A NUCLEOSEDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE FROM RABBITLIVER' Thesis for the Degree or M. 8,: ' MiCHlGAN STATE umvmsm JANE- K. WANG ‘ 1973 - ' .5‘8 “-3 LIBRARY L! Michigan 5"“ 0mm ~ ..,...._..... . ran—mum ._~ ‘9’“ If - m. “. m- .,__.__.‘N‘_““_,._.,.. “1; “t 0,-.." . m ABSTRACT A NUCLEOSIDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE FROM RABBIT LIVER By Jmth.ng ‘ A nucleoside triphosphate perphosphohydrolase had been purified 600-fold from rabbit liver by a procedure combining heat denaturation, ammonium sulfate solubilizat- ion and DEAR-cellulose anion exchange chromatography. The final recovery was approximately 30%. The enzyme catalyzed the hydrolysis of ITP and certain other nucleoside triphos- phates with the stoichiometric liberation of inorganic pyrophosphate and the corresponding nucleoside monophosphates as products. The enzyme catalyzed the hydrolysis at an opti- mum rate at pH 9.75 and required a sulfhydryl compound for activity. dITP was shown to be the most effective substrate for this enzyme. while ITP and XTP were hydrolyzed to a lesser extent. GTP, dGTP. TTP, and UTP were hydrolyzed at less than 1Q% of the rate of ITP. Neither ATP, CTP,dCTP, nor IMP could serve as substrates. Use of sucrose density centrifugation permitted estimation of the molecular weight at about 37,000. Pyrophosphohydrolase appears to be a soluble enzyme occurring in the cytoplasm. Crude extracts of rabbit heart, pancreas, kidney, thymus, bone marrow, lung, spleen, brain and muscle possessed similar perphos- phohydrolase activities. Brain tissue was found to be the most potent source of this enzyme, containing the highest catalytic activity per cell of any tissue tested. A NUCLEOSIDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE FROM RABBIT LIVER By Janet K Wang A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1973 AC1QIOWLEDGEI-1ENT The author wishes to eXpress her gratitude to Dr. Allan J. Morris for his guidance, patience and encouragement throughout the course of this study. She would like to thank Dr. J. L. Fairley, Dr. H. Kitchen, and Dr. W. L. Frantz for serving on her guidance committee. Financial support from the National Institutes of {ealth and the Department of Biochemistry, Michigan State University is appreciated. ii TABLE OF CONTENTS Page INTRODUCCION...................................... 1 MATERIALS......................................... 8 I. Reagents.................................. 8 II. Biological Materials ..................... 9 A. Preparation of Crude Enzyme from Rabbit Liver.......................... 9 B. Preparation of Crude Homogenates and High Speed Supernatants of Different Organs from Rabbit.................... 10 T'ETHODSOOOOOOOOOOOOOQ0.0000000000000000.0000000on. 11 I. Assay of Nucleoside Triphosphate Pyrophosphohydrolase...................... 11 II. Protein Determination..................... 12 III. Sucrose Density Centrifugation............ 12 IV. Identification of Reaction Products by Dowex I Chromatography and PEI cellulose Thin Layer Chromatography................. 13 A. DowexIChromatograpm,r ............... 13 B. PEI Cellulose Thin Layer Chromato- grapWOOOOOOOOOOOOCCOOCI0.00.00.00.00. 14 V. Analytical Disc Gel Electrophoresis....... 1h VI. Separation of Lysosomes, Mitochondria, and Peroxisomes by ISOpycnic Density gradientOOOOOOOOOOOOOOOOOIOOOOOOOOOIOOOOOO 16 VII. Preparation of Rabbit Liver Nuclei........ 17 A. Aqueous Medium........................ 17 Be NOD-aqueous MediumOOOOOOOOOOOO00.00.00 18 C. Glycerol Medium....................... 19 D. Citrate Medium........................ 19 iii Page VIII. Determination of Deoxyribonucleic ACidOOOIOOOOOICOOOOOOOIOOOIOOOOOOO.00.... 2O RESUL'FSOOOOOIOOOOOOOOI...OOOOOOOOOOOOOOOOIOOOOOC 21 I. Purification of Nucleoside Triphosphate Pyr0ph05phohydrolase.................... 21 A. Heat Denaturation Step.............. 21 B. Ammonium Sulfate Solubilization..... 21 C. DEAE Cellulose Chromatography....... 25 II. Properties of Perphosphohydrolase...... 29 A. pH Optimumoococooooooooooocooooooooo 29 B. Substrate Specificities............. 32 C. Substrate Concentration Curve....... 32 D. Requirement for a Sulfhydryl Compound........oo.........o........ 32 E. Determination of Molecular Weight by Sucrose Density Centrifugation... 38 F. Stoechiometry and Reaction Products of ITP hydrolysis by Nucleoside Tri- phosphate Perphosphohydrolase...... 38 G. Comparison of Rabbit Liver, Rabbit Red Blood Cell and Human Red Blood Cell Nucleoside Triphosphate Pyro- phosphohydrolase by Analytical Disc Gel ElectrOphoresis............ 47 III. Occurrence of Nucleoside Triphosphate Perphosphohydrolase.................... 52 A. Consideration of the Possibility that Pyrophosphohydrolase is a Lysosomal enzyme.................... 52 B. Is Perphosphohydrolase Located in the Nucleus...................... 58 IV. Survey of Nucleoside Triphosphate Pyro- phosphohydrolase Activity in Different Organs Of Rabbiteecocoooocooooeoooooeooo 59 DISCUSSION...................................... 61 LIST OF REFERENCESOOOOOOOOIOOOO000000.00.00.0000 65 iv Table LIST OF TABLES Page Purification of Nucleoside Triphosphate Perphosphohydrolase....................... 28 Substrate Specificities.................... 37 Products and Stoichiometry of ITP Hydro- lysis by Nucleoside Eriphosphate Pyro- phosphomdrolasetoiOOOOOOOOOOOOOOOOOOOOOOI. 1+6 Survey of Nucleoside Triphosphate Perphos- phohydrolase Activity in Different Organs Of Rabbitooeooooooooo00000000000000.0000... 60 LIST OF FIGURES Figure Page 1. Ammonium Sulfate Solubilization of the Heat Denatured Supernatant Fraction.......... 2h 2. DEAE Cellulose Column Chromatography on Ammonium Sulfate Fraction.................... 27 3. The Effect of pH on Perphosphohydrolase AetiVityOOOO...OOOOOOOOOOOOOOO000.000.000.000 31 u. Effect of ITP Concentration upon the Observed Perphosphohydrolase Activity....... 3# 5. Effect of Sulfhydryl Compound on the Observed Perphosphohydrolase Activity....... 36 6. Sucrose Density Centrifugation of the Perphosphohydrolase obtained from DEAE Cellulose Chromatography..................... #0 7. Analysis of Reaction Products by Dowex I Chromatography following IIP hydrolysis by Pyrophosphohydrolase...................... #3 8. Comparison of Reaction Products of IIP Hydrolysis by Pyrophosphohydrolase to the Reference Standards on PEI Cellulose Thin Layer Chromatography.................... #5 9. Disc Gel ElectrOphoresis of Perphospho- hydrolase Preparations from Rabbit Liver, Rabbit Red Blood Cell, and Human Red Blood Cell at pH 80600IOOOOOOOOOIOOI...0.0.00.0...OO 49 10. Disc Gel Electrophoresis of Perphospho- hydrolase Preparations from Rabbit Liver, Rabbit Red Blood Cell. and Human Red Blood cell at pH 705.00.COCO...OOOOOOOOOOIOOOOOIOOIO 51 vi Figure 11. 