TEE E§FECT GE HEXéSHLGBOPfiENE 8N ELECTEOR TRANSPORT {N BACILL‘JS MEGATEREUM T5253: ‘or the Degree 0‘ M. S. MICHEGAN STATE UNWERSITY John Joseyh Frederick €974 OVERDUE FINES:- 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records (“I I; m fr. .' ' mo...»- - r Ze‘fi8¥$\f&fzfl.s~a:a.' "area‘s/:3: I 90‘“: ¢ l" ABSTRACT THE EFFECT OF HEXACHLOROPHENE ON ELECTRON TRANSPORT IN BACILLUSUMEGATERIUM By John Joseph.Frederick Hexachlorophene inhibited electron transport in isolated B. megaterium KM membranes. One site of inhibition was at the level of soluble and membrane-associated dehydrogenases (substrate:DCPIP oxido- reductases). Kinetic analysis of the effects of HCP on soluble NADH dehydrogenase (with respect to NADH) and soluble L-malate dehydrogenase (with respect to L-malate), indicated for both enzymes an inhibition of the partially competitive type. Whereas the kinetics of hexachlorOphene inhibition of membrane-associated Limalate dehydrogenase (with respect to Ldmalate) were typical of mixed inhibition, hexachlorophene inhibited membrane—associated NADH dehydrogenase (with respect to NADH) in a noncompetitive fashion. However the high Ki's calculated for HCP inhibition of dehydrogenases indicated that this portion of the electron transport system was not the site of maximal HCP sensitivity of isolated membranes. The effect of HG? on isolated membranes was also investigated by means of difference spectroscopy. The site of maximal HCP sensitivity depended on the cytochrome content of the membranes. When the membranes contained a bl-type cytochrome, an a-type cytochrome, an a3-type cytochrome, but lacked o-type cytochrome, about 8 pg HEP/mg membrane dry wt inhibited electron transport on the substrate side of the b -type cytochrome, l John Joseph Frederick probably by inhibition of electron transfer from dehydrogenases to menaquinone. Addition of excess exogenous menadione restored oxidase activity to these HOP-inhibited membranes. When the membranes contained a bl-type cytochrome, an a3 an a—type cytochrome, as little as 2 ug HEP/mg membrane dry wt inhibited -type cytochrome, an o-type cytochrome, but lacked electron transport on the oxygen side of the bl-type cytochrome, probably at a site between the bl-type cytochrome and the oxidases via subtle disorganization of the electron carriers in the membrane. THE EFFECT OF HEXACHLOROPHENE ON ELECTRON TRANSPORT IN BACILLUSaMEGATERIMM By John Joseph Frederick A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1974 A DEDICATION an Dedicated to my wife, Valerie, whose assistance encouragement, patience and interest put the smile on the Cheshire cat ii ACKNOWLEDGMENTS I wish_to express my sincere thanks and appreciation to my advisor, Dr. Thomas R. Corner, for his interest and encouragement throughout the course.of this work. Thanks also go to the members of my committee, Dr. J. Tiedje, Dr. S. Aust, and Dr. R. Brubaker, for their help and guidance; to Dr. C. San Clemente for the use of his fermenter; and to Dr. T. Akara for the use of his spectrophotometer. I also thank the Department of Microbiology and Public Health for generous financial assistance. A special thanks goes to my family for their constant moral and financial support. Last, but certainly not least, I acknowledge Dr. Rene M. Scherrer whose constant stream of trenchant criticisms helped "... quite much you know." TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . Toxicity for Mammals . . . . . . . . . . . . . . . . . . . . . l Toxicity for Microorganisms . . . . . . . . . . . . . . . . . 2 Respiratory System of B. megaterium . . . . . . . . . . . . . 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . S Organism . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Cultivation of Cells . . . . . . . . . . . . . . . . . . . . . 5 Isolation of Membranes . . . . . . . . . . . . . . . . . . . . 6 Isolation of Soluble Dehydrogenases . . . . . . . . . . . . . 7 Enzyme Assays . . . . . . . . . . . . . . . . . . . . . . . . 8 Determination of P/O Ratios . . . . . . . . . . . . . . . . . 8 Respiratory Inhibition . . . . . . . . . . . . . . . . . . . . 9 Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Effect of HCP and'Menadione on the Rate of Oxygen Uptake by Both Normal and Ultraviolet-irradiated Membranes . . . . 11 Flo Ratios of Membranes . . . . . . . . . . . . . . . . . . . ll Effect of HCP on Membrane-associated L-malate:DCPIP Oxidoreductase . . . . . . . . . . . . . . . . . . . . . . . 14 Effect of HCP on Soluble LéMalateaDCPIP Oxidoreductase . . . . 14 Effect of HCP on Membranerassociated NADH;DCPIP Oxidoreductase 19 iv Page Effect of HCP on Soluble NADfioDCPIP Oxidoreductase.. . . . . . 19 Effect of HCP on.Cytochrome Spectra of Membranes Isolated From Cells That Were Grown in Buffered Peptone . . . . . . . 19 Effect of HCP on Cytochrome Spectra of Membranes That Were Isolated From Cells Grown in Unbuffered Peptone . . . . . . 26 Effect of HCP on the Redox State of the b -Type Cytochrome in l Membranes Isolated From Cells Grown in Unbuffered Peptone . 26 Cytochrome Content of Supernatant Solutions . . . . . . . . . 29 DISCUSSION 0 O O O O O O O O O O C O O O O O O O O O O O O O O O 32 Effect of HCP on Soluble Dehydrogenases . . . . . . . . . . . 32 Effect of HCP on Membrane-associated Dehydrogenases . . . . . 33 Effect of HCP on Electron Transport in Membranes Isolated From Cells of B. megaterium Grown in Unbuffered Peptone . . 33 Effect of HCP on Electron Transport in.Membranes Isolated From Cells of B. megaterium Grown in Phosphate-buffered Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 B I BL IOGRAPIiY O O O O O O O O O O O O O O O 0 O O O O O O O O O O 4 1 LIST OF FIGURES FIGURE Page 1. Sequence of electron carriers in B. megaterium KM . . . 4 2. Effect of HCP and menadione on the rate of Lemalate oxidation by isolated membranes of B. megaterium grown in unbuffered peptone . . . . . . . . . . . . . 13 3. Effect of HCP and menadione on the rate of Lemalate oxidation by isolated membranes of B. megaterium grown in buffered peptone . . . . . . . . . . . . . . l3 4. Effect of HCP on membrane-associated L-malate:DCPIP oxidoreductase (Lineweaver-Burke transformation). . . 16 5. Effect of HCP on the activity of membrane-associated L-malate:DCPIP oxidoreductase . . . . . . . . . . . . 16 6. Effect of HCP on the activity of soluble L-malate:DCPIP oxidoreductase (Lineweaver-Burke transformation). . . 18 7. Effect of HCP on the activity of soluble Ldmalate: DCPIP oxidoreductase . . . . . . . . . . . . . . . . 