FURTHER CHARACTERIZATION OF THE PURINE . ‘ IUCLEOSIIDE PHOSPHORYLASES 0F BACILLUS CEREUS I ‘ SPORES AND ‘VEGETATIVE CELLS A Thesis for the Degree of Ph. «D. MICHIGAN STATE UNIVERSITY ' HELEN LOUISE ENGELBRECHT 1968 A 0-169 I)ate This is to certify that the thesis entitled Further Characterization of the Purine Nucleoside Phosphorylases of Bacillus cereus Spores and Vegetative Cells presented by Helen Louise Engelbrecht has been accepted towards fulfillment of the requirements for Ph.D. degree thicrobiology Layette/J. 1mg Major professoiZ/ z] November 18 1968 LIB R A R Y Michigan State Unix/t1 :ty ‘3.“ o. III/III IIIIIIIIIIIII III IIIIIIIL 31293300515078 ‘ mum ABSTRACT FURTHER CHARACTERIZATION OF THE PURINE NUCLEOSIDE PHOSPHORYLASES OF BACILLUS CEREUs SPORES AND VEGETATIVE CELLS By Helen Louise Engelbrecht The purine nucleoside phosphorylases of Bacillus cereus spores and vegetative cells were each purified to a state of electrophoretic homogeniety. The enzymes had previously been shown to be the products of one cistron and were similar in many properties. They were identical in their pH-activity spectra with optima at 8.3 and 7.7 depending on the method of assay. The molecular weights of the enzymes in the presence of excess phosphate were approximately 110,000. The Michaelis constants for the spore and vegetative cell purine nucleoside phosphorylases (PNPase) for phosphate were 7.3 x 10‘3M and 5.1 x 10‘3M respectively. Both PNPases were affected by sulfhydryl reagents and their activities were enhanced to differing degrees by manganese. The turnover numbers for the spore and vegetative cell enzymes were calculated to be 128 and 186 moles of inosine per mole of enzyme per second, respectively. The PNPase from vegetative cells was more anionic than that from the spores during gel electro- phoresis in low concentrations of phosphate buffer. The Helen Louise Engelbrecht StokeS' radii and sedimentation constants of the vegeta- tive cell enzyme were constant over a wide range of phosphate concentrations. However, these parameters of the spore enzyme were concentration dependent with respect to phosphate ion. The spore and vegetative cell enzymes were identical at phosphate concentrations above the Michaelis constants for phosphate. As a consequence, the molecular weight of the spore enzyme increased from approximately 89,000 to 117,000 while that of the vegeta- tive cell enzyme remained unchanged at 110,000 as the phosphate concentration was increased from zero to 0.05M. The half—life of spore PNPase at 50C was approximately 75 minutes in the absence of phosphate, which decreased to 10 minutes in 0.05M phosphate. The heat resistance of the spore enzyme in phosphate was equal to that of the vegetative cell PNPase. The data support the hypothesis that the vegetative PNPase undergoes modification during sporulation to yield an active enzyme whose state of aggregation and other physical properties varies with the ionic environment. FURTHER CHARACTERIZATION OF THE PURINE NUCLEOSIDE PHOSPHORYLASES OF BACILLUS CEREUS SPORES AND VEGETATIVE CELLS By Helen Louise Engelbrecht A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1968 ACKNOWLEDGMENTS I wish to thank Dr. Harold L. Sadoff, the director of this research for his patience, wise counsel, and con- structive suggestions throughout my graduate education. Appreciation is also extended to Dr. Ralph N. Costilow, Dr. James Fairley, and the members of their research groups for their assistance and the generous use of their laboratory facilities. I am deeply indebted to Mrs. Ruth M. Bullen for her technical assistance. I also extend special thanks to those fellow graduate students and staff here at Michigan State without whose encouragement this thesis might still remain a dream. ii TABLE OF CONTENTS ACKNOWLEDGMENTS. . . . . . LIST OF TABLES . . . . . . LIST OF FIGURES INTRODUCTION. . . . . . . LITERATURE REVIEW . . . . . Purine Nucleoside Phosphorylases Spore Enzyme Heat Resistance MATERIALS AND METHODS. Cells, Spores, and Their Extracts. Preparation of Medium . . Preparation of Inoculum, Cell and Crops. . . Enzyme Extraction . Enzyme Assay. . Xanthine Oxidase Preparation Xanthine Oxidase Assay . . Estimation of Protein. . Acrylamide Gel Electrophoresis. Preparative Gel Electrophoresis Analytical Gel Electrophoresis Sedimentation . . . . Gel Filtration . . Molecular Weight Determinations 0 Purification and Assay of Protease .EXPERIMENTAL RESULTS . . . . Purification. . . . . Gel Filtration Analytical Gel Electrophoresis iii Spore Page 11 vi CDUl-E‘H 10 Functional Properties. . . . . . . . pH Response . . . . . . . . . . Kinetic Parameters . . . PHMB Inhibition and Reactivation . . Ion Effects . . . . . Physical Properties . . . . . . . . Sedimentation. . . . . . . . . . Stokes' Radius . . . . . Calculated Molecular Weights. Thermal Inactivation Proteolysis of the Vegetative Cell Enzyme DISCUSSION . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . REFERENCES . . . . . . . . . . APPENDIX I . . . . . . . . . . . . . APPENDIX II . . . . . . . . . . iv Table LIST OF TABLES Page Purification of purine nucleoside phosphory— lase from vegetative cells of Bacillus cereus. . . . . . . . . . . . . 32 Purification of purine nucleoside phosphory- lase from spores of Bacillus cereus . . . . 35 Effect of phosphate on spore and vegetative cell purine nucleoside phosphorylases in acrylamide gel disc electrophoresis . . . . 39 Effect of sulfhydryl reagents on spore and vegetative cell purine nucleoside phorphory- lases . . . . . . . . . . . . . 50 Effect of calcium on spore and vegetative cell purine nucleoside phosphorylase . . . . . 51 Sedimentation data for vegetative cell and spore purine nucleoside phosphorylase based on human hemoglobin. S2O w = “.2. . . . . 53 3 Stokes‘ radius and statistical analysis of data for spore and vegetative cell purine nucleoside phosphorylases in various buffer systems . . . . . . . . . . . 55 Calculated molecular weights of purine nucleo- side phosphorylases . . . . . . . 