12. Separation of Particles by ISOpycnic Sucrose Density Gradient Centrifugation.............. Activity Profiles using ITP and ATP as Substrates on Fraction from ISOpycnic DenSity Gradient...............o............o vii Page 55 57 LIST OF ABBREVIAPIONS IXP, IDP, IIP, inosine 5' mono-, di-, and triphosphates, respectively. GKP, GDP, GDP, guanosine 5' mono-, di-, and triphosphates, respectively. AMP, ADP, ATP, adenosine 5' mono-, di-, and triphosphates. XIP, xanthosine triphosphate. CIP, cytidine triphosphate. UDP, UTP, uridine 5' di-, and triphosphates. dITP, dGTP, dCTP, dADP, deoxyinosine. deoxy- guanosine, deoxycytidine and deoxyadenosine triphosphates respectively. TTP, thymidine 5' triphosphate. Pi, PPi, orthOphosphate and perphosphate. GSH, glutathione. DTT, dithiothreitol. DEAE, diethylaminoethyl. PEI, polyethylene- imine. EDTA, (ethylenedinitrilo)-tetraacetic acid. DNase, deoxyribonuclease. RNase, ribonuclease. viii INTRODUC I‘ ION Inosinic acid is centrally located in the metabolic interrelationships of purines. Greenberg (1), and later Sch- ulmand and Buchanan (2) reported that in a soluble enzyme preparation from pigeon liver capable of effecting the rapid synthesis of purines from small molecule precursors, the nucleotide inosine 5' monophosphate was the initial purine compound formed. Both of the primary nucleic acid purines, adenine and guanine, are derived in nucleotide form directly from IMP by amination and oxidation reactions (1+.S.6.7.8). The natural occurrence of inosine polyphosphates is still an Open question. The chromatographic analyses for nucleoside polyphosphates in various sources (9,10,11,12) did not show the presence of IDP or ITP. although IMP was often found. It is quite possible that IDP and ITP are rapidly degraded to IMP and thus are found in very low concentration in the sources examined. However, Siekevitz g:_§l. reported that an appreciable amount of ITP was found in rat liver mitochondria (13). Also, ITP is reported to be present in amounts up to 4.5 fig of phosphorus per gram of hemoglobin in human erythrocytes as determined by high voltage paper electrOphoresis (#7). Phosphorylation of nucleosides to nucleotides in the presence of ATP has been considered as an alternative means for addition to the nucleotide pool. Early investigators (1#,15,16,17,18) have reported systems which catalyze the transphosphorylation of nucleoside mono- and triphosphate to give two nucleoside diphosphates. Myokinase (adenylate kinase), which catalyzes the reversible reaction ATP + AMP ;:::22ADP. was among the first to be discovered (19.20). Besides AMP, ATP can phosphorylate GMP, CMP, and UMP, but not IMP. However, all the nucleoside triphosphates, includ- ing ITP, can phosphorylate AMP. Joklik (21) reported that preparations from yeast carry out a myokinase-like reaction on IDP: 21DP:====IMP + ITP. However, the reaction has been tested only in the forward direction. Recently. Agarwal 23_ El- (22) have isolated and identified a guanylate kinase from human erythrocytes that will also act on IMP. The Km value is much higher for IMP than for GMP, thus the reaction preceeds less efficiently when IMP is used as a substrate. The reaction can be represented as follows: IMP + ATP:— ADP + IDP. A transphosphorylation reaction between ATP and IDP to give ITP and ADP was indicated by the findings of Krebs and Hems(23). They reported that in suspensions of pigeon breast muscle both ATP and ITP rapidly incorporated 32F added as inorganic P32-phosphate, the two terminal phosphate groups of ATP and the terminal phosphate group of ITP inter- changed with inorganic phosphate, all at about the same rate. Berg and Joklik (2“) obtained an enzyme from yeast and muscle which carries out the same reaction between ATP and IDP and also showed that UDP could participate in the reaction. The reaction is specific for a nucleoside triphosphate as phos- phate donor and a nucleoside diphosphate as acceptor, hence the name nucleoside diphosphokinase is given to the enzyme. The occurrence of the reversible reaction between ITP and AMP to give ADP and IDP was reported by Krebs and Hems (25) in sheep brain and lysed mitochondria of sheep liver. However, no reaction occurred between ATP and IMP incubated under the same conditions. Plaut (26) concluded that a potent inosinediphosphat- ase existed in rat liver mitochondria which catalyzed the reaction: IDP + HZOF—‘ilbiP + Pi. The enzyme will also hydrolyse GDP and UDP. but is inactive on ADP and GDP. Reichard.gt_gl. (27.28.29) isolated a ribonucleotide reductase from E.coli which is highly specific for ribonucleo- side diphosphates (CDP,UDP,ADP,and GDP), and the rate of their reduction is markedly affected by the presence of low concen- trations of nucleoside triphosphates. However, the possibility that IDP may serve as a substrate was not tested. Recently, Blakley et al. (30,31) reported the isolation of a ribonucleo- tide reductase from Lactobacillus leichmannii which catalyzes the reduction of GTP, ATP, CTP, ITP and to a much smaller extent, UTP. Although the reduction rate of ITP is small compared to GTP, however, the rate is much enhanced in the presence of dTTP. Thus for E.coli and Lactobacillus leichmannii (32.33), it was concluded that the exclusive biosynthetic pathway yielding deoxyribonucleotides involves reduction of the corresponding ribonucleotides. That this pathway Operates at least to some extent in animal tissues is evident from the results of tracer experiments in various mammalian organs (3h), tumor cells (35), and chick embryo (36). Larsson and Neilands (37) have also shown by a labelling eXperiment that direct ribonucleotide reduction is the exclusive biosynthetic pathway of deoxyribonucleotides in regenerating rat liver. As a summary of all the above biosynthetic and degrad- ative pathways for inosine polyphosphates, it can be postu- lated that the formation and transformation of inosine phos- phates and deoxyinosine phosphate may occur according to the following scheme: guanylate nucleoside ribonucleotide kinase diphosphokinase reductase Imps :7131D V ‘ ITP a dITP \_ inosine'fi“\ H20 diphosphatase Wyatt et al. (38) reported that the deoxyribonucleic acid of bacteriOphages T2, TM, and T6, in contrast to the deoxyribonucleic acid of their host, has hydromethylcytosine in place of cytosine. Later, Koerner gt_al. (39) characterized a dCTP-cleaving enzyme which catalyzes the hydrolysis of dCTP with the release of perphosphate and the corresponding deoxy- mononucleotides, thus excluding dCTP as a substrate for the deoxyribonucleic acid polymerase present in cell-free enzyme preparations of E.coli infected with T2 phage. At about the same time, Zimmerman and Kornberg (#0) reported that a single enzyme specific for the deoxycytidyl residue was found in phage infected E.coli which catalyzed the cleavage of both deoxycytidine di- and triphosphates according to the equations: dCDP + H20;::::deMP + Pi dC'I‘P + HZOv—‘deMP + PPi Another similar situation is found in the transform- ation of uridine and deoxyuridine phosphates. It is known that dUTP can substitute for dTTP in the DNA polymerase reaction (#1). However, with rare exceptions, uracil does not occur in DNA. The explanation for this appears to be the existence of a specific and powerful deoxyuridine tri- phosphatase which specifically cleaves dUTP to dUMP and perphosphate (42). Later, Bertani (43) postulated that the formation and transformations of deoxyuridine phosphates occurred according to the following scheme: UMP;===3UDP-———9dUDP;===3dUTP:===deMP + PPi 'The physiological function of deoxyuridine triphosphatase thus appears to be to prevent the accumulation of dUTP in the cell and involves the same principle as that underlying the action of the phage-induced deoxycytidine triphosphatase mentioned above. Recently an inosine triphosphate perphosphohydrol- ase has been purified and characterized in red blood cells of the rabbit (44). The enzyme was found to be most active towards the hydrolysis of ITP, dITP and XTP with the release of inorganic perphosphate and the corresponding mononucleo- tides as products. Since inosine and xanthine do not occur in RNA and deoxyinosine does not occur