18 8. Effect of HCP on the activity of membrane—associated NADH:DCPIP oxidoreductase (Lineweaver-Burke transformation) . . . . . . . . . . . . . . . . . . . 21 9. Effect of HCP on the activity of membrane-associated NADHsDCPIP oxidoreductase . . . . . . . . . . . . . . 21 10. Effect of HCP on the activity of soluble NADH;DCPIP oxidoreductase (Lineweaver—Burke transformation) . . 23 vi FIGURE 11. 12. 13. 14. 15. 16. vii Effect of HCP on the activity of soluble NADHcDCPIP oxidoreductase . . . . . . . . . . . . . . . . . . Effect of HCP on the difference spectra of membranes isolated from cells of B. megaterium grown in phosphate-buffered peptone . . . . . . . . . . . . Reduced + C0 minus reduced difference spectra of membranes isolated from cells of B. megaterium grown in phosphate-buffered peptone . . . . Effect of HCP on the difference spectrum of membranes isolated from cells of B. megaterium grown in unbuffered peptone . . . . . . . . . Reduced + C0 minus reduced difference spectra of membranes isolated from cells of B. megaterium grown in unbuffered peptone . . . . . . . . . . . . . Effect of HCP on the redox state of the bl—type cytochrome in membranes isolated from cells of B. megaterium that were grown in unbuffered peptone Page 23 25 25 28 28 31 INTRODUCTION "...(Alice) had never forgotten that, if you drink much from a bottle marked "poison", it is almost certain to disagree with you, sooner or later." -Lewis Carroll, 1865 For nearly 25 years, hexachlorophene [2,2'amethylenebis (3,4,6—trichlorophenol); HOE] has been widely used as an antimicrobial compound. Its broad spectrum.antimicrobia1 activity against gram- positive and, to a lesser extent, gram-negative microorganisms, compat— ibility with soap formulations, stability, lack of odors or color, and economics made it suitable for incorporation into a large number of consumer products (6). Toxicity for Mammals Several recent reports indicating high HCP toxicity for mammals (5,7,19,21,24,26,28) have prompted the U.S. Food and Drug Administration to prohibit the nonprescription sale of drugs which contain more than 0.75% HCP and to ban the use of HCP in cosmetics except as a preserva- tive in levels up to 0.1%, and then only when other suitable preserva- tives are not available (37). The toxicity of HCP for mammals depends on the dose, method of administration, and organism. Repetitive small oral or topical doses (<50 mg/kg) are not immediately toxic whereas large single doses (>100 mg/kg) are generally fatal (16,20,21,25,26,30). HCP has been shown to inhibit dehydrogenases and oxidases of animal origin and to uncouple oxidative.phosphorylation in.mammalian systems (5,7,19). The cause of death in acute HCP intoxication of rats may be related to hyperthermia brought about by this uncoupling (32). Toxicity for Microorganisms The primary lethal action of HCP in microbes still has not been completely resolved. Some previous suggestions for its mode of action include (i) release and leakage of cytoplasmic materials; (ii) disruption or lysis of the cytoplasmic membranes; (iii) generalized protein denaturation; and (iv) respiratory inhibition. It has been concluded that these first three mechanisms are secondary events to the primary lethal mechanism since they are only observed at concentrations of HCP much higher than the reported minimum.letha1 concentration of 8 ug HCP/mg cell dry weight for Bacillus megaterium (8,23,33). Recently Corner and Gerhardt (unpublished results) have used warburg respirometry to demonstrate inhibition of oxygen uptake in HCP-treated cells of B. megaterium. They found that the rate of oxygen uptake from exogenous or endogenous substrates decreased progressively as the concentration of HCP was increased from 0 to 8 ug HCP/mg cell dry weight. Higher HCP levels did not further depress respiration. Since the concentration of HCP necessary for respiratory inhibition correlated with the value for the minimum.lethal concentration, and since inhibition of endogenous respiration ruled out inhibition of transport systems as the lethal event, they reasoned that the respiratory system was the primary target of HCP. Much earlier Gould at a1. (18) found that HCP inhibited the activity of selected dehydrogenases (succinic, glucose, lactic) and cytochrome c oxidase activity in intact cells of both Eachar£8hfia coli and B. subtilis. In their experiments they held the concentration of HCP constant and varied the biomass to achieve a desired reaction rate. Since they did not report the amount of biomass, comparison of their data with other work is precluded. Attempts to enrich for strains of organisms resistant to HCP revealed that anaerobic or facultative organisms were less sensitive to HCP than highly aerobic organisms and that no striking adaptation to the inhibitory effects of HCP took place (20). A lower sensitivity to HCP of the anaerobic or facultative organisms was interpreted to mean that a sensitive oxidase system was bypassed so that some other less sensitive enzyme system became limiting for growth. Adams and Hobbs have shown that HCP is capable of forming chelates with a variety of metals (1). They found that only Fe++ was capable of suppressing the action of HCP against their test organism, Staphylococcus aureus, and reasoned that the bactericidal properties of HCP may stem from inhibition of certain iron-containing enzyme systems. However, iron salts neither prevented nor reversed HCP inhibition of bacterial growth when Corner and Gerhardt (unpublished results) repeated the experiments of Adams and Hobbs using B. megaterium as the test organism. Since the primary lethal mechanism.of HCP appeared to be respiratory inhibition, the purpose of this investigation was to determine the site(s) of this inhibition in B. megaterium. Respiratory System of B. megatcrium The respiratory system of B. megaterium is localized in the protoplast membrane. The membrane can be isolated by subjecting protoplasts of B. megaterium, prepared by controlled lysozyme treatment, to osmotic shock.followed by differential centrifugation (35,39). These membranes catalyze oxidation of various substrates (2). The respiratory enzyme content of the membranes depends on the growth conditions of the cells before harvest, but one can draw a generalized scheme for the electron transport system of B. megaterium KM (Figure l). CYTOCHROME a DEHYDROGENASES -'- MENAQUINONE -* CYTOCHROME bl -- CYTOCHROME a3}* ( O2 CYTOCHROME 0 Figure 1. Sequence of electron carriers in B. megaterium KM. Redrawn from (29). This scheme does not take into account the possible involvement of nonheme iron or of c-type cytochromes which have been