THE ROLE OF NUCLEOSIDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE, A GENETICALLY VARIABLE ENZYME, IN INOSINE TRIPHOSPHATE METABOLISM IN HUMAN ERYTHROCYTES By Vernon L. Verhoef A DISSERTATION Submitted to Michigan State University in partial fulfiliment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1978 ABSTRACT THE ROLE OF NUCLEOSIDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE, A GENETICALLY VARIABLE ENZYME, IN INOSINE TRIPHOSPHATE METABOLISM IN HUMAN ERYTHROCYTES By Vernon L. Verhoef Preliminary genetic information is presented concerning the inher- itance of nucleoside triphosphate pyrophosphohydrolase (NTPH) activity found in human erythrocytes. Previous data had shown that NTPH activity varied over lOO-fold in the red cells of a human population, but that the specific activity of any one individual was constant for a period of years. To confirm that the specific activity is indeed a genetic trait, NTPH analyses of the red cells of the members of a few selected families are presented. Secondly, to demonstrate that the variation in this trait is not a unique property of red cells, the specific activity in samples of human granulocytes, lymphocytes, or platelets obtained from selected individuals is shown to reflect the relative value of the specific activity of the erythrocytes of each of these people. Finally, to explore the molecular basis of the variation of NTPH specific activity, evidence is presented which shows that the variation cannot be attributed to differences in the Km for its sub- strate ITP or the presence of intracellular effectors. The relationship between NTPH activity and ITP in the red cell is examined by two approaches. First, the accumulation of [M]ITP in Vernon L. Verhoef erythrocytes incubated ifl_vitro with [14C1hypoxanthine is shown to be inversely related to the NTPH activity determined in hemolysates of these erythrocytes. In fact the relationship between these two para- meters follows the relationship of substrate concentration and enzyme activity predicted by Michaelis-Menten kinetics. Second, endogenous ITP, analyzed by high pressure liquid chromatography (HPLC), was detected in an extract of fresh whole blood from an individual with very low NTPH activity. This evidence suggests that NTPH limits the concen— tration of ITP allowed to exist in erythrocytes and that ITP is present at undetectable to very low concentrations in the red cells of the general population. A methylene analog of ITP (IPCPOP) was chemically synthesized in Order to inhibit NTPH activity in hemolysates and thus observe ITP synthesis in a cell-free system. IPCPOP was synthesized by two methods 13c-NMR. and characterized by HPLC, UV absorption, IR absorption and IPCPOP is shown to be a competitive inhibitor for ITP with partially purified rabbit and human NTPH with Ki equal to 3.7 and 5.9 pM, respec- tively. An affinity resin was chemically synthesized by covalently coupling the y phosphate of IPCPOP to Separose-4B by means of a six carbon spacer molecule. This resin is shown to be able to purify NTPH ZOO-fold from hemolysates. ITP synthesis by fresh hemolysates was monitored by HPLC. Manipu- lation of the incubation conditions indicated that IDP and ITP synthe- sis were significantly enhanced by the presence of phosphoribosylpyro- phosphate (PRPP) and inosine. Furthermore, the incorporation of [14C1inosine and [3H]hypoxanthine into IDP and ITP in these cell-free Vernon L. Verhoef systems was stimulated by the presence of unlabelled IMP and inosine, respectively. These ig_vitrg_experiments have been interpreted to indicate that an IMP kinase may not be involved in IDP synthesis but rather that a unique pyrophosphotransferase reaction may convert inosine directly to IDP. The stimulation of IDP/ITP synthesis with PRPP sug- gests that PRPP may be the pyr0phosphoryl donor. A proposed salvage role of IDP/ITP synthesis from inosine and PRPP coupled to NTPH degradation of ITP is discussed in terms of the possible implications of this mechanism in purine metabolism. To my parents; my wife, Nan; and my children, Philip and Renee ii ACKNOWLEDGMENTS I am most grateful to Dr. A. J. Morris for the understanding, encouragement and guidance which he provided during my years at Michigan