in DNA, there must be a biological control mechanism which prevents the accum- ulation of these three nucleotides within the cell. Further- more, it is known that DNA polymerase isolated from calf thymus gland will accept dITP as a substrate in the synthesis of single-chain polydeoxynucleotides (polydeoxyinosine phos- phate) (45). Polyribonucleotides with alternating inosinic acid and cytidylic acid units have also been shown to be synthesized de novo by the Azotobacter vinelandii RNA poly- merase (46). One of the possible reasons that ITP and dITP do not participate in nucleic acid synthesis may involve the existence of a Specific perphosphohydrolase such as the one isolated from red blood cells of the rabbit. This enzyme may reduce the concentration of dITP and ITP, thus making them unavailable for use as substrate by either DNA or RNA polymerases respectively. Hence it was of interest to inves- tigate the absence or existence of such an enzyme in mammal- ian tissues other than the erythrocytes. This thesis reports that a nucleoside triphosphate perphosphohydrolase has been detected in rabbit liver and partially purified from that tissue. The enzyme was found to catalyze the hydrolysis of nucleoside triphosphates with the release of inorganic perphosphate and the corresponding nucleoside monOphOSphates according to the following stoi- chiometric equation: NTP + H20;:::iNMP + PPi (N: purine or pyrimidine nucleoside) ITP, dITP and XTP were found to be the most effective substrates. Several other properties of this enzyme isolated from liver were examined and compared to the enzyme of red blood cells. Efforts have been made to elucidate the cellular distribution of perphosphohydrolase. Particle separation by iSOpycnic sucrose density gradient and the isolation of nuclei by various methods were carried out. Similar pyro- phosphohydrolase activity was detected in other tissues of the rabbit, suggesting that liver and the red blood cells were not the only sources of this enzyme. MATERIALS I. Reagents Ribonucleoside mono-, di- and triphosphates and deoxy- ribonucleoside triphosphates (except dITP) were purchased from P—L Biochemicals, Milwaukee, Wisconsin. Deoxyinosine 5' triphosphate and deoxyribonucleic acid (Type V) were purcha- sed from Sigma Chemical Company, St. Louis, Missouri. Hyflo- supra celite was obtained from Johns-Manville Co., New York. Analytical reagent grade ammonium sulfate was ordered from Mallinckrodt Chemical Works, St. Louis, Missouri. Yeast inor- ganic perphosphate (600 units/mg) was acquired from Nutri- tional Biochemical Corporation, Cleveland, Ohio. Deoxyribo- nuclease I (3000 units/mg, RNase free) was purchased from Worthington Biochemical Corporation, Freehold, New Jersey. The acrylamide, TEMED (N, N, N', N'-Tetramethylethylenedia- mine), BIS (N, N'-methylenebisacrylamide) and ammonium persulfate were purchased from Canal Industrial Corporation, Rockville, Maryland. Cellex-D (DEAE cellulose) and Dowex I resin were obtained from Bio-Rad Laboratories, Richmond, California. Nembutal was purchased from Abbott Laboratory, North Chicago, Illinois. Heparin was acquired from Fisher Scientific Company, Chicago, Illinois. Polyethyleneimine cellulose-coated plastic sheets were purchased from Brink- mann Instruments, Inc., Westburg, New York. Formaldehyde was obtained from Merck Chemical Division, Rahway, New Jersey. Stannous chloride was from Mallinckrodt Chemical Works, New York. Ammonium molybdate was purchased from Baker Chemical Co., Phillipsburg, New Jersey. 2-mercapto- ethanol was obtained from Matheson Coleman and Bell, East Rutherford, New Jersey. Indole was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin. All other com- pounds or enzymes were purchased from Sigma Chemical Company, St. Louis, Missouri. II. Biological Materials A. Preparation of Crude Enzyme from Rabbit Liver A New Zealand white male rabbit was injected intra- venously with a solution containing 2000 I.U. of heparin and 100 mg of nembutal. The animal was killed by exsan- guination by means of a heart puncture and the liver was then perfused through the aorta with one litre of 0.9% saline. The liver was excised, passed through a tissue mincer, and weighed. The minced liver was then homogenized in 3 volumes (w/v) of 50 mM Tris-Cl buffer, pH 7.a (0'0). and 1 mM GSH with a Potter-Elvehjem homogenizer equipped with a Telfon pestle. This crude homogenate was then cen- trifuged at 27,000 x g for 20 minutes to remove cell debris. The supernatant solution was then subjected to centrifugation p. 10 at 45,000 r.p.m. for two hours in order to sediment the ribosomes. The high speed supernatant fraction thus obtained was used as the starting material from which nucleoside triphosphate pyrophosphohydrolase was purified. B. Preparation of Crude Homogenates and High Speed Supernatantof Different—Organs from Rabbit The rabbit was killed as described in A. The liver, thymus, bone marrow, lung, heart, spleen, pancreas, kidney, brain and skeletal muscle were removed, kept at 4°C, and weighed. The tissue were chOpped with scalpels and then homogenized in 5 volumes (w/v) of 50 mM Tris-Cl, pH 7.4, 1 mM GSH with a telfon pestle in a motor driven Potter- Elvehjem homogenizer. The homogenates were allowed to pass through four layers of cheesecloth. The homogenates obtained at this point were referred to as the crude homogenates and were used for DNA analysis. The crude homogenates and centrifuged at 27,000 x g for 20 minutes. The supernatant fractions obtained were again centrifuged at 37,000 r.p.m. for two hours. The high epeed supernatant fractions prepared in this way were used for the analysis of nucleoside tri- phosphate perphosphohydrolase in the respective tissues of the rabbit. METHODS I. Assay of Nucleoside Triphosphate Pyrophosphohydrolase The assay for perphosphohydrolase was carried out by using a coupled assay system ultilizing yeast inorganic perphosphatase as described by Chern g£_al. (44). The enzyme was incubated in 1 ml of reaction mixture containing 50 mM e-alanine buffer, pH 9.5 (37°c). 10 mM MgClZ, 1 mM DTT, one unit yeast inorganic perphosphatase and 0.5 mM ITP. The reaction was usually initiated by the addition of the sub- strate and then incubated at 37°C for 20 minutes. A control reaction mixture lacking perphosphohydrolase was used to correct for the small amounts of orthOphosphate and pyro- phosphate found in commercial preparations of nucleoside polyphosphates used as substrates throughout the study. After incubation, the reaction was stopped by the addition of 2.2 ml of 7.27% cold trichloroacetic acid (TCA) at 4°C. The precipitate thus formed was removed by centrifugation at 2.000 x g for 5 minutes. The supernatant solution was then neutralized to pH 4.29 by the addition of a mixture of 37% formaldehyde and 3M acetate buffer (1:10). The solution was then analyzed for inorganic phosphate according to the 11 12 precedure of Rathbun et al. (48). Inorganic phosphate pro- duced in the coupled reaction was then estimated colorimet- rically by comparison with values obtained using a stand- ard KHZPOA solution. II. Protein Determination Protein concentration was determined by the method of Lowry et al. (49) using crystallized bovine serum albumin as the standard protein. III. Sucrose Density Centrifugation The method of Martin and Ames (50) for the deter- mination of the sedimentation coefficient and the molecular weight of a protein by sucrose density centrifugation was used. Linear sucrose