detected in other strains of B. megaterium (40). The precise relationship between the oxidases, cytochrome a or cytochrome o, to either the bl-type 3 cytochrome or cytochrome a is not known. B. megaterium has several membrane-associated and soluble dehydrogenases that supply electrons to the electron transport chain. L—malate dehydrogenase and NADH dehydro- genase were chosen for study because they are present in relatively large amounts and each exists in both membrane-associated and soluble forms (35). MATERIALS AND METHODS Organism The asporogenous KM strain of B. megaterium was chosen for this work because its sensitivity to HCP has been well characterized (8,23,33), and details of its respiratory chain are known (2,34,12,13,29). Stock cultures were maintained on 2% trypticase soy agar (Baltimore Biological Laboratories, Cockeysville, Maryland) plates at 30 C. Cultivation of Cells Cells of B. megaterium were grown to late exponential phase of growth (15-18 hr) in 2% (wt/vol) Oxoid peptone (Flow Laboratories, Rockville, Maryland) in aerated batch cultures on a gyratory shaker (250 rev/min) at 30 C. These cells were used as inocula for 10 L of medium I?% Oxoid peptone in distilled H20 or 0.01 M sodium-potassium phosphate buffer (pH 7.0) containing 0.3 g Dow antifoam/L] in a fermentor (model MF-14 New Brunswick Scientific Co., New Brunswick, New Jersey) which was operated at 30 C with an aeration rate of 10 L of air per minute and a stirring rate of 250 rpm. When necessary, the pH of the medium.was readjusted to pH 7.0 with concentrated HCl. The cells were harvested during the late exponential phase of growth when the optical density of the culture reached about 2.45. This optical density cor— responds to about 2.4 mg cell dry wt/ml. Growth was monitored by means of a Spectronic 20 spectrophometer (Bausch and Lomb, Rochester, New York) at a wavelength of 700 nm, with 1.17 cm.light-path cuvettes and distilled water used as a blank. The cells were collected by means of continuous flow centrifugation (27,000 x g; 4 C) at a fl w rate of 150 ml/min. A Sorvall RC-ZB refrigerated centrifuge equipped with a Szent-Gyorgi and Blum continuous flow system was used. The wet weight yield of the cells was 4.5 110.1 g/L. The cells were washed twice in cold distilled water and used immediately. Isolation of Membranes The membrane fraction of B. megaterium was prepared by treat- ment with mucOpeptide N acetylmuramylhydrolase (E.C. 3.2.1.17; lyso- zyme) followed by osmotic shock according to a modification of the method of Broberg and Smith (2). Each gram (wet wt) of washed cells was suspended in 2.5 ml of 0.5 M sucrose solution buffered with 0.01 M sodiumrpotassium phosphate buffer (pH 7.0). Approximately 45 g (wet wt) of cells were used in each isolation. Crystalline lysozyme (30,000 units activity/mg) dissolved in 15 ml of the buffered sucrose solution was added to the cell suspension to give a final concentration of 0.01 g lysozyme/g cell wet wt. The resultant suspension was incubated at 25 C until conversion to single protOplasts was complete as judged by viewing with a phase contrast microscope (about 60 min). The protoplasts were sedimented (25,000 x g; 20 min) in a Sorvall RC-ZB refrigerated centri- fuge at 4 C and then lysed osmotically by suspending the pellet of protoplast residue in 150 ml of 0.01 M phosphate buffer (pH 7.0) containing 0.5 mg deoxyribonucleate oligonucleotidohydrolase (E.C. 3.1.4.5; deoxy- ribonuclease l). The mixture was shaken at 25 C for 15 min. Complete lysis of the protoplasts occurred under these conditions as judged by microsc0pic examination. Prot0p1ast ghost membranes were centrifuged at 25,000 x g_for 30 minutes. The buff-colored upper part of the pellet containing the membrane fraction was suspended in.0.1 M N-2-HydroxyethlpiperazineeN'-2-ethanesu1fonic acid-KOH.buffer, pH 7.65 (HEPES buffer). This procedure.was repeated at least three times and each time the buff-colored membrane pellet was scraped off the heavier white pellet of poly-thydroxybutyrate (PHB) granules. The final membrane suspension, which still contained some PHB, was stored at -30 C. When needed, an apprOpriate amount of the frozen membrane preparation was suspended to the desired concentration in 0.1 M HEPES buffer (pH 7.60), distributed to lS-ml screwhcapped test tubes and immediately refrozen in an ethanol-dry ice bath. Isolation of Soluble Dehydrogenases The soluble enzymes were contained in the supernatant obtained by direct lysis of protoplasts in hypotonic solution. To 88 g of washed cells (wet wt) were added 100 ml of 0.1 M HEPES buffer containing 0.250 g lysozyme and 0.05 mg deoxyribonuclease. This mixture was shaken at 250 rpm in a gyratory shaker at 30 C for 2 hr. The protoplasts were further disrupted by alternately freezing and thawing the preparation. Conversion to protoplast ghosts was at least 95% complete as judged by phase microscopy. The membrane fraction was sedimented (25,000 x g; 30 min) and the yellowish supernatant was decanted and stored at 4 C. The buff-colored part of the pellet containing the membranes was scraped off the heavier white PHB layer, resuspended in 50 ml of 0.1'M HEPES buffer, and recentrifuged at 25,000 x g for 30 min. The supernatant was again decanted and pooled with the previous one. The pooled super- natants were further clarified by centrifugation at 25,000 rev/min with an average centrifugal force of 75,000 x g) for 2 hr at 4 C in a prepara— tive ultracentrifuge (International Equipment Co., model B-60) equipped with a swinging bucket rotor Qmodel 83-110). The yellowegreen supernatant was removed with a Pasteur pipette and stored at -30 C. When needed, an appropriate amount of the soluble lysate was suspended to the desired concentration in 0.1 M HEPES buffer, distributed to 15dml screw-capped test tubes, and quickly frozen in an ethanol-dry ice bath. Protein concentrations were estimated by the buiret method (17) using bovine serum albumin as a protein standard. Enzyme Assays Dehydrogenase activity was determined by the method of Storck and wachsman (35) which involves the reduction of 2,6-dichlor0phenol- indolphenol (DCPIP). Each 1.0 cm-lightpath cuvette contained 0.15 umoles DCPIP, 15 umoles MgSO 30 umoles NaCN (freshly adjusted to pH 7.60 4, with HCl), 150 umoles HEPES buffer, and HCP dissolved in 0.1 N NaOH as indicated (final volume = 3.0 ml). Substrate, either reduced dihydro— nicotinamide adenine dinucleotide (NADH) or potassium L-malate, was added to the reference cuvette. The reaction was started by simultaneous injection of 0.25 ml of either the