57 Figure LIST OF FIGURES Analytical acrylamide gel electrophoresis of vegetative cell purine nucleoside phos- phorylase of B. cereus obtained by prepara- tive electrophoresis. . . . . . . . . Analytical acrylamide gel electrophoresis of spore purine nucleoside phosphorylase of B. cereus obtained by preparative electro— phoresis. . . . . . . . . . . Comparison of mobility in analytical gel electrophoresis of spore (sPNPase) and vege— tative cell (vPNPase) purine nucleoside phos— phorylases in various buffer systems . Log-log plot of the effect of phosphate on the relative mobility of the purine nucleo- side phosphorylases in disc gel electrophore— sis . . . . . . . . . . pH response of the purine nucleoside phosphory- lases using the colorimetric assay at 25C. . pH responses of the purine nucleoside phos— phorylases using the spectrophotometric assay at 37C . . . . . . . . . . Lineweaver-Burk plot of the spore and vege— tative cell purine nucleoside phosphorylases. Lineweaver-Burk plot of spore and vegetative cell purine nucleoside phosphorylases . Hill plots of the purine nucleoside phos- phorylases showing a slope of 1.0 for both enzymes with respect to phosphate concen— trations. . . . . . . . . . . . vi Page 34 37 38 A0 A4 A5 “7 us Figure 10. ll. 12. Page Thermal inactivation of the purified purine nucleoside phosphorylases at 50C . . 58 Thermal inactivation of (a) spore and (b) vegetative cell purine nucleoside phos- phorylases at 600. . . . . . . . . . 59 The effect of protease on vegetative cell purine nucleoside phosphorylase . . . . . 61 vii INTRODUCTION Members of the Bacillaceae are Gram positive bac— teria. They are capable of forming endospores which are usually heat resistant and metabolically dormant. The process of sporulation is of interest because it is a well—documented case of morphogenesis which can be viewed superficially as a case of abortive cell division. The spore structure is quite complex and differs from that of the cell. Spores are composed of an inner core (proto- plast) which contains ribosomes, nucleic acids, and enzymes, which are bounded by a typical membrane. Outside the membrane is a cortical layer of peptidioglycan compo- sition. It appears electron transparent by most commonly used fixation techniques for electron microscopy. The cortex is surrounded by numerous proteinaceous lamellar layers composing the spore coat. Many spores also have an exosporium composed of protein and lipid which appears as a loose fitting coat. While the spore is able to :remain in a dormant state for long periods of time, it is aLLso capable of rapid germination and outgrowth to form vwagetative cells under proper external conditions. Many of the proteins found in the spores are similar catalytically, to those of vegetative cells. Such is the case for the spore and vegetative cell purine nucleoside phosphorylases of Bacillus cereus. The synthesis of these enzymes was shown to be controlled by a single genomic unit (19). Therefore, it would be expected that the pro— teins in the two forms would be identical. However, the enzymes have strikingly different properties in the intact cells or spores. Unlike the vegetative cell enzyme, the spore phosphorylase is heat stable and dormant in Elig- These differences in properties may be the result of the unique ”packaging" in the spore or may reflect some struc— tural modifications of the enzyme which occurs during sporulation. A partial understanding of the basis of the differences in protein properties could be obtained by purifying the enzymes and studying them under identical conditions. Enzymes are the most readily studied proteins of spores because of their catalytic activity. The purine necleoside phosphorylases are present in spores and vege- ‘tative cells in quantities great enough to permit exten— sive purification and they are stable during ammonium stzlfate fractionation, gel filtration, and preparative gefil electrophoresis. Gardner and Kronberg (19) reported triat the highly purified spore enzyme differed in subtle evagrs from the vegetative cell enzyme in pH response, SEdimentation velocity, and substrate specificity. There- fore, it was decided that the enzymes merited further characterization to determine if they were in fact identi- cal or similar enzymes. The purpose of this thesis was, therefore, to char- acterize the purine nucleoside phosphorylases from spores and vegetative cells and to determine if the enzymes were identical or only similar. The enzymes were purified to electrophoretic homogeniety and their physical chemical and functional properties were studied. LITERATURE REVIEW Numerous and extensive review articles have appeared in recent years concerning various aspects of sporulation and germination in the Bacillaceae (26, A6, 60, 82, 86). These publications are evidence for the widespread interest and efforts being expended in understanding this system of unicellular morphogenesis. The process may be visualized as an abortive cell division in which one product subse- quently develops into a resting cell form. Little is understood of the various biochemical changes which occur during sporulation. In the exponential growth of cells, there is a deple- tion of carbohydrates and a building up of metabolic pro— ducts, principally acetate in Bacillus cereus (2A, 27, 62). There is a shift from an almost completely Embden-Meyerhoff metabolism to the tricarboxylic acid and diacetyl oxidation cycles (A3). At this time there appears a synthesis, in a temporal sequence, of sporulation-specific enzymes, par- ticularly protease (7). This enzyme is thought to promote protein turnover in that part of the sporulating cell which becomes the sporangium. During the course of protein turn— over, there is loss of some catelytic properties and the A 1083 of solubility of the electron transport system (1“). There is an apparent cessation of DNA synthesis, but early in sporulation, new macromolecular synthesis involves lipid synthesis as part of membranes to encase DNA. Later, the carbohydrate and protein structural materials are synthe— sized. The last acts of the sporulation process are the synthesis of small spore—specific molecules such as dipicolinic acid which accounts for up to 15% of the dry weight of the spore. At this time, the uptake of divalent cations such as manganese and calcium also occurs. Purine nucleoside phosphorylase (PNPase) is normally repressed in exponentially growing vegetative cells, but is derepressed at the time of sporulation. Alternatively, PNPase can be induced in cells by addition of inosine (19). It