gradients of 5-20% sucrose in 50 mM Tris-Cl, pH 7.0, 1 mM GSH and 1 mM MgC12 in a total volume of 4.6 ml were prepared in the cellulose nitrate tubes. These were kept at 4°C for 2-3 hours. A sample of 0.1 ml was care- fully layered onto the top of the gradient, and the material was then centrifuged at 50,000 r.p.m. in a SW-SO swinging bucket rotor for 16 hours at 4°C. Rabbit hemoglobin (M. w 65,000) (51) and pancreatic DNase I (M. W. 31,000) (52) were used as reference markers. After centrifugation, the tubes were punctured at the bottom and a volume of 0.2 ml per fraction was collected. The fractions were analyzed for DNase I and perphosphohydrolase activities. Hemoglobin was 13 measured by the absorption at 415 nm. DNase I activity was followed by the increase in UV absorption at 260 nm due to the depolymerisation of DNA by DNase I according to the method of Kunitz (53). IV. Identification of Reaction Products by Dowex I Chromatography and Polyethyleneimine Cellulose Thin Layer Chromatography A. Dowex I Chromatography Chromatographic analysis of the reaction products by perphosphohydrolase was performed according to the method of Zimmerman and Kornberg (40), as modified by Chern and Morris (44). A l x 12 cm Dowex I resin (100-200 mesh, Cl' form) column was prepared, washed with 0.3 N HCl and then washed with distilled water until the pH of the effluent was about 5-6. A recording UV analyzer (Instrument Specialties Company, Inc., Lincoln, Nebraska) was used to monitor the absorbance of the column effluent at 254 nm. An automatic fraction collector (ISCO, Nebraska) was used to collect the eluate fractions (8.8 ml each). The sample was applied to the column and elution was started with 0.02 N HCl. Fractions were collected until Pi and IMP were eluted from the column. The eluant was then changed to 0.1 N HCl and elution continued until PPi, IDP and ITP were washed from the column. The perphosphate peak was identified with the aid of yeast inor- ganic pyrophosphatase in producing measurable Pi. The identity 14 of each nucleotide was confirmed by chromatographic com- parison to reference standards on PEI cellulose thin layer chromatography. Fractions containing each nucleotide were pooled and the absorbance at 248.5 nm in 0.1N HCl was determined and compared to ITP in 0.1N HCI as reference standard. 3. PEI (polyethyleneimine)Cellulose Thin Layer Chromatography Nucleotide separation by ion exchange chromatography on PEI cellulose thin layer was carried out according to the procedure of Randerath and Randerath (54). A 20 x 20 cm PEI cellulose precoated plastic sheet (0.1 mm thick, cellulose MN 300 polyethyleneimine impregnated, Brinkmann Instruments) was washed with water by ascending chromatography and then dried. The samples together with reference standards were Spotted onto the plate and develOped with 1.6 M LiCl for 2 hours by ascending chromatography. Nucleotides were visual- ized with the aid of an ultra-violet lamp. V. Analytical Disc Gel Electrophoresis Polyacrylamide gel electrOphoresis was carried out essentially as described by Davis (55). The acrylamide and bisacrylamide were recrystallized from chloroform and acetone as described by Loening (56). The 7% acrylamide gel was prepared as follows: one part of solution A (1N HCl, 48 ml; 15 Tris, 36.6 gm; TEMED, 0.23 ml; and water to 100 ml) was mixed with two parts solution C (acrylamide, 25 gm; BIS, 0.735 gm; and water to 100 ml) and one part of water. The polymerization reaction was then initiated by the addition of 4 parts of catalyst (ammonium persulfate, 0.14 gm; and water to 100 ml) at 4°. Without delay, the mixture was added to gel tubes (0.6 cm diameter) to a depth of 7.5 cm, and a small volume of water was carefully layered on the tOp of the gel solution. When polymerization was completed, a stacking gel (large pore gel) with one-quarter of the height of the running gel was polymerized on the tOp of the running gel. The stacking gel was prepared as follows: one part of solution B (1N HCl, 48 ml: Tris, 5.98 gm; TEMED, 0.46 ml: and water to 100 ml) was mixed with two parts of solution D (acrylamide, 14 gm: BIS, 0.25 gm: and water to 100 ml) and 4 parts of water. Polymerization reaction was started by the addition of 1 part of catalyst (ammonium persulfate, 0.14 gm: and water to 100 ml). The protein sample was pre- pared in 2.5% of sucrose and 1 mM 2-mercaptoethanol. 3yl of 0.05% bromOphenol blue was added as a tracking dye to pro- duce a leading band of dye for locating the front during electrOphoresis. The gels were subjected to a current of 5 ma per gel cylinder with 0.025M Tris-glycine (pH 8.6) buffer containing 1 mM 2-mercaptoethanol as electrode buffer. ElectrOphoresis in 7% gel using 0.05M Tris-Cl, pH 7.5, 1 mm 2-mercaptoethanol as electrode buffer was also carried 16 out using the above procedure. After electrOphoresis, gels were stained for pro- teins with Coomassie brilliant blue in trichloroacetic acid for one hour as described by Chramback gt_§;.(57). Pyro- phosphohydrolase in polyacrylamide gel was histochemically stained by a modification of the lead conversion methods deveIOped by Gomori (58,59,60). After electrOphoresis, the gel was first incubated in 50 mmnp-alanine, 1 mM DTT, 10 mM mgCl 20 mm CaClZ, and 0.5 mM ITP for two hours at 4°, it 29 was then transferred to a water bath at 37°. Due to the hydro- lysis of ITP by perphosphohydrolase, calcium pyrophosphate precipitated as a white band which intensified after 1 hour of incubation. VI. Separation of Lysosomes,_Mitochondria and PeroxiSomes‘byOISOpygnIciDenSIty Gradient Particles were separated by iSOpycnic sucrose density gradient centrifugation in a B-30 zonal rotor as described in detail by Tolbert (61). Female Sprague-Dawley rats of about 150 gm were used and the livers perfused with grinding medium. From 20-50 gm of tissue was chOpped into very small pieces and homogenized by one pass in a loose-fitting power- driven Potter-Elvehjem homogenizer with a solution of 7% sucrose and 20 mM glycylglycine at pH 7.5. The homogenate was filtered through 4 layers of cheesecloth. The particles were sedimented between 100 and 10,000 x g for 20 minutes, 17 and resuspended in grinding medium before application over the sucrose gradient at the core area of the zonal rotor. The gradient were deveIOped for 3 to 4 hours at 30,000 r.p.m. Gradient fractions of 10 ml were collected and analyzed for perphosphohydrolase, cytochrome c oxidase (62), catalase (63), and acid phosphatase (64) activities. VII. Preparation of Rabbit Liver Nuclei A. queous Medium The method of Chauveau (65) for the isolation and purification of liver nuclei was performed. 5 gm of rabbit liver was forced through a tissue mincer to remove most of the connective tissue. The extract was then suspended in 50 ml of 2.4M sucrose, 3.3 mM CaClz prepared in 50 mM Tris- Cl buffer, pH 7.4, 1 mM GSH, and was homogenized in a power- driven Potter-Elvehjem homogenizer using 6-10 strokes. The suspension was centrifuged at 26,000 r.p.m. for one hour. The nuclei pellet was resuspended in 5 ml of 1M sucrose, 1 mM CaClZ, prepared in 50 mM Tris-Cl, pH 7.4, rehomogenized and then centrifuged at 3,000 x g for 5 minutes. The pellet materials were stained with Wright stain and found to be free of cytOplasmic and intact cells contamination as judged by visual examination in the light microsc0pe. 18 3. Non-aqueoufl_Medium Isolation of nuclei in non—aqueous medium was a modification of the method of Allfrey g£_§l.