membrane suspension or the soluble lysate, diluted appropriately in 0.1 M HEPES buffer, to both the sample and reference cuvettes. DCPIP reduction was followed at 600 nm in a recording spectrophotometer (Model DB—G, Beckman Instrument Co.) equipped with thermal spacers through which water at 25 C was circulated. Initial velocities were calculated from slopes of the traces during the first 5 to 7 seconds of the assay during which time the rate is linear. The extinction coefficient of DCPIP was taken to be 21 L uM-lcm-1 (27). Determination of FIG Ratios Oxygen uptake at 30 C was measured by standard Warburg tech- niques (36). The reaction mixture consisted of 170 umoles glucose, 50 umoles MgClz, 50 umoles potassium phosphate, 1 umole.ADP, 0.1 mg ATP: D-hexose 6-phosphotransferase (EC 2.7.1.1) (400 units), 82 mg membranes €dry~wt), and water tO'a final volume of 2.8 ml. The center well of the flask contained 0.1 ml 20% w/v KOH. After 20 min of equilibration, the stopcocks were closed and endogenous respiration was followed for 20 min. The reaction was started by tipping in 75 umoles of Lemalate and from 1 to 1,000 umoles of HCP in 0.1 ml 0.1 M NaOH from the side wells. Control flasks were immediately removed and the reaction stopped by pipetting 0.5 ml aliquots into 9.5 ml cold 10% trichloroacetic acid (TCA). The remaining flasks were incubated for an additional 30 min, and the reaction stopped in the same manner. TCA—precipitated protein was removed by centrifugation and a 1.0 ml aliquot of the supernatant was used for inorganic phosphate determination according to the method of Fiske and SubbaROW’(l4). Respiratory Inhibition Oxygen uptake was followed polographically by means of a teflon covered Clark-type oxygen electrode in a standard bath assembly (models YSI 5331 and YSI 5301, respectively, Yellow Springs Instrument Co.) and a modified Beckman oxygen analyzer (model 777, Beckman Instruments, Inc.) equipped with a Sargent model SR recorder. The temperature was maintained at 25 C by use of a constant temperature circulator (model FK, Haake Instruments, Inc.). When necessary, 0.05 ml aliquots of HCP (dissolved in 0.1 M NaOH) or 2 methyl-l,4-naphthaquinone (Menadione) (dissolved in ethanol), or both were injected into the membrane suspension by means of long-tipped micropipettes. The oxygen analyzer was calibrated to read percent 0 in the suspension by settingng zero percent 02 with the 2 probe under a stream of argon, 100 percent 02 with the probe under a 10 stream of 02, and checking atmospheric 02 (20.9%). Irradiation of the membrane preparation with ultraviolet light was performed with a long wave (365 um) blacklight CUVSL 13, Ultra Violet Products Inc., San Gabriel, California). Membranes suspended in 0.1 M HEPES buffer were placed in a glass Petri dish on a magnetic stirrer and irradiated at a distance of 1.5 cm for 20 min while being slowly stirred by a teflon-coated magnetic stirring bar. Spectra Difference spectra were obtained with a Shimadzu model MPS-SOL spectrophotometer (American Instrument Co., Inc., Silver Springs, Maryland). RESULTS Effect of HCP and Menadione on the Rate of Oxygen.Uptake by Both Normal and Ultraviolet-irradiated Membranes I501ated B. megaterium membranes contained an electron transport system that was capable of oxidizing L—malate and transferring these electrons to oxygen (Figure 2A, 2B and Figure 3A, 3B). Hexachlorophene inhibited oxygen uptake by isolated B. megaterium membranes. This inhibition was partially overcome by addition of 80 umoles menadione/mg membrane dry wt to the membrane suspension (Figure ZB and Figure 3B). This amount of exogenous menadione was about 20 times the reported concentration of natural menaquinone in protoplast membranes of B. megaterium (29). Irradiation with ultraviolet light has been shown to inactivate Micrococcus Zysodcikticus naphthaquinones (11,15). B. megaterium membranes which had been irradiated for 20 min with ultra- violet light respired very slowly on L-malate. Addition of menadione to these membranes increased the rate of L-malate oxidation to the levels observed with unirradiated membranes (Figure 2C and Figure 3C). Menadione- restored Lemalate oxidase activity could be inhibited by HCP and stimulated by additional exogenous menadione (Figure 3C). Exogenously added mena- dione did not cause oxygen uptake when substrate was absent (Figure 3D). P/O Ratios of Membranes The P/O ratios of all membrane preparations were zero. Brief sonication or heating of the membrane suspensions did not increase the PIC ratios. 11 Figure 2. Figure 3. 12 Effect of hexachlorOphene and manadione on the rate of L-malate oxidation by isolated membranes of B. megaterium grown in unbuffered peptone. Oxygen uptake was followed polographically at 25 C. Each sample chamber contained 0.0679 g (dry wt) of membranes in 2.5 ml HEPES buffer. The membrane suspensions were stirred under a stream of 02 for 3 min immediately before the teflon-covered oxygen electrode was inserted. Curves A and B represent untreated membranes, curve C represents membranes that were irradiated with ultra- violet light to destroy natural quinones (see Materials and Methods). Symbols: (M) 25 pmoles L—malate; (E) 100 p1 ethanol; (H) 1.0 mg hexachlorOphene in 100 pl 0.1 N NaOH (14.7 pg HCP/mg membrane dry wt); (Q) 5.6 pmoles manadione in 100 pl ethanol. Effect of hexachlorophene and manadione on the rate of Lemalate oxidation by isolated membranes of B. megaterium grown in phosphate—buffered peptone. Oxygen uptake was followed polographically at 25 C. Each sample chamber contained 0.1470 g (dry wt) of membranes in 2.5 ml HEPES buffer. The membrane suspensions were stirred under a stream of 02 for 3 min immediately before the teflon—covered oxygen electrode was inserted. Curves A, B and D represent untreated mem- branes, curve C represents membranes which were irradiated with ultraviolet light to destroy natural quinones (see Materials and Methods). Symbols: 0!) 25 pmoles L—malate; (Q) 11.6 pmoles menadione in 100 p1 ethanol; (H) 1.0 mg hexachlorophene in 100 pl 0.1 N NaOH (6.8 pg HCP/mg mem- brane dry wt); (H/Z) 0.5 mg HCP in 50 pl 0.1 N NaOH. Percent Oxygen a. m 8 8 o o 5 (10 MINUTE) FIGURE 3 14 Effect of HCP on Membrane-associated Lemalate:DCPIP Oxidoreductase HCP inhibited the.membrane—associated form of L-malate:DCPIP oxidoreductase (E.C. 1.1.99.