The nature of the derivatives accumulated. J. Biol. Chem. 243t I 8 8 I - I 8 8 5 . 16. Heppel, L.A. 1967. Selective release of enzymes from bacteria. Science 156: 1451-1455. 17% Hirsch, J.G., and M.E. Fedorko. I968. Ultra-structure of human leucocytes after simul-taneous fixation with glutaraldehyde and osmium tetraoxide and "post fixation" in uranyl acetate. J. Cell Biol. 3.8: 615-627. 18* Holm-Hansen, 0 . , and C.R. Booth. 1966. The measurement of ATP in the ocean and i t s ecological significance. Limnol. Oceanog. I l l 510-519 19. Kaback, H.E. 1968. The role of the phosphoenol-pyruvate-phosphotransferase system in the transport of sugars by isolated membrane preparations of Escherichia c o l i . J. Biol. Chem. 243: 3711-3724. 93 20. Kaback, H.R., and T.P. Deuel. 1969. Proline uptake by disrupted membrane preparations from Escherichia c o l i . Arch. Biochem. Biophys. 132: 118-129. 21. Kaback, H.R., and L.S. Milner. 1970. Relationship of a membrane-bound D-(-)-lactio dehydro-genase to amino acid transport in isolated bacterial membrane preparations. Proc. Natl. Acad. Science 66: 1008-1015. 22.. Kaback, H.R. 1970. Transport. Ann. Review of Biochem. 3£: 561-598. 23. Kay, W.W. 1968. Amino acid transport and pool formation in Pseudomonas aeruginosa. PhD. Thesis, University of British Columbia. 24. Kay, W.W., and A.P. Gronlund. 1969a. Isolation of amino acid transport-negative mutants of Pseudomonas aeruginosa and cells with repressed transport activity. J. Bacteriol. 98:116-123. 25. Kay, W.W., and A.P. Gronlund. 1969b. Influence of oarbon or nitrogen starvation on amino acid transport in Pseudomonas aeruginosa. J. Bacteriol. 100: 276-282. 26. Kay, W.W., and A.P. Gronlund. I9690. Proline transport by Pseudomonas aeruginosa. Biochim. Biophys. Acta 193: 444-455. 27* Kepes, A., and G.N. Cohen. 1962. Permeation. In I.C. Gunsalus and R.Y. Stanier, ed., The Bacteria, vol. 4, Academic Press, Inc., New York: 179-221. 28, King, E.O., M.K. Ward, and D.E. Raney. 1954. Two simple media for the demonstration of pyo-cyanin and fluorescin. J. Lab. Clin. Med. 44s 301-307. 29, Kundig, W., S. Ghosh, and S. Roseman. 1964. Phosphate bound to histidine in a protein as an intermediate in a novel phosphotransferase system. Proc. Natl. Acad. Science 52: 1067-1074. 94 30. Lowry, O.H., N.J. Rosebrough, A.L. Parr, and R.J. Randall. 1951* Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193* 265-275. 31. Medveczky, N., and H. Rosenberg. 1969. The binding and release of phosphate by a protein isolated from Escherichia ool i . Biochim. Biophys. Acta 192t 369-371. 32. Medzihradsky, P., M.H. Kline, and L.E. Hokin. 1967. Studies on the characterization of the sodium-potassium transport adenosine-triphosphatase. I. Solubilization, stabilization, and e s t i -mation of apparent molecular weight. Arch. Biochem. Biophys, 126: 331-342. 33. Nossal, N.G., and L.A. Reppel. 1966. The release of enzymes by osmotic shock from Escherichia  c o l i in exponential phase. J. Biol. Chem. 241: 3055-3062. 34. Pardee, A.B. 1966. Purification and properties of a sulfate binding protein from Salmonella  typhimurium. J. Biol. Chem. 241t5886-5892. 35. Pardee, A.B. 1967. Crystallization of sulfate-binding protein (permease) from Salmonella  typhimurium. Science 156: 1627-1628. 36. Pardee, A.B. 1968. Membrane transport proteins. Science 162: 632-637. 37. Pease, D.C. 1964. Histological techniques for Electron Microscopy, 2nd ed., Academic Press, New York. 38. Penrose, W.R., G.P. Niohoalds, J.R. Piperno, and D.L. Oxender. I968. Purification and properties of a leucine-binding protein from Escherichia c o l i . J. Biol. Chem. 243t 5921-5928. 39% Piperno, J.E., and D.L. Oxender. 1966. Amino acid-binding protein released from Escherichia c o l i by osmotic shock. J. Biol. Chem. 2411 5732-5734. 95 40. Piperno, J., and. D.L. Oxender. 1968. Amino acid transport systems in Escherichia c o l i K12. J. Biol. Chem. 243: 5914-5920. 41. Sapico, V., T.E. Hansen, R.W. Walter, and R.L. Anderson. 1968. Metabolism of D-fructose in Aerobacter aerogenes: analysis of mutants . lacking D-fructose-6—phosphate kinase and D-fructose 1,6 diphosphatase. J. Bacteriol. 26: 51-54. 42. Smith, L. I968. Bacterial respiratory chain systems of bacteria. In Biological Oxidations, T.P. Singer, ed., Interscienoe Publishers, New York: 55-122. 43. Stanley, P.E., and S.G. Williams. 1969. Use of the liquid scintillation spectrometer for determining ATP by the luciferase enzyme. Anal. Biochem. 29_: 381-392. 44. Strasdine, G.A. 1961. A study of oxidative phosphorylation in Pseudomonas aeruginosa. PhD. Thesis, University of British Columbia. 45. Stryer, L. 1968. Fluorescence spectroscopy of proteins. Science 162: 526—533. 46. Wasserman, R.H., and A.N. Taylor. 1966. Vitamin D3~induced calcium-binding protein in chick intestinal mucosa. Science 152: 791-793. 47. Wilson, O.H., and J.T. Holden. I969. Arginine transport and metabolism in osmotically shocked and unshocked cells of Escherichia c o l i W. J. Biol. Chem. 244: 2737-2742. 

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