(66). One gram of rabbit liver was forced through a tissue mincer and then suspended in 50 mM Tris-Cl (pH 7.4) in a lyOphilizing flask. 'The cell suspension was rapidly frozen as a thin film on the wall of the lyOphilizing flask by immersing and spinning the flask in a COB-acetone slurry. The flask was then connected to a lyOphilizer (VirTis Research Equipment, New York) for 48 hours. The dried cell mass was then ground in a porcelain dish for 2 minutes with 5 ml of petroleum ether. The cell suspension was then centrifuged at 900 x g for 20 minutes. The sediment was resuspended with the help of a Vortex mixer in 7 ml of cyclohexane-CClu mixture (specific gravity 1.30), and then centrifuged at 2,000 x g for 40 minutes. The sediment obtained in the above step was resuspended in 7 ml of cyclo- hexane-CC14 of specific gravity 1.31, centrifuged at 2,000 x g for 40 minutes. The above process was repeated by suSpen- ding the respective sediments in 7 ml of cyclohexane-CClg of specific gravity 1.32, 1.33, and 1.335. and centrifuged as before. The pale white pellet obtained at specific gravity 1.335 was then suspended in 5 ml of cyclohexane-CCIA mixture of specific gravity 1.39, and centrifuged at 2,000 x g for 50 minutes. The supernatant fraction, which contained the nuclei, was carefully decanted and dried under vaccumn. 19 C. Glycerol Medium Preparation of liver nuclei in a glycerol medium was performed by a modified method of Schneider (67). One gram of minced rabbit liver was suspended in 15 ml of 70% glycerol, 1 mN EDTA at pH 8.4. The suspension was homogenized in a Potter-Elvehjem homogenizer by means of 20 strokes against a stationary pestle. The homogenate was filtered through 4 layers of cheesecloth. Effective separation of the nuclei from the homogenate was accomplished by the use of the foll- owing layering technique. The homogenate was simply poured down the side of a 15 ml centrifuge tube containing 1 ml of suspending glycerol medium, and centrifuged at 2,700 x g for 10 minutes. The sidiment of nuclei was washed twice with 10 ml of original medium by centrifuging at 2,700 x g for 10 minutes. The final sediment was suspended in 1 ml of original medium. D. Citrate Medium Preparation of nuclei in citrate medium was performed using a modification of the method of Higashi g£_gl.(68). The connective tissue of rabbit liver was removed by passing through a tissue mincer. The tissue was then homogenized with 1:9 volumes (w/v) of 2.5% citrate, 1 mm GSH in a motor- driven Potter-Elvehjem. After centrifuging at 600 x g for 10 minutes, the pellet was again homogenized with 5 ml/gm 20 of original tissue of 0.25M sucrose containing 1.5% citrate and 1 mi GSH. The homogenate was then layered on 2 volumes of 0.88H sucrose containing 1.5% citrate, 1 mm GSH and cen- trifuged at 900 x g for 10 minutes. VIII. Determination of Deoxyribonucleic Acid (69) This colorimetric procedure is based on a reaction between indole and the deoxysugar of DNA. Two ml of the solution to be tested (in 1N NaOH) was measured into a throu- ghly cleaned test tube. 1 ml of 0.0Q% indole and 1 ml of concentrated HCl were added and the mixture was shaken well. The test tube was then placed in a boiling water bath for 10 minutes. The tube was then cooled under running water and centrifuged to remove precipitated protein. The solution was then extracted three times with 4 m1 of chloroform and cen- trifiged to give a completely clear water phase. The intensity of the yellow color which deve10ped was measured at 490 nm against a blank treated in an identical manner but using 2 ml of 1N NaOH as the sample. A standard curve was prepared using a commercial preparation of calf thymus DNA analyzed under the same conditions. 21 RESULTS I. Purification of Nucleoside Triphosphate Pyrophosphohydrolase A. Heat Denaturation Step The high speed supernatant fraction prepared from the rabbit liver homogenate as described under the section of "Biological Material" was heat denatured at 65°C for 5 minutes with continuous swirling. The suspension was cooled immediately to 5°C. The precipitate formed was removed by centrifugation at 12,000 x g for 20 minutes. The supernatant fraction was further purified by the ammonium sulfate grad- ient described in the next section. lost of the inorganic perphosphatase was destroyed by this heat denaturation step. As shown in Table 1, a 3-fold increase in specific activity was obtained. B. Purification by Ammonium Sulfate Solubilization Further purification of perphosphohydrolase by the method of King (70) ultilizing a reverse ammonium sulfate gradient to solubilize the precipitated protein was carried out. The classical method of protein separation by ammonium 22 sulfate precipitation could be performed in the reverse manner. Namely, the proteins were first precipitated in the presence of a carrier and then they were solubilized with a decreasing salt gradient. An advantage of the reverse procedure is that it makes possible for one to choose the best recovery of the desired protein with the least amounts of contaminants. Hyflo-supra celite was added to the heat denatured supernatant fraction (1 gram celite to 100 mg protein) and mixed thoroughly. The suspension was brought to 805 saturation by the slow addition of powdered ammonium sulfate. After 30 minutes of stirring, the suspension was used to prepare a column of 3 x 2.8 cm. Elution was performed with a decresing gradient of 805-0% saturation of ammonium sulfate prepared in 50 mm Tris-Cl buffer, pH 7.4 (4°). 1 mm GSH. The flow rate was adjusted to 100 ml per hour. A volume if 15 ml per fraction was collected and aliquots of fractions were analyzed for nucleoside triphosphate perphosphohydro- lase activity. The enzyme was eluted at the region of 45-50% ammonium sulfate saturation (Figure 1). Fractions around the perphosphohydrolase peak were pooled and concentrated by the addition of powered ammonium sulfate to 70% saturation. After 30 minutes of stirring, the precipitate was collected by centrifugation and dissolved in 20 ml of 50 mm Tris-Cl (pH 8.1) containing 1 mm GSH. In some cases, the pooled fra- ctions was concentrated by presure dialysis against the same buffer. As shown in Figure 1, the perphosphohydrolase acti- 23 .umpmsovonmoupomam chomHHo m mch: 8: 0mm pm moswnuomnm how wouhHmcm one: mCoHpowhw mpmcpmpH< .muonpmz :H cwnHmommc mm coauomumq mm; couscoum mpmsmmonm oHsm :muocH no mHmmHmc< .HE H on gowns cam mHH Es m.o .mpmus cssHoo Mo as mmo.o .mmMpmsamozaouza OHGanocH pwmmh mo pHc: H .990 26 H .NHoms :s 0H .Am.m xav mchwHMnQ Es om mchHmpcoo mumprs coHpowmu m :H .cHs om mom comm pm COHPQDSocH an mmeouchnocmmonmohhm pom coshHmcm mum; Aonpomum pom as nHV wCOHpoth uopoonm .mmo SE H cam 3.5 mm .HoumHum as on CH mpmmHSm 55H:osem no COHPMMSpwm Rosmom mo pcmH unmuw m :pHs uoPSHm mm: ms>NCm egg .coHpomuH undamcpmmsm cmuSPMCmc pawn any mo :oHpmNHHHpsHom umMHSm EzHcOes< .H wusmHm 24 ( 4_ ) wu 08219 aoueqmsqv fi fi A j O L“ .. i C C . Q ‘8 p—I L J l l l l I o o o O O O o {—0—} (salowrf) p93np0Jd amudsoudon FRACTION NUMBER 25 vity was separated from the main protein peak and the hemo- globin peak (around fraction 10) as indicated by the profile of absorption at 280 nm. C. DEAE Cellulose Chromatography Anion exchange chromatography carried out in DEAE cellulose column was applied to the purification of pyro- phosphohydrolase. The enzyme solution from ammonium sulfate gradient was dialysed against 50 mm Tris-Cl (pH 8.1) and 1 mm GSH. The sample was applied to a DEAE cellulose