-; L-malate dehydrogenase). When the concentration of DCPIP was held constant, the kinetics of inhibition of L-malate dehydrogenase with respect to Lemalate were typical of mixed inhibition as defined by Webb (38) (Figure 4). In such cases both the maximum velocity (Vmax), and the apparent affinity of the enzyme for its substrates are reduced. Moreover, a plot of velocity vs. HCP concentration indicated that the inhibition was of the completely mixed type since a finite amount of HCP could completely inhibit enzyme activity (Figure 5). The inhibitor constant (Ki) of HCP for the membrane—associated L-malate dehydrogenase was 23.7 pg HCP/mg membrane dry wt (see appendix A for calculations). Preincu- bation of the membranes with HCP for up to 3 min did not affect the initial velocity. Effect of HCP on Soluble Lemalate:DCPIP Oxidoreductase Kinetic analysis of the effect of HCP on soluble Lemalate dehydrogenase, with respect to Lemalate, indicated that the inhibition was of the competitive type (Figure 6). Moreover, a plot of velocity vs. HCP concentration indicated that the inhibition was of the partially competitive type (Figure 7). In such cases inhibition does not increase indefinitely with inhibitor concentration, but increases to a definite limit when all of the susceptible sites on the enzyme are combined with the inhibitor and can then increase no further. The K1 of HCP for soluble L-malate dehydrogenase.was 11.04 pg HCP/mg protein (see appendix B for calculations). FIGURE 4. FIGURE 5. 15 Effect of HCP on the membrane associated L-malate:DCPIP oxidoreductase (Lineweaver—Burke transformation). DCPIP reduction was followed spectrophotometrically at 600 nm. Sample and reference cuvettes contained 0.15 pmoles DCPIP, 15 pmoles MgSO 30 pmoles NaCN, 150 pmoles HEPES buffer, 4, and HCP dissolved in 0.1 N NaOH as indicated. The reaction was started by simultaneous addition of 0.25 ml of the mem- brane suspension (0.3548 mg membrane dry wt) to both cuvettes. A11 lines shown were fitted by regression analysis. Symbols: (0) No HCP; (A) 14.09 pg HCP/mg membrane dry wt; ([1) 28.18 pg HCP/mg membrane dry wt. Effect of HCP on the activity of membrane-associated Lemalate: DCPIP oxidoreductase. The reaction conditions were as in Figure 4 except that the concentration of Lemalate was held constant at 5.63 pmoles/mg membrane dry wt. l6 250 ml) 8 % K, (Lmoles DCPIP reduced/mnufe/SO U! 0 v I -o.‘6 10:4 1012 l 0 10:2 ‘02 ‘ 05 505‘ 1.0 _ I/S (pmoles L—malaleflrg membrawe cry WI) FIGURE 4 § rninufe/ 3.0 ml 8 > L l . 1 A _n . u . O 10 20 30 4O 50 [U (pg Iexachlomphene/mg membrane dry wt) FIGURE 5 FIGURE 6. FIGURE 7. 17 Effect of HCP on the activity of soluble L—malate:DCPIP oxidoreductase (Lineweaver-Burke transformation). DCPIP reduction was followed spectrophotometrically at 600 nm. Sample and reference cuvettes contained 0.15 pmoles DCPIP, 15 pmoles Mg304, 30 pmoles NaCN, 150 pmoles HEPES buffer, and HCP dissolved in 0.1 N NaOH as indicated. The reaction was started by simultaneous addition of 0.25 ml of the soluble lysate containing 2.732 mg protein, to both cuvettes. All lines shown were fitted by regression analysis. Symbols: (0) No HCP; (A) 10.98 pg HCP/mg protein; (D) 27.45 pg HCP/mg protein. Effect of HCP on the activity of soluble L—malate:DCPIP oxidoreductase. The reaction conditions were as in Figure 6 except the concentration of L-malate in the sample cuvette was held constant at 0.915 pmoles L-malate/mg protein and the HCP concentration was varied as indicated. l8 600 .. l - + ' 2500' "’ 1 O. . .. . é '- qr. ' A --400- «r €- . .. 3300' e ' ° g ' . €200- 3100- -4 -é -é -1 o 1‘ é 5 Z 5 V5 ( pmoles L-mala’re/hng prolein) FIGURE 6 10 r e . . e - 9 1 8 I F 7 4. 9. Q 6 J to I R a s (n '5 4 4 .02 c 3 .E 3 '3 E 2‘ «z W 1. J > I J 1 J A_ A l_kJ l l A J 0 1o 20 30 4o 50 60” [I] (“9 hexachlorophene/mg protein) FIGURE 7 19 Effect of HCP on.Membrane-associated NADH:DCPIP Oxidoreductase Hexachlorophene also inhibited the.membrane-associated NADHaDCPIP oxidoreductase (E.C. 1.6.99.3; NADH dehydrogenase) but in a noncompetitive fashion with respect to NADH (Figure 8). .A plot of velocity vs. HCP concentration indicated that the inhibition was of the partially noncompetitive type (Figure 9). Effect of HCP on Soluble NADH:DCPIP Oxidoreductase Kinetic analysis of the effects of HCP on soluble NADH dehydrogenase, with respect to NADH, indicated that the inhibition was of the partially competitive type (Figure 10 and Figure 11). The Ki of HCP for soluble NADH dehydrogenase was 42.47 pg HCP/mg protein (see appendix B for calculations). Effect of HCP on Cytochrome Spectra of Membranes Isolated From Cells That Were Grown in Buffered Peptone B. megaterium membranes isolated from cells grown in very similar conditions were found to differ in their cytochrome content. Reduced minus oxidized difference spectra from cells grown in buffered peptone (0.1 M sodium-potassium phosphate buffer pH 7.0) contained peaks at 557 nm, 527 nm, and 425 nm (Figure 12, curve B). Such peaks are characteristic of cytochrome b1 (2,29). When HCP was added to the sample cuvette, increases in the bl—type cytochrome peak heights at 557 nm and at 425 nm were observed (Figure 12, curve C). The extent of reduction of this bl-type cytochrome by Lemalate and HCP was greater than the non—enzymatic reduction produced by dithionite. Kroger and Dadak (29) reported that B. metgaterium has a fumarate reductase in equilibrium with cytochrome b which_allows fumarate to act as an 1 electron acceptor during the anaerobic state. Addition of fumarate to FIGURE 8. FIGURE 9. 20 Effect of HCP on the activity of membrane—associated NADH: DCPIP oxidoreductase (Lineweaver-Burke transformation). DCPIP reduction was followed spectrophotometrically at 600 nm. Sample and reference cuvettes contained 0.15 pmoles DCPIP, 15 pmoles MgSO 30 pmoles NaCN, 150 pmoles HEPES, 49 and HCP dissolved in 0.1 N NaOH as indicated. The reaction was started by simultaneous addition of 0.25 ml of the mem- brane suspension (0.7550 mg membrane dry wt) to both cuvettes. All lines shown were fitted by regression analysis. Symbols: (0) No HCP; (A) 9.933 pg HCP/mg membrane dry wt. Effect of HCP on the activity of membrane-associated NADH: DCPIP oxidoreductase. The reaction conditions were as in Figure 8 except the NADH concentration was held constant at 0.0529 pmoles NADH/mg membrane dry wt. Vv (pmoles DCPIP reduced/minufe/SO ml) ). “moles DC PEP reduced v( 21 60: 50F 40"- 30- 20" 10- - -30 -2o ‘-1'o I o ‘ 1lo#2b' 36' 41015.0 60 V5 (pmoles NADH /mg membrane dry wt) FIGURE 8 O l A l 4 5 I I j n 1104 I 1_ J 115 1 A A 1 20 [I] (pg hmachlonophene/mg membrane dry wt.) FIGURE 9 FIGURE 10. FIGURE 11. 