appears at approximately the same time as glucose dehydrogenase (2), an "early" enzyme in the sporulation process. The role of PNPase in the sporulation process is not understood. Purine Nucleoside Phosphorylases Nucleoside phosphorylases have been studies because of their ability to produce nucleosides and were thought to be in the pathway of nucleic acid synthesis. A number of the early studies were prompted by the discovery that crude hemolysates of animal tissue, particularly red b1°0d cells were able to degrade such compounds as adeno- sine (13, A2). Purine nucleoside phosphorylases (PNPase) were found in human erythrocytes (88), rat liver (33), Escherichia 32;; (56), yeast (30), and the Bacillaceae, particularly B. cereus (A8, A9, 50). Kalckar (33, 3A, 35, 36) isolated the first nucleo- side phosphorylase from rat liver. He showed that equi- molar ribose—l-phosphate and hypoxanthine produced equi- molar inosine and phosphate. The equilibrium of the reaction was shown to favor inosine with 70-80% conver- sion. The reaction was driven to completion if the inor- ganic phosphate was removed. The rat liver enzyme was specific for the production of inosine and guanosine. Manson (56) found that the purine nucleoside phos- phorylase enzyme of'ESCherichia 32E} was able to cleave guanosine and inosine. The enzyme in yeasts was able to cleave inosine, guanosine, and nicotinamide riboside (30). In the case of the Bacillaceae, the interest in PNPase was primarily the result of finding that adenosine was important in the initiation of germination and that the cleavage of purine ribosides did in fact occur in the spore. Lawrence (A8, A9) first described the cleavage of adenosine and the recovery of adenine and free ribose by Bacillus cereus. Ribosidase activity, bound to the spore debris, was directed toward adenosine and inosine according t0 tine observations of Powell and Hunter (70). Krask and Fulk (A7) found that extracts of B. cereus 2. contained nucleoside phosphorylase which cleaved adenosine or ino- sine yielding ribose-l—phosphate. They also presented evidence that a deaminase acted on adenosine prior to phosphorolysis. Purine nucleoside phosphorylase of B. cereus was highly purified and characterized by Gardner and Kornberg (19). These investigators achieved specific activities of 3,790 and A,590 umoles inosine cleaved per hour per milli- gram protein for the spore and vegetative cell enzymes, respectively. They concluded that the enzyme isolated from spores (sPNPase) and vegetative cells (vPNPase) was the same in those physical and catalytic properties which they studied. The enzyme was capable of using deoxyino- sine, deoxyguanosine, and 6-mercapt0purine riboside as well as inosine and guanosine as substrates with varying degrees of efficiency. The molecular weight was estimated to be 80,000 based on their observed sedimentation con- stant of A.85. The enzyme had a Km of 1.1-1.A x 10-“ with :respect to inosine. There were some quantitative differ— eences in pH response of the vegetative cell and spore enzymes and differences with respect to their abilities to use guanosine as substrate. Spore Enzyme Heat Resistance In order to achieve heat resistance, an ER glxg stability is required of all spore enzymes essential for the viability of the spore. Most enzymes from spores are stable i2 KEXQ but they are labile lg XEEEE' Thus it appears superficially that spores are stable because of the "packaging" of their labile components. A number of partially purified spore enzymes have been shown to be intrinsically stable or capable of stabilization by environmental factors. The NADH oxidase of Clostridium botulinum (22) and the catalase of B. cereus (76) appeared to be stable on extraction from spores. The heat resis- tance of glucose dehydrogenase can be varied over a million-fold range depending on the conditions of pH and ionic strength (78). At pH 6.5 and in 3M solutions of sodium chloride, the enzyme is as stable l2 vlggg as it is in the spore. Those conditions which produced the greatest heat resistance also brought about disaggregation of the molecules. The heat resistance of fructose-l, 6-di—phosphate aldolase of spores of B. cereus was 2M solutions of calcium chloride enhanced 10—15 fold by 10' (80). It is significant that the calcium ion concentra- tion in spores is 1.5M, assuming equal calcium and water distribution throughout the spore volume. These kinds of observations suggested that heat resistance of proteins in spores may be related to their stIWHIture and the heat resistance of spores was achieved through these molecular structures fitting into a unique cellular structure or environment. MATERIALS AND METHODS Cells, Spores, and Their Extracts Bacillus cereus is a member of the Bacillaceae and forms endospores upon completion of exponential growth. The organism employed in this research was originally iso- lated at the University of Illinois and has been variously known as Bacillus terminalis (8A) and Bacillus cereus 1 (8). This strain produces an enzyme which lyses the sporangia so that free spores are easily obtained. Preparation of Medium Sporulation levels of 95% or greater were achieved in semi-synthetic growth medium, G medium (29, 8A). The modified "G" medium contained the following materials per liter: K2HPOu, lg; (NHA)ZSOA’ Ag; MnSOu-H2O, 0.1g; MgSOu, 0.8g; yeast extract, 2g; glucose, Ag; ZnSOu, 0.01g; (DuSOu°5H20, 0.01g; CaCl2, 0.1g; and FeSOu-7H20, 0.001g. IDQW Corning antifoam AF was also added at the rate of 1 Inilliliter per liter. The organism grew and sporulated Inell in either large or small quantities at 30C. For convenience in extraction and preparation of enzyme, one hundred liter batches of vegetative cells and spores were produced. 