column (2.5 x 1.h cm) previously equilibrated with the same elution buffer. Elution was performed using a linear NaCl gradient of O - 0.2N prepared in the same elution buffer (Figure 2). A volume of 2 ml per fraction was collected. The fractions were observed to be very low in protein concentration which could not be detected by UV absorption at 280 nm. The fract- ion containing pyr0phosphohydrolase activity were pooled and concentrated by pressure dialysis against 50 mM Tris-Cl, pH 7.0 and 1 mm GSH. The enzyme preparations were then stored under liquid nitrogen. The enzyme activity was found to be stable for long periods by this method of storage. The recovery of enzyme in this step was approximately 58%. The results of each individual purification step are summarized in Table 1. A final specific activity of 130 units of activity/mg protein and an overall purification 26 .H musmHm CH omthommp mm ommHonuazogamonmoumm mom cmumHmcm who; Anomo HE NV chHpomhm empsHm .xmo as H can AH.m saw Ho-mHuH as om :H zm.o - 0 mo pamempm Homz ummcHH a :qu pmpSHm mm: oHasdm on? .chHpumum oPMMHzm Echossm so anamuMOpmsomno sssHoo mmOHaHHmo m 0:» map somspon mucoummmHu on» ma ummwmumxm sz>Hpom exp ecu .mmpmmumDSm mm me< was mHH :pop mch: vohmmmm one; mCOHpomuw osp mmmnm *** chuopa me you sz>Hpom mm cmmwmpaxm zPH>Hpom OHHHommm .<.m we .Amconpms oomv mCOHchsoo hmmmm numccmpm pops: mopscHs om mom meH soak copmuonHH Ham mo mmHosa.mm commmumxo sz>Hpo< t 0.000 omH mm.o o.mm 0mm a.ms o.NH mmpm mmOHsHHmo moomu eth>Hpom as non Hs :Hmponm R Hmvop th>Hpom mesHo> wmmHohchnonamonaouhm mvmnamoanue mpHmooHozz Ho :oHmeHmHnsm .H mHnma 29 from the crude of about 660-fold was obtained. The further studies described below employed this enzyme preparation. II. PrOperties of Nucleoside Triphosphate Pyrophosphohydrolase A. pH gptimum Two buffer system were used to determine the Optimum pH of pyrophosphohydrolase activity on ITP hydrolysis. The results are shown in Figure 3. A sharp pH Optimum at 9.75 was obtained with F-alanine buffer. No activity was observed above pH 11.0 for g—alanine, but considerable activities were observed between 7.0 and 9.0 for both Tris-Cl and p-alanine buffers. Since yeast inorganic perphosphatase was used in the coupled assay system, it was important to determine that this enzyme was not a limiting factor in the assay system in the range of pH used. Chern (71) had shown that a drastic decrease of activity of inorganic perphosphatase above pH 9.5 using sodium pyrophosphate was observed. How- ever, using one unit of yeast inorganic perphosphatase (which was the amount used in the coupled enzyme assay), the activity remaining still exceeded that of the total perphosphohydrolase activity observed. Hence it was con- cluded that perphosphatase was not limiting in the assay system. Therefore the data obtained are considered to be 30 .H mustm CH coanommc mm new: mm: m8»nCm mHpom mmmHouozno: mozaohhm .ApH>Hpom mmMHoudznocmmOSQOHha Co mm mo pommwm one .m mudem 31 l J 1 50' 8 R E”. (salowrf) peonpOJd aieudsoudOJ/(d 10— 12 ll 10 32 reliable for the pH optimum of perphosphohydrolase. B. Substrate Specificity The results of different nucleoside mono-, di- and triphosphates tested as substrates of pyrophosphohydrolase are shown in Table 2. Of all the compounds tested, dITP was shown to be the most effective substrate, while ITP and XTP were hydrolyzed to a lesser extent. GTP, dGTP, TTP, UTP and IDP were hydrolyzed at less than 10% of the rate of ITP, while ATP, CTP, dCTP, and IMP do not serve as substrates for pyrophosphohydrolase. All assays were corrected for the small amounts of Pi and PPi present in commercial prepar- ation of nucleoside polyphosphates used as substrates. 0. Substrate Concentration Curve The result of variation of substrate concentrations on pyrophosphohydrolase activity was shown in Figure 4. A substrate concentration of 5 x 10‘“M was found to be Opti- mum. At higher concentration of substrates, inhibition was observed. D. Requirement for a Sulfhydryl Compound The effect of a sulfhydryl compound-~dithiothreitol, on the hydrolysis of ITP by perphosphohydrolase was studied. It was shown in Figure 5 that the activity of enzyme was dependent on the presence of a sulfhydryl compound in the 33 .mpmupwnsm mm mBH mch: H masmHm CH nopH uaommv mm cospowumm mums mommHmc< .th>Hpom mmmHouoacongmozaohhm cm>pmmno map Goa: COHpmmquocoo mHH mo vacuum .: ehsmHm 31+ 50 o --1 o (\J Ln ".-H' o H Ln qc:> l 1 I 1 \JA 0 C: C q- R N "" (salowrf) peonpwd eieudsoudon ITP Concentration (mM) 35 .sz>Hpom mmeochzonmmonmopzm um>momno one so pcsoasoo thvh:MH:m Mo pomwmm .n whfime 36 1 1 1 + 1 l = ER 8 R 8 53 (selowrf) paonpOJd eieudsoudOJ/(d 10 DTT Concentration (FUND 37 Table 2. Substrate Specificity. All substrates analyzed were 0.5 mm in the assay mixture (see Figure 1). Substrate Relative Activity ITP 100 dITP 152 XTP 99 ATP 0.3 CT? 1.1 dCTP 0.7 GTP 8.0 dGTP 6.0 TTP 3.0 UTP 9.0 IDP 4.0 IMP 0.6 38 assay mixture. Hence at all stages of purification and analyses of perphosphohydrolase, a sulfhydryl compound, either 032 or GSH, was present in the buffer or the react- ion mixture. E. Determination of Molecular Weight by Sucrose Density Centrifugation The procedure for the estimation of molecular weight of perphosphohydrolase was described under the section of "Methods". The sedimentation profile of the activities of nucleoside triphosphate perphosphohydrolase and the two reference markers--rabbit hemoglobin and pancreatic DNase I are shown in Figure 6. An estimated molecular weight of 37,000 to 39,000 was obtained for perphosphohydrolase using the relationship that the sedimentation distance is prOpor- tional to the (molecular weight)2/3. This molecular weight is based on the assumption that all three protein molecules have similar molecular shapes in the solutions used for centrifugation. F. Stoichiometry and Reaction Products of ITP Hydrolysis bnyucleoside Tfifihosphate Pyrgphosphdhydrolase A large scale reaction mixture (50 ml) containing 50 mm p-alanine, pH 9.5 (37°), 10 mm MgClZ, 1 mm DTT, 1.5ml of DEAE purified enzyme, and 40‘}moles of ITP was incubated at 37°C for 20 minutes, and the reaction was stOpped by heating to 80°C for 3 minutes. The sample was then applied 39 AmHHmpew pom muonumz emmv .pcoHUMhm mmouosm momnm m ngz .s.a.m ooo.om pm muse: 0H mom vso cmHuumo mm: COHpmwsmanseo .zzmmmwOpmSOHEQ cesHoo omOHSHHeo mNCm o: :pHsv coHpmnsocH Homecoo m Mo eHHmou COHpSHe on» mekuvmsHHH mm ouzmHm oomHohoznonmmonaouhm mo monomoua on» CH .CHE om mom COHpm unsocH mcHBOHHom mposooum COHpomoH may mo oHHmoum COHpSHm map mopmmpms uHHH <5 ousmHm .mmmHouchnonamocmohha hp mezHoHoz: meH wcHSOHHom zsmmu umopmsoumo H xozoa an mposoohn COHpoMmp Ho mHthmc< .m ehsmHm 43 (_o_) (WLU) uonenueouog {—0—} (WLU) uoneraouog $05 :2 cement 00 0 00_ 00H 0~_ 00H 00 00 J] H u \‘ 1 Ja - u - _ a x \\ .flwmm m x \ . “ am / a , . . o / \ a“ L “C no. / \ mnHur , c / c , \ 1 9H , 0 c4 1 03H; \ x (x I. m..— Efli Hum fifiq X How a Nod 1.” .m .3522 c262... 00H 0: 02 00H 00 00 00 0N H T a J A I u \ (s ,, .0 x, r. a l ASH iJr.‘ , H H .. , a -2 . r _ H m0 g , a . l .0; . . a . r v — 0a 3th . __ 13 .. a , a m4 m5 \Jka .. 0.~ T Hum z H.0 1 Hum 28.0 L <' (...-—) wu pgz 12 eouquosqv (.