22 Effect of HCP on the activity of soluble NADH:DCPIP oxido— reductase (Lineweaver—Burke transformation). DCPIP reduction was followed spectrOphotometrically at 600 nm. Sample and reference cuvettes contained 0.15 pmoles DCPIP, 15 pmoles MgSO 30 pmoles NaCN, 150 pmoles HEPES buffer, 4, and HCP dissolved in 0.1 N NaOH as indicated. The reaction was started by simultaneous addition of 0.25 ml of the soluble lysate containing 0.1300 mg protein, to both cuvettes. All lines shown were fitted by regression anal- ysis. Symbols: (0) No HCP; (2:) 19.23 pg HCP/mg protein; (B) 38.46 pg HCP/mg protein. Effect of HCP on the activity of soluble NADH:DCPIP oxido- reductase. The reaction conditions were as in Figure 10 except the concentration of NADH in the sample cuvette was held constant at 2.461 pmoles NADH/mg protein and the HCP concentration was varied as indicated. 23 100 . - ‘ ‘ ' E) 90- 1- ‘ A 2 C] 5 '80- «~ 0. ”i 15 70' " ' ‘ "E . A o \ 60- ~- . A ' 8 a u 50- s- . , ‘ A o. ‘ . 40- -- . ‘ 08 I . .‘n. £2 30- -- [it 0° ‘ v 20' " of," ‘ 3 0.. ,. / 10' ’ " Io -o5 o 023 1b T5 ab '25 b3 1 L4 1 4 a 4 J J 4 o 2401410‘610 60 100120140 1eok1so [1] (pg hexachlorophene/mg protein) FIGURE 11 FIGURE 12. FIGURE 13. 24 Effect of hexachloroPhene on difference spectra of membranes isolated from cells of B. megaterium grown in phosphate buffered peptone. Both the sample and reference cuvettes contained 0.442 g membranes (dry wt) suspended in 2.5 ml HEPES. Symbols: (A) baseline from oxidized minus oxidized difference spectrum; (B) reduced minus oxidized spectrum obtained after the addition of 25 pmoles sodium L-malate to the sample cuvette; (C) difference spectrum obtained after injection of 1.0 mg HCP in 100 p1 0.1 N NaOH to the reduced sample cuvette. Reduced + CO minus reduced difference spectrum of membranes isolated from cells of B. megaterium grown in phosphate buffered peptone. Sample and reference cuvettes contained 0.372 g membranes (dry wt) suspended in 2.5 ml HEPES. The membranes were reduced with 25 pmoles Ldmalate. Symbols: (A) reduced minus reduced membrane baseline; (B) difference spectrum after bubbling the sample cuvette with C0 for 30 seconds. 25 (fil() Tv'rVr'U'roVVT'lviji‘IiIfT' 005‘ if; 0.00‘ Atrrxtrmvae - 0"0 J J l L l A L 1 A l L 1 A A L L A l 350 400 450 500 ‘526 wavelength (NT!) FIGURE 12 0.10vvvv'VIvv'rTvfiIITrv'vilI'II *- 1 F —O1O . . l I . . . . l l - I . l 1 . L L ' l A L A a J a L in A 350 400 450 500 550 600 650 VkmnflengthIEWn) FIGURE 13 26 HCP-treated.membranes did not result in reoxidation of the bl—type cytochrome. Reduced plus carbon.monoxide (CO) minus reduced difference spectra of these membranes contained a peak at 425 nm, a shoulder at 419 nm, and a trough at 443 to 445 nm (Figure 13, curve B). The peak at 425 nm and trough at 443 to 445 nm.are characteristic of cytochrome a3, and the shoulder at 419 nm is probably the Soret peak of cytochrome o (2). Effect of HCP on Cytochrome Spectra of Membranes Isolated From Cells That Were Grown in Unbuffered Peptone Reduced minus oxidized difference spectra of membranes isolated from cells grown in unbuffered peptone contained 557 nm, 527 nm, and 425 nm peaks of a bl-type cytochrome, as well as peaks at 600 nm and at 443 nm (Figure 14, curve B). Addition of HCP to the L-malate reduced sample cuvette caused a decrease in the peak height of the bl-type cytochrome 557 nm, 535 nm, and 425 nm peaks, and in the a-type cyto- chrome 443 nm Soret peak (Figure 14, curve C). Reduced +»CO minus reduced difference spectra of these membranes contained a small 425 nm peak and a 445 nm trough characteristic of cytochrome a3 (Figure 15, curve B). Effect of HCP on the Redox State of the b1-Type Cytochrome in.Membranes Isolated From Cells Grown in Unbuffered Peptone Kroger and Dadak have calculated the content of B. megaterium ATTC No. 14581 bl-type cytochrome from the absorption difference at the 557.5 nm peak and the 575 trough between the fully oxidized and fully reduced states using the difference extinction coefficient - 17.5 mM—lcm-l (2,9). The (ered-on)557.5nm-(ered—eox)575nm difference spectra reported for the b -type cytochrome in their strain 1 FIGURE 14. FIGURE 15. 27 Effect of HCP on the difference spectra of membranes isolated from cells of B. megaterium grown in unbuffered peptone. Both the sample and reference cuvettes contained 0.0403 g membranes (dry wt) suspended in 2.5 ml HEPES. Symbols: CA) baseline from oxidized minus oxidized difference spectrum; (B) reduced minus oxidized spectrum obtained after addition of 25 pmoles sodium L-malate to the sample cuvette; (C) difference spectrum obtained after injection of 0.6 mg HCP in 0.1 M NaOH to the reduced sample cuvette. Reduced + C0 minus reduced difference spectra of membranes isolated from cells of B. megaterium grown in unbuffered peptone. Sample and reference cuvettes contained 0.205 g membranes (dry wt) suspended in 2.5 ml HEPES. The membranes were reduced with 25 pmoles L-malate. Symbols: (A) reduced minus reduced membrane baseline; (B) difference spectrum after bubbling the sample cuvette with CO for 30 seconds. 28 088834 Wavelength (nm) FIGURE 14 I V V ‘7 r ' ' r I V 1' V f V f I' I I I 0.10 -0.10 '550 650 600 m m m Wavelength (rm) FIGURE 15 29 of B. megaterium was similar to that obServed for the KM strain of B. megaterium so the above difference extinction coefficient was used to calculate the Bhtype CytochrOme redox state of the KM strain of B. megaterium. The extent of reduction of the b-type cytochrome in the presence of excess Lemalate.was greater than the.monenzymatic reduction produced by-dithionite and was considered to be fully reduced state. The aerobic state without substrate was considered to be the fully oxidized state. The redox state of the bl-type cytochrome was calculated from the absorption difference between the fully oxidized state and the reduced state, produced by excess substrate and various amounts of HCP, and was expressed as percent reduction of the fully reduced state (Figure 16). Cytochrome Content of Supernatant Solutions The supernatant solutions of the original protOplast lysates, from which the various membrane preparations were obtained, also were examined by means of dithionite reduced minus oxidized difference spectra. In the case of the supernatant solution associated with the membranes lacking cytochrome a, the spectrum contained only a very broad peak around 435 nm. The spectrum of the supernatant solution associated with the.membrane which contained cytochrome a showed a trough at 410 nm, a broad flav0protein peak around 460 nm (4), and peaks at 557 nm and at 425 nm characteristics of the bl-type cytochrome. FIGURE 16. 