10 ll Bfififiaration of Inoculum, 9.311 and Spore CrOps To obtain the levels of sporulation described, it was necessary to attain 3 liter volumes of exponentially growing cells. This was achieved by deveIOping the fol- lowing schedule of transfers. The stock culture was maintained in the sporulated state at AC on nutrient agar. A fresh nutrient agar slant was inoculated from stock and incubated for six to eight hours. Fifty milli- liters of "G" medium in~a 500 milliliter Erlenmeyer flask were inoculated from the slant and the flasks were shaken for two hours. Two similar flasks were inoculated with 5 ml from the first flask. After two hours, the 110 milliliters of culture were introduced into three liters of "G" medium in a New Brunswick fermenter (New Brunswick Scientific Co. New Brunswick, N. J.). An active inoculum for the 100 liter culture was attained after two hours of growth. Log phase vegetative cells were prepared in the 100 .liter stainless steel fermenter (Stainless Steel Products C30.) and harvested after five to six hours. The culture =3porulated and lysed to produce free spores in approxi— rnately twenty hours. In both cases, the medium was ooom . HE\5 HE mssomoopm .eoe oHoom >Heoa -ohm e Hopoe .efipsm .ooom .mzomoo.msaafiomm mo mHHoo o>Hpmuowo> Song ommHmpocomond opflmooaoss ocfipso mo COHpMoHMHnsmII.H mqm50pdospw H H mH.H Hm OOH www.mm mom oom HommHHmzv emslme one uodhpxm ovsno Homes A.wE .OHpmm Imopmv \mpficzv AHE\wEV ago mpHcD mwmww oapmm .>Hpo< :Hop |>ooom Hence HE\3 HE onzpooopm .MHsdm .oomm loam R .msohoo msHHHomm mo monomm Bosh ommHHHOQQmogo opfiwoofloss ocfinso mo coameHmHHSmll.m mqm<9 36 was obtained in analytical gel electrophoresis, correspond- ing to the one enzyme activity staining band (Figure 2). Analytical Gel Electrophoresis Marked differences were noted in the specific activi- ties of the spore and vegetative cell enzymes. Because this could be a reflection of major structural differences, the enzymes were tested for their relative mobilities in analytical gel electrophoresis. The Rm or relative mobil- ity was defined as the distance traveled by the enzyme sample relative to the distance traveled by the bromphenol blue (anionic) marker. Homogenous enzyme samples prepared by preparative gel were run in duplicate followed by stain- ing for both protein and active enzyme. The enzymes were run in 0.05M Tris-glycine pH 8.3 and 0.002M potassium phos- phate pH 8.3. The enzymes had the same Rm value in Tris, but the vegetative cell enzyme was more anionic in the presence of phosphate ions. In order to determine if the mobility of the enzymes was a function of the phosphate concentration during acrylamide electrophoresis the following buffers were employed: potassium phosphate pH 8.3 at 0.0005M, 0.001M, 0.002M, and 0.01M and the standard Tris-glycine buffer pH 8.3 (Figure 3). As shown in Table 3 and Figure A, the cell and spore phosphorylases exhibited changes in their electrophoretic mobilities which were dependent on the phosphate concentration, differences in relative mobility 37 .5". c ‘1." Figure 2.--Analytical acrylamide gel electrophoresis of spore purine nucleoside phosphorylase of B, cereus obtained by preparative electrophoresis. Approximately 30 ug of enzyme were used per tube. The buffer was stand— ard Tris-glycine, pH 8.3. The upper buffer contained bromphenol blue as a marker to show migration in the gels. Tube #5 was stained with Amido Schwarz protein stain. Tube #6 was stained for enzyme activity using triphenyl tetrazolium chloride. Tube C was the control containing no enzyme. (The migration markers were decolorized in the staining process.) v ”3‘... ‘LJ'A5.31£ Figure 3.—-Comparison of mobility in analytical gel electrophoresis of spore (sPNPase) and vegetative cell (vPNPase) purine nucleoside phosphorylases in various buffer systems. The proteins were stained for enzyme ac— tivity. The gel column and bromphenol blue markers were measured prior to development for enzyme activity and the column was remeasured after staining. #1 and 2 vPNPase and sPNPase in 0.05M Tris-glycine, #3 and A vPNPase and sPNPase in 0.0005M potassium phosphate, #5 and 6 vPNPase and sPNPase in 0.002M potassium phosphate, and #7 and 8 vPNPase and sPNPase in 0.01M potassium phosphate. All columns were run at pH 8.3, with 2.5 ma current per gel column. TABLE 3.-—Effect of phosphate on spore and vegetative cell purine nucleoside phosphorylases in acrylamide gel disc electrophoresis.* Cofiggifigzgion Rm (Spore PNPase) Rm (Veg PNPase) No phosphate 0.98 0.98 0.0005 M 0.75 0.98 0.001 M 0.69 0.92 0.002 M 0.72 0.86 0.01 M 0.66 0.66 *Approximately 30 pg of protein were placed on each gel column. The upper buffer included bromphenol blue as a marker. The current on each gel was 2.5 milliamperes. The gels were removed from the electrophoresis apparatus and the length of the gel and the distance traveled by the marker band was measured. After staining for enzyme activity, the distance traveled by the enzyme and the length of the gel were measured. From this information, the relative mobility of the enzymes was calculated. .-m-_js *- "M A0 .mfimosozdospooao How omen CH mommahhoco Imozo opfimooaosc CCHLBQ one eo mpHHHQoE o>HpmHop one so oumzdmogo mo poommo ohp mo poHQ moHlmoqtl.: mhswflm “#0.; ETD— o: as an o... an o. o s n A1 were observed. The spore enzyme migrated at approximately the same rate in all concentrations of phosphate. However, in the absence of phosphate, the migration of the spore enzyme increased to a value equal to that of the vegetative enzyme. It appeared that the mobility of the spore enzyme was not affected by the phosphate in the same way as the vegetative cell enzyme. Functional Properties pH Response The effect of the hydrogen ion concentration on enzymes was studied using the colorimetric assay at 25C and the spectrophotometric assay at 37C. The assays were identical to those described in the materials and methods except the buffers described by Good g£_ai. (21) were used. 2-(N-Morpholine)Ethanesulfonic acid-H O pK 6.15 2 and N-Tris(hydroxy-methyl)Methyl—2-Amino-Ethane Sulfonic Acid (TES) pK 7.5 were used in combination at 0.1M to obtain buffers over the range of pH 5.0 to 8.6. This system permitted a wide range of pH values while prevent- ing any possible phosphate concentration effect on the assays which would have occurred using Tris-phosphate buf- fers over this range. Phosphate was used in the spectro- photometric assay in substrate amounts (0.01M). The assays at 250 showed a pH optimum of 7.7 based on three assays at each pH for each enzyme (Figure 5). A2 a.» .mmmmm homo Hoe LCBHHHHHHE pod moflcz om hfioomEonsdom mo th>Hpom cm goes pom: mp3 QQQAH sm>o mpH>Hpom onHomdm mo mEmmcm .omm pm Hmmmm oHLomEHLoHoo one wsflms mommaxs0£dmocd mpflmooaozc CCHLSQ mgu mo monsoomos Ldll.m mhswflm :n o.» ex no «.0 co 0.... Nu . It .