--_) LUU pgz 19 eouquosqv an .aEmH >3 mm wchs UoHHHucooH one: mooHpooHosz .muso: m now Hqu 20.H CH com0H0>oo handpwopmsonno wsHvCeomw an neSMH :an mmOHSHHoo Hmm Co moumvsmpm monouoweu es» ov ommHouommonmmosaonhm mp mHmzHouomn mmH Ho mposvoua COHpomeu Ho somHnmasoo .m eudem IMP IDP Sampm ITP 46 Table 3. Products and stoichiometry of ITP hydrolysis by Nucleoside Triphosphate Perphosphohydrolase. ”moles fimoles .hmoles Compound (plus enzyme) (minus enzyme) (difference) IMP 36.20 0 + 36.20 IDP 3.11 3.16 - 0.05 ITP 0 35.80 - 35.80 PPi 36.59 0.05 + 36.1# Pi 1.69 1.76 - 0.065 Unknown 0.69 0 + 0.69 47 G. Comparison of Rabbit LiverL_Rabbit Red Blood Cell and Human Red Blood Cell NuEleoside Triphosphate'Pyrophos- phohydrolase by Analytical Disc Gel Electrophoresis The rabbit red blood cell perphosphohydrolase used was prepared by the method of Chern gt_§l.(##). The human red cell pyr0phosphohydrolase of over 1,000 fold purification was prepared by the procedure of Morris (72). Polyacrylamide gel were prepared and samples were subjected to electrOphoresis as described under the section of "Methods“. Figure 9 shows the profiles of protein bands stained with Coomassie brilliant blue and the corresponding perphosphohydrolase activity bands in the polyacrylamide gel with Tris-glycine buffer at pH 8.6. All three enzymes were shown to be non-homogeneous with more than eight protein bands. The rabbit and human red blood cell enzymes show only one pyrophosphohydrolase activity band as indicated by the white Ca-perphosphate precipitate (see "Methods"). However, for the rabbit liver perphosphohydrolase tested, two white bands were observed. The lower band can be visualized after 20 minutes of incubation, but the upper band can only be seen on longer incubation at 37°C. It was also found that when gels prepared in the same manner were incub- ated in reaction mixtures containing ATP, GTP, or glucose-1- phosphate as substrates, only the upper band was visible. However, it should be pointed out that using the coupled enzyme assay system for nucleoside triphosphate pyr0phosph0hydrolase, very little activity was found when ATP, GTP or glucose 1- phosphate werépsed as substrates instead of ITP. One may thus 48 AmHHmpoo mom muozpoz momv opmgmmonaouzmnmo Ho momma mpHms mm Hnosou mo venues conue>cOo omoH one an uonHPCooH mm: mpH> :Huom ommHouozcozamonmouhm .Amuonpox memv osHp psmHHHHnn onmmsooo cpH3 vaHHmsmH> who; women cHepoug one .w.m mm em HHoo oOOHn new moss: and .HHmo UOOHQ emu panmu .um>HH pHpnmu scum chvaummoua emmHoHoznozmmonmoumm we mHmeuonmoupoeHe Hem omHQ .m ondem 49 A B A B A B F l: _ _ Rabbit Liver Rabbit RBC Hu man RBC NT PH NT PH NTPH A. Pyrophosphohydrolase Activity Stain B. Coomassie Brilliant Blue Stain 50 AmHHmpme pom meoapmz 0000 .m.s mm #0 HHmo eooHn ooh cuss: cam HHeo UOOHn ooh pHnnmn .ue>HH pHnnmu Scum muowpmummmna ommHosoznonamonmoumn mo mHmohonmoupooHe How omHQ .oH ousmHm 51 A A A a H h-l Rabbit Liver Rabbit RBC Human RBC NT PH NT PH NT PH A. Pyr0phosphohydrolase Activity Stain B. Coomassie Brilliant Blue Stain 52 conclude that the upper band is not the result of hydrolysis of ITP by a contaminating phosphatase in the pyrophosphohy- drolase preparation. Rather, it is probably some binding between a protein and the phosphate group of the substrates tested. Figure 10 shows the profiles of the protein bands and the perphosphohydrolase activity band of all three enzymes in polyacrylamide gels with Tris-Cl at pH 7.5 as electrode buffer. The resolution of the protein bands is less distinct and the mobility of the protein molecules is much slower as compared to electrOphoresis at pH 8.6 (1.1 cm/hour at pH 7.5 as compared to 5.4 cm/hour at pH 8.6). As shown in Figures 9 and 10, the pyrophosphohydrolase activity bands of rabbit liver, rabbit red blood cell and human red blood cell migra- ted at the same rate during electrophoresis. III. Occurence of Nucleoside Triphosphate Pyrophosphohydrolase within the Cell A. Consideration of the Possibility that Pyrgphosphohydro- lasePis a Lysosomal_Enzyme Since nucleoside triphosphate pyrophosphohydrolase is a hydrolytic enzyme, it might be located in the lysosome of the cell where most hydrolytic enzymes are located to carry out their degradative functions. Investigations were under- taken to elucidate this possibility. Lysosomes were separat- ed from mitochondria and peroxisomes and other cell elements as described under the section of "Methods". The results are 53 shown in Figure 11. The mitochondria band, marked by cyto- chrome c oxidase, and the peroxisomal band, marked by cata- lase are well separated. The lysosomal activity, marked by acid phosphatase, is located between the mitochondria and peroxisomes. From this gradient profile of rat liver homo- genate, it is observed that a major portion of perphospho- hydrolase activity was found in the cytosol or top soluble fraction of the gradient. A small portion of pyr0phospho- hydrolase was also presented in the lysosomal band and followed closely in distribution with acid phosphatase activity. However, further analysis on the gradient fractions using ATP as substrate (Figure 12) revealed a similar dis- tribution of activity around the lysosomal region which was absent in the cytosol. Also, fractions around the lysosomal region were found to be active towards hydrolysis of GTP, UTP, GTP, and XTP, while fractions from the cytosol were found to hydrolyze only ITP and XTP to a significant extent (dITP not tested). In view of the fact that the pyrophospho- hydrolase characterized has little effect on the hydrolysis of other nucleoside triphosphates except ITP, XTP and dITP, it was concluded that the activity which coincides in dis- tribution with acid phosphatase is the product of some non- specific phosphatase located in the lysosome. It was con- cluded from these results that pyr0ph03phohydrolase is locat- ed in the cytosol. 54 Figure 11. Separation of particles by iSOpycnic sucrose density gradient centrifugation. The gradients were deve- lOped for 3 to 4 hours at 30,000 r.p.m. Gradient fractions of 10 ml were collected. Perphosphohydrolase activity was analyzed as described in Figure 1. Acid phosphatase, cyto- chrome c oxidase and catalase were used as marker enzymes for lysosome. mitochondria and peroxisome respectively. (see Methods for more details). 55 A! let iv CEEEEBE £20.00. 000206 0 0502025 . nw — D H 0. au. 0. o. . 7 5 4 1 6 30‘ 2.0‘ 0-1.50 celsésevassé 30.200 3 3 2 9m 1.. lo .1 0 llOll lLla at All... . I IJT Jul [I'll-u H ilk \k?\| .I ll Hut lHHAIFHlIllItLY \IIIIII “my-nil III 1 I'I.l """" I l”: a. . ..... l... ,0 DH..- """""" *‘lll'll' 30:33 mm a: 050: 33:00. 0022020EmofieE 035$. / Tlollv 4 2 0. 3 6. 4. 2 l .I. l 0 o o 0 - bl . b _ - pl Alllv £23 ssmssE 02 0252. 4 2 3 . . . .w m. mu .w as O l 1 P - '1 60 50 20 30 40 Fraction Number (10 ml per fraction) 10 56 Figure 12. Activity profiles using ITP and ATP as substrates on fractions from isopycnic density gradient as described in Figure 11. The same assay conditions as described in Figure 1 were used for both ITP and ATP. 57 Alliv 30:33. mm a: 9:0: 53:00. 020.20”. nu. . r . _ P D 8 6. 4. 2 1 0 0 0 0 Allollv 20:33 00 a: 050: 33:00. 3:23. 