State. Special thanks also to Dr. F. Rottman who has supported and encouraged me in very many ways. I would like to thank the members who served on my guidance committee: Dr. J. Wilson, Dr. R. Ronzio, Dr. D. McConnell and Dr. J. Higgins. I appreciate the patience and guidance of J. O'Connor during experiments involving organic syntheses, and I thank Dr. R. Barker for making equipment available for my use. I thank the colleagues in our laboratory, S. A. Fuller, C. Vary, H. Hershey and w. Chaney for helpful discussions and for frequently donating their blood for this work. I am especially grateful to S. A. Fuller who allowed me to use his data for Figure 3 and Table l2 of this dissertation. I also thank the many other people especially D. Young for willingly donating blood for these studies. Financial support was received from NIH, Michigan State Agricul- tural Experiment Station and the Department of Biochemistry, Michigan State University. iii TABLE OF CONTENTS List of Tables .......................... List of Figures ......................... List of Abbreviations ...................... I. II. III. Introduction ........................ Literature Review ..................... A. Transport and Turnover of Preformed Purines in the Red Cells ......................... B. Enzymes Involved in Red Cell Purine Metabolism ..... l. Purine Nucleoside Phosphorylase (PNP) ....... 2. Phosphoribosyltransferase (PRT) .......... 3. PRPP Synthetase .................. 4. Kinases ...................... 5. Deaminases ..................... 6. Phosphatases .................... 7. Other Enzymes ................... C. Physiological Properties of Purine Nucleosides ..... D. Diseases Associated with Enzyme Defects in Purine Meta- bolism ......................... l. Gout ........................ 2. Lesch-Nyhan Syndrome ................ 3. Xanthinurea .................... 4. Severe Combined Immunodeficiency .......... 5. Other Diseases ................... E. Blood Storage ..................... F. ITP Metabolism ..................... l. Accumulation of ITP in Erythrocytes ........ 2. Synthesis of ITP .................. 3. Endogenous ITP Concentrations ........... 4. Degradation of ITP ................. Reagents, Materials and Methods .............. A. Reagents ........................ l. Commercial ..................... 2. Distillation of Solvents .............. 3. Purification of ITP and PRPP ............ iv Page vii ix xi 3O IV. B. C. Results A. 4. Special Reagents .................. a. Triethylammonium bicarbonate buffers ...... b. Synthesis of methyl-tri-nroctylammonium hydrox- ide ...................... c. Preparation of [3H]ITP from [3H]ATP by deamina- tion ...................... d Preparation of IPCPOP from APCPOP by deamination Materials ....................... l. 2. 3. Commercial ..................... Special Preparation of Glassware .......... Biological ..................... Methods ........................ 1. Analytical ..................... a. NTPH, phosphate, hemoglobin, and protein analy- 51s ...................... Staining and counting procedures for blood cells Paper chromatography and visualization of spots UV, IR and 13C-NMR spectrometry ........ High pressure liquid chromatography ...... Liquid scintillation analysis ......... . Computer analysis of data ........... ynthetic Procedures ................ Synthesis of IPCP ............... Synthesis of IPCPOP .............. Synthesis of Sepharose-4B covalently attached to IPCPOP ..................... General Procedures ................. a. Preparation of hemolysates ........... b c MLQ'thOU' 00’!!! Blood cell separations and sonication procedures Henderson's methods for the accumulation of [ 4C]ITP in intact erythrocytes ........ Analysis of nucleotides in blood or lysates Determination of fin and Ki for NTPH ...... Affinity chromatography of NTPH ....... Cell-free synthesis of ITP ........... (0"th Genetic Variability of NTPH .............. l. 2. 3. 010143 Population Distribution .............. Inheritance of NTPH in Two Families ........ Mixing Study with Hemolysates of Red Cells of Selected Individuals ................ The Michaelis Constant for ITP of NTPH in Hemoly- sates of Selected Individuals ........... A Comparative Study of NTPH Activity Found in Ery- throcytes, Granulocytes, Lymphocytes and Platelets . NTPH Activity in the Red Cells of l3 Patients with Muscular Dystrophy ................. Relationship Between NTPH and the Accumulation of [14C]ITP in Intact Erythrocytes ............ Page 34 34 73 VI. C. Analysis of Endogenous Levels of ITP in Whole Blood l. Preparation of the Internal Standard, [ 3H]ITP 2. Resolution of Nucleotides by HPLC ......... 3. Analysis of Endogenous ITP Levels in the Blood . . D. Synthesis of Methylene Analogs of ITP ......... l. Synthesis of IPCP ................. a. Synthesis of APCP ............... b. Deamination of APCP .............. 2. Synthesis of IPCPOP ................ a. Preparation of the IPCP-imidazolide and its reaction with orthophosphate .......... b. Purification and characterization of IPCPOP . . l) UV Absorption ............... 2) HPLC .................... 3) IR Absorption ............... 4) 13C-NMR Absorption ............. 3. Synthesis of IPCPOP Covalently Attached to Sepha- rose-4B ...................... E. Application of a,B-Methylene-ITP Analogs to the Study of NTPH .......................... l. Inhibition of NTPH with IPCPOP ........... 2. Affinity Chromatography of NTPH .......... F. Kinetic Differences Between Human and Rabbit NTPH . . . G. Cell- free Synthesis of ITP ............... Stimulation of Cell- free Synthesis with PRPP . . . . Correlation of IDP/ITP with the Presence of Inosine and Hypoxanthine . ................ Incorporation of [14C]Inosine and [3H]Hypoxanthine into IDP/ITP .................... Effect of NTPH Activity on IDP/ITP Accumulation . . Other Conditions Affecting Cell-free IDP/ITP Synthe- s1s ........................ 0145 on N-J Discussion ......................... A. The Genetic Variability of NTPH ............ B. The Metabolic Consequences of the Genetic Variation of NTPH .......................... C. The Physiological Role of ITP and NTPH ......... Summary .......................... Appendix A Theoretical Relationship Between Endogenous ITP In Human Red Cells and NTPH Specific Activity ..... References . . . . . ....................... vi 121 124 124 130 130 133 133 136 137 148 150 154 LIST OF TABLES Table I Page 1 Study of NTPH Activity in Mixed Hemolysates .......... 64 2 The Michaelis Constant (Km) of NTPH of Human Red Cells Lysates from Selected Individuals ................... 65 3 Characterizatidn and NTPH Analyses of Populations of Erythro- cytes ............................. 67 4 Characterization and NTPH Analyses of Populations of Granulo- cytes ............................. 68 5 Characterization and NTPH Analyses of Populations of Lympho- cytes ............................. 70 6 Characterization and NTPH Analyses of P0pulations of Platelets 72 7 NTPH Activity in the Red Cells of Patients with Muscular Dys- trophy ............................ 77 8 Retention Times for Standard Nucleotides by HPLC Analysis . . . 88 9 Identification and Characterization of Synthesized APCP . . . . 96 10 Comparison of the UV Absorption Spectral Characteristics of IPCPOP and ITP ........................ 100 11 The 15.08-MHz, Decoupled 13C-NMR Chemical Shifts and Coupling Constants ........................... l07 12 Purification of NTPH by Affinity Chromatography ........ 116 13 Kinetic Constants for Human and Rabbit NTPH .......... 118 14 Stimulation of Cell-free ITP Synthesis with PRPP ....... 123 15 Correlation of Cell-free IDP/ITP Synthesis with Elevated Con- centrations of Hypoxanthine and Inosine ............ 126 16 Cell-free Incorporation of [14C]Inosine and [3H]Hypoxanthine into IDP/ITP ......................... 128 17 Effect of NTPH Activity on the Cell-free Synthesis of IDP/ITP . 130 vii Table 18 Effect of Extensive Charcoal Treatment of Hemolysates on Cell- free IDP/ITP Synthesis .................... viii Page LIST OF FIGURES Figure Page 1 Proposed Role of NTPH in ITP Metabolism in the Red Cell . . . . 5 2 Interconversion of Purine Nucleotides in the Red Cell ..... 9 3 A Random Survey of NTPH Specific Activity in the Red Cells of a Caucasian Population ..................... 61 4 NTPH Specific Activity in the Red Cells of Members of Two Fami- lies ............................. 63 5 Correlation of NTPH Activities in Granulocytes and Erythrocytes 74 6 Correlation of NTPH Activities in Lymphocytes and Erythrocytes 75 7 Correlation of NTPH Activities in Platelets and Erythrocytes . 