‘llflm‘ “hon“ . "4 “‘\\‘ Av \ D0> OS‘N “ \t ‘t \ AV O§\H NS \\ ‘N 1‘\~\N\~\L .\ N\ \_\ A'» ~ . A. s _\ It \ .\ I I \ In N Iwnv% 8 A an co. A3 'Ihe assays at 370 showed a pH optimum at 8.3 based on two assays at each pH for each enzyme (Figure 6). The optimum pH appears to be a function of the method used to test for the enzyme. In the spectrophotometric system, no activity could be detected at the low pH values. This was due to the pH sensitivity of the xanthine oxidase in the MES buffer system. The two enzymes were similar with respect to their activity and pH response. Kinetic Parameters Each of the purified purine nucleoside phosphorylases was assayed with varying concentrations of inosine and constant excess phosphate. They were also assayed with varying concentrations of phosphate and constant excess inosine at pH 7.5. The spore purine nucleoside phosphorylase had a Vmax of 182 units at 370 based on Lineweaver-Burke plots (52) for inosine as the substrate and a Km of 7.3 x lO-SM (Figure 7). The vegetative cell purine nucleoside phosphorylase at 37C had a Vm of 192 units based on the Lineweaver- ax Burke plots for inosine as substrate. The Km was deter- mined to be 6.7 x lO-SM for the vegetative cell enzyme (Figure 7). At 370, the spore purine nucleoside phosphorylase had a Vm of 188 units and Km of 7.2 x 10-3M with phos- ax phate as limited substrate. The vegetative cell purine I'l-II" AA .ooo.H Lo>o mpfi>flpom onHoon w zpflz sopoHHHHHE sod woes: omH pocflmBCOo onQEwm oEzmzm .owm pm zommm oHpooE0pocqosuoon who wch: mowszsochosg opflmomflozc CCHHSQ ecu mo womCOQmoL mQII.© mszmflm “1 CV % v. 3 ‘ .LOE‘I " . omA 00>. N. K. I o . m5 w§ \tt ”Ill . oo— A5 '2 cl AV I, I w ,I [,1 III I, I, I l ’1 r I I I, ‘ . .9 II”, '/4' Spa... 5 10 I5 20 m" M Inosine Figure 7.—-Lineweaver—Burke plot of the spore and vegetative cell purine nucleoside phosphorylases. The enzymes were measured with the spectrophotometric assay at various concentrations of inosine and excess phosphate. vPNPase: Vmax = 192 units, Km = 6.7 x 10'5, sPNPase: V = 182 units, Km = 7.3 x 10-5. max - .‘L‘I— A6 nucleoside phosphorylase had a Vmax of 180 units with a Km of 5.1 x 10‘3M with respect to phosphate (Figure 8). Because of the pronounced effect of phosphate in the electrophoretic mobilities of the enzymes in the electro- phoretic mobilities of the enzymes, Hill plots were con- structed to determine if more than one substrate molecule bound per molecule of enzyme. The slopes of the Hill plots are shown in Figure 9 to be 1. Therefore, the enzymes do not appear to be allosteric with respect to phosphate. This does not, however, eliminate the possi- bility of the one phosphate molecule binding at more than one place on the enzyme or in certain cases, more than one phosphate binding to the enzyme. PHMB Inhibition and Reactivation Sulfhydryl group involvement at or near the active site of the enzyme, may be demonstrated by blocking them with mercury causing a loss of enzyme activity. Upon the addition of thiols such as mercaptoethanol or dithio- threitol (Cleland's reagent) the thiol groups of the enzyme are regenerated and the activity is recovered (10). Vegetative cell and spore purine nucleoside phos- phorylases were treated with various concentrations of p—hydroxymercuribenzoate (PHMB) to determine an inhibitory concentration of the reagent. The enzymes were subse- 2 quently treated with an equal volume of 10— M PHMB and A7 xoe .ZmuoH x mH.e u ea .wwH u > ”ohoEZEm .amIOH x H.m u ax omH n me> ”ommmzm> .ocfimocfi mmooxo new opmgdmogo e0 accepmsp Icoocoo msoHpm> pm ammmm oflsuoEOBOQQOLuoodm och spas monommoe mgms moEcho one .mommamsozdmOLQ ooflmooaosc CCHHSQ Haoo o>Hp Imoowo> new macaw mo poag oxssmlso>mozocflqll.m oszmflm 2233.... 3.2 e n N _ when» a 00> I I," .8 ' ’l’, - II I I I 6 _ (It . " I, t ’1’ I] 4 ' "I A I - ‘1’ I‘ll '2 I I‘ll - "II o y. I veg v d", log V-_V 0 ' "Ill ‘V. Spore ' - I ’I - 2 ‘l‘ 1" ' l .- ~ "I, I -.4 ’I’ll I «I; I; -.6 - "I” .. ‘Ul —.8 J: A 1‘ 1 1 A A A A A L -30 » -2.0 -l-0 loo 5 Figure 9.—-Hill plots of the purine nucleoside phos- phorylases showing a slope of 1.0 for both enzymes with respect to phosphate concentrations. A9 assayed after five minutes. No activity remained in either crude enzyme preparations or in highly purified fractions from the preparative acrylamide gel column. The enzyme activity was recovered by treatment with Cleland's reagent and mercaptoethanol as recorded in Table A. From these observations, it can be concluded that there are sulfhydryl groups present in the active site of the enzyme. Ion Effects A number of metal ions were tested for their effect on the activities of highly purified vegetative cell and spore PNPases. No effects were observed in the cases of sodium (lO'lM), magnesium (10—2M), zinc (10-3M), cobalt (IO-3M), ferrous iron (lo-MM) or potassium (lo-lM). A 1.8 fold increase in activity was observed in the spore enzyme in the presence of 10-3M manganese. A 1.1 fold increase was observed in the case of the vegetative cell enzyme treated with lO-3M manganese. Calcium ions have an inhibitory effect on the activity of the enzymes. The averages of five sets of assays are presented in Table 5. Physical Properties Sedimentation The sedimentation behavior of the vegetative cell and spore purine nucleoside phosphorylases in the presence TABLE A.——Effect of sulfhydryl reagents on spore and vegetative cel purine nucleoside phorphory- lases.* Vegetative Cell Spore Enzyme Enzyme Units/ml % Units/ml Recovery Recovery Original activity 3 100 8 100 PHMB Treatment 0 0 0 O Cleland's reagent 2.76 92 A.96 62 Mercaptoethanol 5 100 2.A8 31 *Spore and vegetative cell PNPases of specific activity greater than 3,000 units/mg were treated with an equal volume of 10' M parahydroxymercuribenzoabe and assayed after five minutes at room temperature. The treated enzymes were divided into two 0.1 ml samples. One was treated with 1M Cleland's reagent. The other was treated with 1.28 moles of mercaptoethanol. The pre- parations were allowed to incubate five minutes at room temperature then assayed. -———— I TABLE 5.--Effect of calcium on spore and vegetative cell purine nucleoside phosphorylase.