20 10 Fraction Number (10 ml per fraction) 58 B. Is Pyrgphosphohydrolase Located in the Nucleus? Several methods of preparation of liver nuclei were carried out as described in the section of "Methods". How- ever, no conclusive result can be drawn due to the difficul- ty of obtaining a clean preparation of nuclei without any cytoplasmic contamination. In the preparation of liver nuclei in aqueous medium, the nuclei isolated are morphologically distinct and free of cytoplasmic contamination, but the activity was found entire- ly in the cytosol fraction. However, the fact that pyrophos- phohydrolase activity was not found in the nuclei may be due to one of the following reasons: when cells are broken in an aqueous medium, there is the possibility of loss of water soluble components from the nucleus, or there may be an exchange reaction of soluble enzyme between the nucleus and the soluble phase of the homogenate. Hence a non-aqueous method of isolation of nuclei which retains the water soluble components of nuclei was carried out. In the non-aqueous preparation of nuclei, perphos- phohydrolase activity was found in both cytoplasmic and nuclear fractions. However, nuclei prepared in organic solvents were broken into fragments and morphologically non-identifiable. There may have been cytoplasmic and whole cell contaminations which were not distinguishable. The total pyrophosphohydrolase activity in the nuclei fraction was 5 units as compared to about 200 units in the cytoplas- 59 mic fraction. Glucose-6-phosphate dehydrogenase was chosen as a cytoplasmic enzyme marker (73) and was found mostly in the cytoplasmic fraction as was predicted (total activity of 2.5 units in cytOplasmic fraction as compared to 0.05 units in nuclear fraction). Thus, it is possible that the 5 units of pyr0phosphohydrolase activity is due to cyt0plasmic contaimination. Two other methods of preparation of nuclei were tried. The nuclei isolated from both the glycerol medium and citrate medium were found to be contaiminted with cell membranes and cytoplasmic constituents, and pyrophosphohy- drolase activity was found to be much higher in the cyto- plasmic fraction than was in the nuclear fraction. Hence, no conclusion can be drawn as to whether perphosphohydro- lase indeed occurs in the nucleus from these eXperiments. IV. Survey of Nucleoside Tri hosphate Perphospho- hydrolase Activity in D1 erent Organs of Rabbit Crude homogenates and high speed supernatants of different organs of rabbit were prepared as described under the section of "Methods”. They were analyzed for pyr0phos- phohydrolase activity and DNA content. Taking the value of 7.2 x 10'12 gm of DNA per cell for rabbit liver (74), the amount of pyr0phosphohydrolase per cell was calculated. The results were summarized in Table 4. 60 .mHHoo esp mchhH an mpH>Hpom ommHonohnonamozaouza on» mcHNhHmcm menu was meooaHo mHHoo mo Hones: esp mchnsoo an pochpno mm; 05Hm> mHne * *0.0 . HHoo eoon 00m o.omH w.om mHomSfi 0.000 o.Hm cHdam o.mum m.Hm zmceHx o.mNN m.on mmmpocmm 0.00 0.0 cmmHmm o.HHN m.wm thom o.Hmm m.nm MSSH 0.mm m.m sounds esom 0.0mm m.Nm uneasy 0.:N: 0.3m am>HH AmnoH xv HHeo\oosuom Ham mmHosa. Hpo< mmMHOthnosamozaoahm epmnmmonaHnB oonooHosz ho ho>hsm .3 eHnma 61 DISSCUSSION A nucleoside triphosphate perphosphohydrolase which is most active in catalyzing the hydrolysis of ITP, dITP and XTP with the release of inorganic pyrophosphate and the corr- esponding nucleoside monophosphates was first detected and purified in rabbit erythrocytes (44). An investigation to detect the possible occurrence of a similar enzyme in rabbit liver is the subject of this report. It has been possible to demonstrate that such an enzyme does exist in rabbit liver. Furthermore, crude extracts of rabbit heart, pancreas, kidney, thymus, bone marrow, lung, spleen, brain and skeletal muscle were all found to possess similar perphosphohydrolase activity, with brain having the highest activity on a per cell basis. The significance of the latter finding cannot be defined at this stage. The nucleoside triphosphate pyrophosphohydrolase was purified to 600-fold from rabbit liver by a procedure combin- ing heat denaturation, ammonium sulfate solubilization and DEAE-cellulose chromatography. The crude homogenate of rabbit liver was found to be contaminated with phosphatase and pyro- phosphatase which interfere with the coupled enzyme assay system used. The fraction was therefore assayed using both 62 ITP and ATP as substrates and the difference between the two values was taken as perphosphohydrolase activity. However, it was found that the total activity obtained by this method for crude homogenate was less than the total activity in the high speed supernatant fraction obtained using the same procedure (Table 1). This has been taken to indicate that the differ- ence in activity produced by ITP and ATP probably gives only a relative estimate of perphosphohydrolase activity in the crude homogenate. The activity assayed in the high Speed supernatant fraction was found to be mostly perphosphOhydro- lase. Heat denaturation Of the high speed supernatant fract- ion at 65°C removed most of the inorganic pyrophosphatase activity, the specific activity of the perphosphohydrolase was increased 3-fold with a 50% recovery. The reverse ammon- ium sulfate gradient produced a 6-fold purification with 80% yield, the perphosphOhydrolase activity was efficiently separated from the hemoglobin peak and other preteins by this procedure. DEAR-cellulose chromatography was used to further purify the enzyme to give a final yield of 33%. The partially purified nucleoside triphosphate pyro- phosphohydrolase from rabbit liver was found to possess similar specificities and characteristics to that of red blood cell enzyme. Studies of the effect of pH on perphos- phohydrolase activity shows that they both possess a pH Optimum at 9.75. They both have an absolute requirement of sulfhydryl compound for activity. Substrate concentration 63 of 5 x 10'“ M was found to be Optimum for both enzymes when ITP is used as substrate. Identification and quantitation Of the reaction products on Dowex I column show that the liver and red cell enzymes catalyze the same reaction, that is, they catalyze the perphosphorolytic cleavage of ITP to yield stoichio- metric amounts of IMP and PPi. As far as substrate specificity is concerned, ITP, dITP and XTP are found to be the most effective substrates for both enzymes, although the degree of hydrolysis was somewhat different: for red blood cell enzyme, ITP, 100%; dITP, 103%: XTP, 71%, for liver enzyme (Table 2), ITP, 100%. dITP, 152%: XTP, 99%. Hydrolytic rates towards GTP, dGTP, UTP, TTP, GTP, and ATP are about the same for both perphOspho- hydrolase. An estimated molecular weight of 37,000 using the method Of sucrose density centrifugation with two reference markers was obtained for both liver and red cell perphOs- phohydrolase, which adds one more line of evidence that they are the same protein. Disc gel electrOphoresis at pH 8.6 and the histochem- ical identification method for perphosphohydrolase activity on polyacrylamide gel revealed that the rabbit liver and red blood cell pyrophosphohydrolase migrate through the gel at the same rate, as does the human red blood cell perphos- 64 phohydrolase. Thus each of these protein has the same elect- rOphoretic mobility at pH 8.6 and 7.5. The separation of lysosomes from mitochondria and peroxisomes revealed the fact that perphosphOhydrolase is not a lysosomal enzyme, but that it is primarily located in the cytosol. One possible function of perphosphohydrolase is that it catalyzes the hydrolysis of ITP, dITP and XTP in order to prevent the accumulation of these nucleotides so that they will not be available for nucleic acid synthesis. If this hypothesis is correct, then there is the possibility that this enzyme is a nuclear enzyme. However, as shown in the results, attempts to identify the pyrophosphohydrolase as a nuclear enzyme were unsuccessful and the results were inconclusive. 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