30 Effect of HCP on the redox state of the bltype cytochrome in membranes isolated from cells of B. megaterium which were grown in unbuffered peptone. Cytochrome content was calculated from the difference spectra using the difference extinction coefficient (Ered-eox)557.Snm-(€red-on)575nmf l7.5 mM-lcm-l. The 100 percent reduced state was produced by adding 25 pmoles L—malate to the sample cuvette. The indicated amounts of HCP were added to the sample cuvette in 0.1 NaOH. All lines were fitted by regression analysis. Circles and triangles represent different batches of meme branes. Symbols: (0) 0.1082 g membranes (dry wt) in 2.5 ml HEPES; (A) 0.2005 g membranes (dry wt) in 2.5 ml HEPES. 31 Percen’r reducfion 100' I I r fi I I I I I I I I I I I I I go.- \ .. I. . .. A 80- — 70p _ _ + . ~ 60- ' A ~ I- .4 50' 4 P g '4 40- o . - I . . 30r- - .- . .. 20- a 1o~ .. 1 L 1 L 1 l 1 l L l L l l 1 1 0 1 2 3 4 5 6 7 8 9 pg hexachloroPhene/mg membrane dry wt FIGURE 16 DISCUSSION Effect of HCP on Soluble Dehydrogenases HCP inhibited both.membrane-associated and soluble dehydro- genases. Kinetic analysis of the effects of HCP on soluble NADH dehydrogenase (with respect to NADH) and soluble L—malate dehydrogenase (with respect to L-malate), indicated for both enzymes an inhibition of the partially competitive type. The interpretation of these kinetics is that HCP inhibited the association of substrates with the active site of the enzymes but did not affect the rate of transfer of electrons from substrates to DCPIP once the substrate-enzyme-inhibitor-DCPIP complex was formed. The exact nature of the interaction of HCP with the dehydrogenases could not be determined. Analysis of the plots of enzyme activity vs. HCP concentration for the soluble dehydrogenases (Figure 7 and Figure 11) indicated that HCP probably did not act as a substrate analog in reversible equilibrium with the substrate binding sites of the dehydrogenases. Since HCP binds to proteins and synthetic polypeptides (18,22) a working hypothesis could be that HCP inhibited by binding to the soluble dehydrogenases, either altering their tertiary structures or partially blocking their substrate binding sites, the result of such interaction being altered enzymes with lowered substrate affinity.‘ The observation that the Ki for soluble NADH dehydrogenase was fourfold greater than that for soluble Limalate dehydrogenase may reflect the relative concentration of the two enzymes in the cytoplasm rather than different affinities for HCP. 32 33 Effect of HCP on Membrane-associated Dehydrogenases HCP inhibited membrane-associated Lemalate dehydrogenase and membrane-associated NADH dehydrogenase. The kinetics of inhibition of membrane-associated L—malate dehydrogenase, with respect to L-malate, were typical of mixed inhibition (Figure 4). The usual interpretation of such inhibition is that HCP inhibited both the association of L—malate with the dehydrogenase and the ability of the dehydrogenase to catalyze the transfer of electrons from L-malate to DCPIP once the substrateeenzyme—inhibitor-DCPIP complex was formed. However, since the dehydrogenase requires two substrates, Lamalate and DCPIP, HCP inhibition of association of DCPIP with the dehydrogenase could be the reason for the apparently lower catalytic ability of the membrane— associated Lemalate dehydrogenase. The kinetics of inhibition of membrane-associated NADH dehydrogenase, with respect to NADH,were typical of non-competitive inhibition (Figure 8). The interpretation of these kinetics was that HCP inhibited the ability of the dehydrogenase to catalyze the transfer of electrons from NADH to DCPIP once the substrate-enzyme-inhibitor- DCPIP complex was formed. Again, HCP inhibition of the association of DCPIP with the dehydrogenase could have been the reason for this apparently lower catalytic ability of the membrane-associate NADH dehydrogenase. Effect of HCP on Electron Transport in.Membranes Isolated From Cells of B. megaterium Grown in Unbuffered Peptone HCP appeared to disrupt the flow of electrons from substrate to oxygen in isolated B. megaterium membranes. The site of maximum HCP sensitivity appeared to depend on the cytochrome content of the membranes. 34 When membranes contained a b -type cytochrOme, an a-type;cytochrome, an 1 a3-type cytochrome, and lacked detectable cytochrome o, HCP caused both the bl—type cytochrome and the a—type cytochrome to become oxidized (Figure 14). This indicated that HCP inhibited electron transport on the substrate.side of the bl—type cytochrome. Extrapolation of the line relating of percent reduction of the b —type cytochrome and HCP 1 concentration to 0% reduction (the fully oxidized state, or the aerobic state without substrate) indicates that at a concentration of HCP within the range of 6 to 9 ug/mg membrane dry wt, HCP completely inhibited the flow of electrons to the bl-type cytochrome (Figure 16). Since the Ki for the membrane-associated L-malate:DCPIP oxidoreductase is 23.7 mg HCP/mg membrane dry wt, and because the flow of electrons from L-malate to DCPIP is completely inhibited by about 50 ug HCP/mg membrane dry wt (Figure 5), Lemalate dehydrogenase may not be the only site of inhibition between substrate and the b -type cytochrome. Kroger 1 and Dadak (29) have presented evidence that a pool of membrane bound menaquinone, which is present in sixfold molar excess of the bl-type cytochrome, is functionally linked to each of the dehydrogenases and is probably the only component which.mediates electron flow from the dehydrogenases to the cytochromes. Addition of menadione, which resembles menaquinone but lacks the 35 carbon side chain, partially restored oxidase activity in HCP-inhibited membranes (Figure ZB and Figure BB). HCP may bind to or inhibit menaquinone.and exogenous menadione could then bypass this inhibition. Alternatively, since the kinetics of inhibition of membrane- associated L-malate dehydrogenase indicated that HCP might inhibit by preventing association of DCPIP with the dehydrogenase, HCP inhibition 35 of oxidase activity might occur by inhibition of association of the physiological electron acceptor, menaquinone, with the dehydrogenase. The 20-fold excess of soluble.menadione may have then overcome this inhibition. B. megaterium has been reported to have two cytochrome oxidases, cytochrome a3 and cytochrome 0, which can be reduced by substrate and bind CO (2,3). When both_cytochromes are present in equal amounts, cytochromea3 catalyzes a large fraction of the respiration and is relatively insensitive to C0 (4). The relative amounts of these cyto- chromes vary with the growth stage of the organism (2). Since the