* Spore PNPase Vegetative Cell Melar PNPase Calcium Units/ml %R:;:IHIEZ Units/ml %RQEEIXI:Z 10—6 119 100 127 100 10_5 119 100 127 100 10'” 119 100 127 100 10-3 103 86 100 82 5 x 10‘3 A“ 37 32 25 10'2 5.5 13 2'5 2 *Spectrophotometric assays as reported in Materials and Methods were run with 0.5 ml instead of 0.6 m1 0.1M phosphate and the amount of calcium in 0.1 ml necessary to give the proper molarity was added to the cuvette. The reactions were started by addition of the enzyme. The specific activities were 600 units per mg and 2,162 units per mg for the spore and vegetative cell enzymes respectively. 52 and absence of phosphate was determined by sucrose density gradient centrifugation (57). These experiments were undertaken because of the effects of the phosphate ion on the mobilities of the phosphorylases during electrophoresis. Sucrose gradients were prepared for each enzyme in 0.05M Tris-H01 0.05M Tris-H01 and 0.001M potassium phosphate 0.05M Tris-H01 and 0.01M potassium phosphate 0.05M Tris-H01 and 0.05M potassium phosphate buffers, all at pH 7.5. Two to five sedimentation runs per phosphate concentration were run for each of the two enzymes. On the basis of fourteen determinations, it was found that the sedimentation of the vegetative cell purine nucleoside phosphorylase did not change as a function of phosphate concentration. The data were analyzed and shown to be consistent at the 95% confidence interval. On the basis of five determinations, it was also shown that the spore enzyme has the same sedimentation rate as the vege- tative cell enzyme in 0.05M Tris-H01 and 0.05M potassium However the spore enzyme and vegetative phosphate pH 7.5. cell enzyme differ in their sedimentation properties in 0.001M and 0.01M phosphate buffer (Table 6 and Appendix I). Stokes' Radius In order to further analyze the effects of phosphate ions on the spore and vegetative cell enzymes, gel 53 TABLE 6.—-Sedimentation data for vegetative cell and spore purine nucleoside phosphorylase based on human hemoglobin. 20,w A.2 Spore PNPase Vegetative PNPase Buffer S20,w Average Average S20,w 0.05M Tris—Hcl 5.A, 5.5l 5.A5 5.7 5.6, 5.8 pH 7.5 ' 0.001M Potassium 6.0, 6.0 6.17 A.9 A.7, A.8, A.9, Phosphate 6.5 5.1, 5.2 pH 7.5 0.01M Potassium 5.7, 5.8 5.9 5.1 5.0, 5.0 Phosphate 6.0, 6.1 5.1, 5.2 DH 7.5 0.05M Potassium A 9, 5.0 5.1 A.8 A.5, A.8, Phosphate 5.1 5.1 pH 7.5 5A filtration experiments were performed (1). The Stokes' radii for the enzymes were calculated from these experi- ments in 0.05M Tris-HCl 0.001M potassium phosphate and 0.05M Tris-H01 0.01M potassium phosphate and 0.05M Tris-HCl and 0.05M potassium phosphate, at pH 7.5 using human hemoglobin (a = 3.08) and cytochrome c (a = 1.A2) for markers. The results are given in Table 7 and Appendix II. The data show that the Stokes' radii of the vegeta- tive cell and spore purine nucleoside phosphorylases are significantly different in 0.05M Tris—HCl pH 7.5 and in 0.05M Tris-HCl and 0.001M phosphate, pH 7.5. The Stokes' radius of the spore enzyme remains constant in these two buffers with an a value of 3.9 nm. The vegetative cell enzyme is the same under all conditions tested at the 99% and 90% confidence levels. The spore enzyme in 0.05M potassium phosphate is the same as the vegetative cell enzyme at the 99% and 95% levels. The critical phosphate level for the configurational change in the spore enzyme is between 0.001M and 0.01M phosphate. Calculated Molecular Weights The molecular weights of the vegetative cell and spore PNPases at the various phosphate concentrations were (:alculated from their respective sedimentation constants 55 TABLE 7.--Stokes' radius and statistical analysis of data for spore and vegetative cell purine nucleoside phosphorylases in various buffer systems. sPNPase vPNPase Buffer a Values av av a values 0.05M Tris—H01 3.6, 3.7, 3.86 5 2 5.1, 5.1, 5.2, pH 7.5 3.9, “.0, 5.2, 5.“ A.1 0.001M Potassium 3.8, 3.8, 3.87 5.0 5.0 Phosphate A.0 pH 7.5 0.01M Potassium A.7, A.9 A.8 5.0 5.0 Phosphate pH 7.5 0.05M Potassium 5.A, 5.6, 5.68 5.1 A.9, 5.1, 5.2 Phosphate 6.0 I pH 7.5 __.I'~:- 'L . 56 and Stokes' radii assuming a partial specific valume of 0.725 cm3/g. These values were as follows: The molecu- lar weight of the vegetative cell enzyme is 110,000. The molecular weight of the spore enzyme in 0.05M Tris-HCl is 87,000, in 0.001M phosphate is 99,000, and in 0.01 and 0.05M phosphate is 117,000 (Table 8). Thermal Inactivation Another possible difference in the vegetative cell and spore purine nucleoside phosphorylases is their rela- tive ability to withstand elevated temperatures over various time periods. Samples of the enzymes were assayed, placed in a regulated water bath, and sampled at designated time intervals. The enzyme remaining at various times was noted as a function of temperature and the composition of the suspending medium. At 50C in Tris buffer, the purified vegetative cell enzyme had a half life of 10 minutes while the enzyme from spores had a half life of approximately 75 minutes (Figure 10). In the presence of 0.05M phosphate, the heat stabil- ity of the spore enzyme was reduced to that of the vegeta- tive cell PNPase. ility of the enzyme was also studied t in 0.05M The thermal stab at 60C with respect to the calcium ion conten Tris-HCl pH 7.5 (Figure 11). At this temperature, the half-life of the vegetative cell enzyme in Tris buffer was A minutes while that of the spore enzyme was 57 TABLE 8.--Calculated molecular weights of purine nucleo- side phosphorylases. Buffer sPNPase vPNPase 0.05M Tris-H01 87,000 110,000 0.001M P0: 99,000 110,000 0.01M P0: 117,000 110,000 0.05M P0: 117,000 110,000 V.‘ 58 10°F“~ \ 9O "\ ‘~ \ \ x s. - 80 I ‘ “ I \ “\ \‘ I ““““‘ 70 p \\ I ““““‘ o - \ £6 \‘ O Veg .5 \ I Spore :50 - ‘2 \° ' “‘— o ‘Eg!> O O C " <2QQZ-' 40 r "we 0 5 TO 15 20 25 30 35 40 Minutes Figure 10.—-Thermal inactivation of the purified purine nucleoside phosphorylases at 500. The enzyme was tested in the absence of phosphate and calcium in 0.05M Tris-RC1 buffer pH 7.5. The specific activities of the two enzyme samples was greater than 3,000. O ivity %Act‘ ' lVlI’y‘h % Act h) C) 5 IO 15 ‘20 5 IO 15 20 Minutes Minutes Figure ll.