76 8 NTPH Activity in Red Cells Stored in Heparin for 5 Weeks . . . 78 9 The Relationship Between [14C]ITP Accumulation in Intact Red Cells and NTPH Specific Activity ............... 80 10 DEAE-Sephadex Purification of [3H]ITP ............. 82 ll Radiochemical Purity of the Internal Standard, [3H]ITP . . . . 83 12 UV-absorbing Impurities in the Internal Standard, [3H]ITP . . . 85 13 HPLC Analysis of Endogenous Nucleotides in a PCA Extract of DY Blood ............................. 87 14 HPLC Analysis of Endogenous Nucleotides in a PCA Extract of VLV Blood ............................. 90 15 HPLC Analysis of Endogenous Nucleotides in a PCA Extract of SF Blood ............................. 92 16 Purification of APCP by DEAE-Sephadex Column Chromatography . . 95 17 HPLC Analysis of Deaminated APCP ............... 97 18 Characterization of the IPCP-imidazolide by Paper Chromato- graphy ............................ 98 ix Figure Page 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 DEAE-Sephadex Purification of IPCPOP ............. 99 HPLC Analysis of Synthesized IPCPOP .............. 101 IR Absorption Spectra of ITP and IPCPOP ............ 103 The 15.08 MHz, Decoupled 13c-NMR Spectra of (A) ITP, (B) IPCPOP, and (C) the Expanded Methylene Region of IPCPOP . . . . 105 Characterization of N-triflouroacetyl-6-aminohexane-l-phosphate by Paper Chromatography .................... 108 Characterization of the Imidazolide of N-triflouroacetyl-6- aminohexane-l-phosphate by Paper Chromatography ........ 108 Purification of y-(N-triflouroacetyl-6-aminohexyl)-IPCPOP by Dowex Column Chromatography .................. 109 HPLC Analysis of (A) y-(N-triflouroacetyl~6-aminohexyl)-IPCPOP and (B) y-(6-aminohexyl)-IPCPOP ................ 110 Bio-Gel P2 Column Chromatography of y-(6-aminohexyl)-IPCPOP . . 112 Structure of y-(6-aminohexyl)-IPCPOP ............. 113 Competitive Inhibition of Partially Purified Human NTPH with IPCPOP ............................ 114 Effect of IPCPOP on the Apparent Km of NTPH .......... 115 Lineweaver-Burk Plot for the Determination of the Km for GTP of Rabbit and Human NTPH ..................... ll7 HPLC Analysis of Hypoxanthine, Inosine, IMP, GMP, IDP, ITP, and ATP .............................. 120 Standard Curve for the Quantitation of ITP by HPLC ...... 121 Two Models for the Role of NTPH in the Metabolism of ITP . . . . 140 Theoretical Relationship Between Endogenous ITP in Human Red Cells and NTPH Specific Activity ............... 153 LIST OF ABBREVIATIONS Enzymes: ADase, adenosine deaminase; APRT, adenine phosphoribosyltrans- ferase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; NTPH, nucleoside triphosphate pyrophosphohydrolase; PNP, purine nucleoside phosphorylase. Reagents: APCP, a,B-methyleneadenosine 5'-diphosphate; APCPOP, G,B- methyleneadenosine 5'-triphosphate; CDI, 1,1'-carbony1diimidazole; DCC, dicyclohexylearbodiimide; DMF, dimethylformamide; 2,3-DPG, 2,3- diphosphoglycerate; DTT, dithiothreitol; HAP, 6-amino-l-hexanol phos- phate; HMPA, hexamethylphosphoremide; Hyp, hypoxanthine; IPCP, 3,8- methyleneinosine 5'-diphosphate; IPCPOP, a,8-methyleneinosine 5'- triphosphate; PCA, perchloric acid; PRPP, 5-phosphoribosylpyrophosphate; RlP, a,D-ribose l-phosphate; RSP, D-ribose 5-phosphate; TCA, trichloro- acetic acid; TEAB, triethylamnonium bicarbonate; TFA-HAP, N—triflouro- acetyl-6-amino-l-hexanol phosphate. Miscellaneous: ACD, acid citrate dextrose; BSS, buffered salt solution; CID, combined immunodeficiency; IR, infrared; Hb, hemoglobin; HPLC, high pressure liquid chromatography; NMR, nuclear magnetic resonance; PRP, platelet rich plasma; UV, ultraviolet. xi CHAPTER I INTRODUCTION Prior investigations in this laboratory of ifl_vitrg globin syn- thesis by rabbit reticulocyte lysates led to the observation that a GTP hydrolase activity was present in these lysates. This activity was purified 2000-fold and characterized as a nucleoside triphosphate pyrophosphohydrolase (NTPH) which catalyzes the general reaction, NTP + H20 + NMP + PPi, where NTP and NMP refer to nucleoside triphosphate and monophosphate, respectively (1). The best substrates for NTPH were ITP, dITP and XTP. GTP was