reduced + C0 minus reduced difference spectra of the membranes grown in unbuffered peptone lacked cytochrome 0 peaks or troughs, cytochrome a3 probably catalyzed all of the oxidation and was apparently less sensitive to HCP inhibition than the dehydrogenases and the quinones were. Effect of HCP on Electron Transport in Membranes Isolated From Cells of B. megaterium Grown in Phosphate-buffered Peptone When the membranes contained a bl-type cytochrome, an a3-type cytochrome, and an o—type cytochrome but lacked detectable cytochrome a, addition of HCP to the L-malate-reduced membranes caused an increase in the bl-type cytochrome reduction (Figure 12). HCP has been reported to be a potent uncoupler of oxidative phosphorylation in mammalian systems (5,7,31). Uncoupling increases both oxygen uptake and the apparent reduction of the cytochromes. But HCP inhibited oxygen uptake in these membranes and since the PIO ratio of these membranes was zero even without HCP, uncoupling cannot be the reason for the apparent increase in bl—type cytochrome reduction. 36 An alternate explanation for the increase in bl—type cytochrome reduction is that HCP inhibited electron transport on the oxygen side of the cytochrome. The exact site of inhibition could not be determined but previous experimentation has indicated that it could be at cytochrome oxidase since HCP has been shown to inhibit the oxidation of mammalian cytochrome c by rat tissue preparations (5,19) and by intact cells of Bacillus subtilis (18). However the experiments of Gould et al. with B. subtilis involved the inhibition by HCP of ascorbate-reduced mammalian cytochrome c—induced oxygen uptake by intact cells of B. subtilis. Since it has been shown that even broken cell preparations of B. subtilis have only slight ability to oxidize this cytochrome (34), their results may be interpreted in other ways. For example, the HCP could be reacting with the mammalian cytochrome c to prevent its reduction by ascorbate. Unless the a3-type cytochrome in.membranes isolated from cells that were grown in buffered peptone is more sensitive to HCP than the same a -type cytochrome in the membranes from cells grown in unbuffered 3 peptone, it could presumably catalyze all of the oxidation even if the o-type cytochrome oxidase were totally inhibited by HCP. The site of maximum HCP sensitivity in these membranes isolated from cells grown in unbuffered peptone was probably between the b -type cytochrome and the 1 oxidases. Since HCP is a surfactant and has been shown to physically . disrupt membranes at high concentrations (2>30 pg HCP/mg cell dry wt) (8,23,33), perhaps part of its action derives from.disorganization of the cytochromes in the cell membrane, In the membranes which_contained an a-type cytochrome.between the bl-type cytochrome and the oxidases, this disorganization apparently did not stop the flow of electrons. 37 Summary HCP inhibited both soluble and membraneeassociated dehydrogenases. Analysis of the inhibition kinetics indicated that HCP inhibited the association of substrates with the active site of the soluble dehydro- genases but did not affect the rate of transfer of electrons frmm substrates to DCPIP once the substrate-enzyme-inhibitor-DCPIP complex was formed. In the case of the membrane—associated dehydrogenases, HCP appeared to inhibit both the ability of these dehydrogenases to transfer electrons from substrates to DCPIP once the substrate-enzyme-inhibitor- DCPIP complex was formed, and the association of Lemalate with L-malate dehydrogenase. In membranes, the site of HCP inhibition of electron transport appeared to depend on the cytochrome content. When the membranes contained a bl-type cytochrome, an a-type cytochrome, and an a3-type cytochrome, about 8 ug HCP/mg membrane dry wt inhibited electron transport on the substrate side of the b -type cytochrome, 1 probably by inhibition of electron transfer from dehydrogenases to menaquinone. When the membranes contained a bl-type cytochrome, an a3-type cytochrome, an o-type cytochrome, but lacked an a-type cyto- chrome, as little as 2 pg HCP/mg membrane dry wt inhibited electron transport on the oxygen side of the b -type cytochrome, probably at l a site between the b -type cytochrome and the terminal oxidases. 1 This inhibition may have occurred by means of subtle disorganization of the electron carriers in the membrane. The concentration of HCP necessary to completely inhibit electron transport in isolated membranes is less than one tenth of the minimum lethal concentration for B. megaterium cells. This 38 difference may be due to a protective effect of bacterial cell walls, or to a degradative effect on the membrane-bound electron transport system during isolation. 'More information on the effect of HCP on electron transport in cells of B. megaterium is now needed. APPENDIX APPENDIX A Ki's have been calculated by the methods described by Webb (38) and by Dixon and Webb (10). R1 = inhibitor constant. Km 8 Michaelis constant. I = inhibitor concen- tration. K = K (1+I/K ). V 8 maximum velocity. p m m m v = velocity. S = substrate concentration Completely mixed inhibition: The curve of the double reciprocal plot of l/v vs. l/S has a slope of: K I m vm K; For the uninhibited reaction (I = 0), the slope can be simplified to Kh/Vm: K I m SLOPEinhibited Vm Ki = 1+.E. (AA-1) . K SLOPEuninhibited Kin i V" m In the case of the membrane-associated L-malate:DCPIP oxido- reductase (Figure 4), when I a 0 the slope of the least squares regression line is 53.196. When I = 14.098 ug HCP/mg membrane dry wt, the slope of the regression line is 84.724. Substituting these values into equation AA-l and solving for K 84.724 14.098 -—————-= 1+ 53.196 K i: 1 K1 = 23.7 ug HCP/mg membrane dry wt. 39 APPENDIX B Partially competitive inhibition: The K for partially competitive inhibition can be calculated from.the 1 expression: I K Ki = 1., —2- gas-1) Y KP K.m K m Where K'm is the Michaelis constant in the presence of excess inhibitor. This quantity can be obtained by plotting KP against 1/I and extrapolating the curve to infinite inhibitor concentration. For soluble L-malate:DCPIP oxidoreductase (Figure 6), Km = 0.5472, I 8 10.980, Kim = 2.07, and K.p when I = 10.980 is 0.9473. Substituting these values into equation AB-l: 10.980 .5473 K1 = .9473 1- 2.070 - 11.049 pg HCP/mg protein. .5472 '1 For soluble NADH:DCPIP oxidoreductase (Figure 10), Km = 2.799, I - 19.230, Kfm a 14.900, and KD when I = 19.230 is 3.6754. Substituting these values into equation AB-l: 19.230 3.6754 . 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