--Thermal inactivation of (a) spore vegetative cell purine nucleoside phosphorylases at The effect of calcium on the vegetative cell enzyme shown. A = Control. No calcium I = 5 x lO‘uM, o = and v = 5 x 10-3M calcium. and (b) 600. is 10-3M, 60 approximately 30 minutes. Calcium ions have relatively little effect on the heat resistance of the spore enzyme. However, a 50% loss occurred in the stability of the vegetative cell enzyme at 5 x 1073M calcium. Proteolysis of the Vegetative Cell Enzymg In order to determine if the vegetative cell purine nucleoside phosphorylase could be converted to the spore form by limited proteolysis the vegetative enzyme was treated with purified protease of Bacillus cereus. Pro- tease was prepared as reported in Materials and Methods. As shown in a previous section, the Stokes' radius of the spore enzyme 3.9 nm while the vegetative cell enzyme had a radius of 5.1 nm in 0.05M Tris-HCl, pH 7.5. On this basis, if the vegetative cell enzyme was converted to the spore form, a change in Stokes' radius would be observed after limited proteolysis. Three samples of vegetative cell enzyme were treated with various concentrations of protease then placed on a calibrated G—200 Sephadex column. The a values were 5.0, 5.0A, and A.9. These values were the same or similar to the vegetative cell value of 5.1. Therefore, in spite of the drop in activity as shown by all three samples (Figure 12), the vegetative cell form of the purine nucleoside phosphorylase does not appear to be converted to the spore form by treatment with sporulation—specific protease. 61 / f % Activ 20 2 4 6 Hours. Figure l2.—-The effect of protease on vegetative cell purine nucleoside phosphorylase. A = no protease, Y = .02 units protease, o = 0.03 units protease, I== 0.0A units protease. Purified enzyme samples, 0.3 ml (SA 5,000) con- taining approximately 100 u/ml were incubated at A0 for six hours. Samples were assayed at 30 minute intervals for two hours then every hour. DISCUSSION The purpose of the research described in this thesis was to further characterize the purine nucleoside phos- phorylases (PNPase) from vegetative cells and spores of Bacillus cereus. From their specific activities and molec- ular weights, the turnover numbers of the spore and vege- tative cell enzyme were calculated to be 128 and 186 moles of inosine per mole of enzyme per second respectively. The presence of manganese resulted in a pronounced stimu- latory effect on the spore PNPase, but very little effect on the vegetative cell enzyme. Taking into account the respective 1.8 and 1.2 fold stimulation by manganese, the turnover number of the two phosphorylases become identical. Although Gardner and Kornberg (19) showed that the synthe— sis of the enzymes were directed by one genomic unit, the results presented in this thesis show that the enzymes are different in some properties. A major difference appeared in the effect of phos- phate ions on the structure of the two enzymes. This was first observed in disc gel electrophoresis where the mobilities of the two enzymes in the absence of phosphate or in excess phosphate were the same. The movement of an 62 63 enzyme in acrylamide disc gel electrophoresis is known to be controlled by the Stokes' radius of the molecule and the molecular net charge. In the absence of phosphate, the Stokes’ radius of the spore PNPase was smaller than that of the vegetative cell enzyme. Therefore, the vege- tative cell enzyme must be more negatively charged. At low phosphate concentrations (below 10'3M), the Stokes' radius of the spore enzyme was only 80% of that of the vegetative cell enzyme. Since no change of size was observed in the vegetative cell enzyme, the changes in its relative mobility over the concentration range of phosphate studied must have been due to a decreasing nega- tive charge on the molecule. The vegetative cell enzyme probably became less anionic in high phosphate concentra- tions by exposure of positive groups. In high concen- trations of phosphate, the size of the spore enzyme increased. There was an unknown charge effect on the spore enzyme, but it was equal to the charge effect on the vegetative cell enzyme in 0.01M phosphate. The Stokes' radius of an enzyme is that value for a hypothetical spherical molecule displaying similar hydrodynamic prOperties determined by the ability of the molecule to diffuse a certain distance at an average velocity under the same experimental conditions. The Stokes' radius of the spore enzyme was shown to be signifi- cantly different from that of the vegetative cell enzyme 6A 311 low concentrations (10-3M) or in the absence of phos- phate. At higher concentrations of phosphate (greater than 1072M), the spore and vegetative cell enzymes appeared to have the same Stokes' radius. Thus, there appeared to be a critical level of phosphate required for the conversion of the spore enzyme to a form similar to the vegetative cell enzyme. This level corresponded well to the Km for phosphate as substrate (7.15 x 1073M). The significance of the changes which occur near the Km for phosphate may be due to substrate binding to the enzyme. However, on the basis of the Hill plot, there was nothing unique with respect to the phosphate binding at the active site. A further effect of phosphate was shown by the changes in heat resistance of the spore enzyme. The spore enzyme was shown to be more heat stable in the absence of phosphate. In the presence of phosphate, the heat resis- tance was equal to that of the vegetative cell enzyme. The same effect was shown using the analog arsenate instead of phosphate (Sadoff, unpublished results). The information obtained from the sedimentation con- stants and the Stokes' radius determinations permitted an approximation of the molecular weights. The vegetative cell enzyme had the same molecular weight regardless of its environment with respect to phosphate ions (110,000). The spore enzyme (88,000) appeared to increase its 65 molecular weight by 30,000 in the presence of phosphate concentrations higher than the Michaelis constant. The molecular weight of the spore enzyme (117,000) was simi- lar to that of the vegetative enzyme (110,000). The difference is within the error of the estimate. The intermediate value (99,000) represented a weight average molecular weight between the two forms. An explanation for the apparent changes in molecu- lar weight of the spore enzyme in the presence of phos- phate was developed and is based on the ability of the molecule to disaggregate. It is proposed that the vege- tative cell and spore enzymes are each composed of four subunits of approximately 30,000 molecular weight. The PNPases as initially synthesized in cells or spores are identical. This is consistent with their control of syn- thesis by one cistron. However, during the development of the spore, some structural modification (viz. proteo- lysis, hydrolysis, or removal of a functional group) occurs which permits the molecule to exist in a lower state of aggregation. In the absence of phosphate and arsenate, the predominate form consists of three subunits and has a higher intrinsic heat resistance than the aggre— gate containing four subunits. In this respect the spore enzyme resembles the glucose dehydrogenase of Bacillus cereus which is more heat resistant when in a disaggre— gated state (78). In higher phOSphate concentration, a 66 'Pedistribution of subunits occurs which yields the four subunit spore PNPase. It is identical to the vegeta- tive enzyme except for its lower turnover number. The addition of manganese to the four subunit form of the spore enzyme results in a molecule which has identical activity to the vegetative cell PNPase. Although this hypothesis is dependent upon unusual stoichiometry which has never been previously reported for any other enzyme, it is consistent with the data. A 50% loss of heat stability occurred when the vegetative cell enzyme was in 5 x 10-3M calcium chloride. Relatively little effect was observed on the heat resis- tance of the spore enzyme at any calcium concentration. Both enzymes were inhibited by 10-2 M calcium chloride. Thus the dormancy of the spore PNPase may be the result of the high calcium concentration in spores. The two PNPases were expected to have similar phy- sical and chemical properties because they were products of the same cistron. Both enzymes were inhibited by p-hydroxymercuribenzoate and reactivated by sulfhydral reagents showing the presence of —SH groups at the active sites of the enzymes. The effect of the change in pH on the two enzymes was also shown to be very similar. There appeared tote a particularly negative effect of the morpholine in the Good's buffers at low pH as shown by low activities in the spectrophotometric assays. This 67 could be an effect on the xanthine oxidase rather than on the PNPases. The Km of the two enzymes with respect to both phosphate and inosine were shown to be very similar. The experiments involving limited proteolysis of vegetative PNPase were an attempt to convert that enzyme into the spore form. The differences between the vege— tative cell and spore PNPases were not due to proteolytic cleavage of the vegetative enzyme. It may be concluded that despite all similarities observed in these enzymes, including their genetic con- trol by a single cistron, the enzymes appear to be dif— ferent aggregations of the same kinds of material. The effect of the phosphate may be a result of the structural modification of the active center, revealing E- amino groups from lysine or histidyl groups thus giving the molecule a net positive charge. SUMMARY The purine nucleoside phosphorylases of Bacillus cereus spores and vegetative cells were each purified to a state of electrophoretic homogeniety by ammonium sul- fate fractionation, gel filtration, and preparative gel electrophoresis. The enzymes had previously been shown to originate from one cistron and were similar in many properties. They were identical in their pH-activity spectra with optima at 8.3 and 7.7 depending on the method of assay. The molecular weights of the enzymes in the presence of excess phosphate were approximately 110,000. The Michaelis constants for phosphate were 7.3 x lO-3M and 5.1 x 10—3M for the spore and vegetative cell PNPases. The Michaelis constants for inosine were 7.3 x 10‘5M and 6.7 x 10-5M for the spore and vegetative cell PNPases. Both PNPases were inactivated by mercura- tion and reactivated by Cleland's reagent or mercapto- ethanol (Sulfhydryl reagents). The activities of the enzymes were enhanced in the presence of manganese, though to different extents. The turnover numbers for the spore arni vegetative cell enzymes were calculated to be 128 and 3186 moles of inosine per mole of enzyme per second, 68 69 I‘e'Specttively. In low concentrations of phosphate, the vegetative cell enzyme was more anionic than the spore enzyme during gel electrophoresis. The Stokes' radii of the enzymes were determined by the method of Ackers (1) using calibrated Sephadex G-200 columns. The sedi- mentation constants were obtained from the mobilities of the enzymes by centrifugation in sucrose density grad- ients. The Stokes' radii and sedimentation constants of the vegetative cell enzyme preparations were constant over a wide range of phosphate concentrations. These parameters of the spore enzyme were concentration— dependent with respect to phosphate. The molecular weight of the spore enzyme increased from approximately 90,000 to 120,000 while that of the vegetative cell enzyme remained at 110,000 as the phosphate ion concentration was increased from zero to 0.05M. The spore and vegeta- tive cell enzymes were identical at phosphate concentra- tions above the Michaelis constants for phosphate. The half-life of the spore PNPase at 50C was approximately 75 minutes in the absence of phosphate, but decreased to 10 minutes in 0.05M phosphate. This was equal to the stability of the vegetative cell PNPase in the presence or absence of phosphate. 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APPENDICES APPENDIX I Statistical Analysis of S Results 20,w Confidence Levels 99% 95% 90% A11 vPNPase 5.1 i 9 5 l i .7 5 l i All vPNPase+ 5.1 i .9 5.1 i .5 5.1 i 0.05M Tris-HCl and 0.05M P0: 0.05M Tris and 0.05M 5.16 i .59 5.16 i .36 5.16 1 PO“ sPNPase 0.001M P0, sPNPase 6.2 i 1.6 6.2 i .7 6.2 i 0.001M P0,I vPNPase 5.0 i .A6 5.0 i .28 5.0 i 0.01M P0, sPNPase 5.9 i .49 5.9 i .26 5.9 r 0.01M P0“ vPNPase 5.1 i .29 5.1 i .16 5.1 i 81 .27 .A8 .21 w.o«>o.m m.ow No.m m.mh.>o.m Hm.op >.m mh.o H >.m N.H h >.m mom Smo.o o.m mo.oa m.: sm.H H m.: m.m H m.e mom zHo.o 82 o.m Hm.oa sm.m ms.o A sm.m H.H H Hm.m mom zHoo.o HH.OH m.m mH.ow N.m mm.oa m.m 00.0“ mw.m mw.o H ww.m m:.H H mw.m Homlmfihfi Emo.o Rom umm Ram mom mmm xmm mHo>oq cocopfimcoo homesm whom2m> omBmme some oaHoom .moxorm no mHoAHoe< HeoHcmeocm HH NHQmem< 15‘:- IIIIIIIIIIIIIIIII