hydrolyzed at 10% the rate of ITP; and IDP, IMP and ATP were not hydrolyzed at all. The purified enzyme required at least 10 mM MgC12, a sulfhydryl reagent and a pH of 9.75 for optimal activity. The molecular weight of NTPH was estimated to be 37,000 daltons by sucrose density sedimentation. A survey of NTPH in various tissues of the rabbit indicated that activity was present in the thirteen tissues examined and was highest in terms of activity per cell (determined by NTPH and DNA analyses) in brain, liver and kidney but lowest in erythrocytes (2). NTPH purified from rabbit liver exhibited the same characteristics as that previously purified from reticulocytes. No isozymes were detected by disc gel electrophoresis in NTPH preparations from rabbit liver or erythrocytes or preparations from human erythrocytes. NTPH activity has also been detected in the red cells of twelve species including those of human. A survey of the specific activity (units/mg hemoglobin) in erythrocytes obtained from various human indi- viduals indicated that the activity differed more than lOO-fold from one individual to another yet was constant in any one individual over a period of years. A survey of 6000 subjects by Vanderheiden in 1969 gave evidence for the genetic transmission of an "inosine triphospha— tase," the activity of which inversely correlated with the presence of ITP in red blood cells (3). He was inadvertently measuring a coupled reaction between NTPH and endogenous inorganic pyrophosphatase (4). These observations suggested that NTPH activity in the human erythro- cyte may be an inherited trait. Further work on NTPH was carried on by S. A. Fuller, a graduate student in genetics, and myself. The data presented here support the thesis that NTPH at a genetically defined activity determines the level of ITP that is allowed to exist in human erythrocytes; and furthermore, that this enzyme may complete a previously unrecognized salvage cycle in which inosine, a metabolic waste product, is converted to IMP, a metabolite which plays a central role in the biosynthesis and catabo- lism of AMP and GMP. Preliminary observations were made in an attempt to determine the genetic or molecular basis for the remarkable enzyme variation from one individual to another. A population distribution study performed by S. A. Fuller and NTPH analyses of the members of two families indicate that the specific activity is an inherited trait but that its mode of inheritance may be rather complex. A mixing study using red cell lysates of selected individuals with greatly different NTPH activities gave no evidence for intracellular activators or inhibitors. The Km for ITP of NTPH of hemolysates prepared from red cells of selected individuals was independent of the specific activities of these individ- Uals and thus variation of the Km of NTPH does not account for the variation of NTPH specific activity in the red cell. Finally, the NTPH activity per cell found in granulocytes, lymphocytes, or platelets was directly correlated with the activity per cell found in erythrocytes. These studies show that the specific activity for NTPH of an individ- ual's red cells is associated with NTPH specific activity in other blood cell types. These experiments support the conclusion that NTPH specific activity in all tissues is inherited in a defined manner and that variation of the Km for ITP or the presence of intracellular fac- tors are unrelated to the origin of the wide variation in NTPH levels found in the human population. In a cooperative study with J. F. Henderson's laboratory at the University of Alberta (Edmonton, Alberta, Canada) it was possible to examine the relationship between NTPH activity of erythrocytes and the ability of erythrocytes incubated in_vitrg to accumulate [14C]ITP from [14C]hypoxanthine (5). The relationship between these two parameters closely fits a theoretical relationship predicted by employing Michaelis- Menten kinetics for the relationship of an enzyme activity with the concentration of its substrate. Furthermore, ITP could be detected in the perchloric acid extract of fresh blood of one individual, a person \vho had a very low level of NTPH activity. These experiments support ‘Vanderheiden's inverse correlation between "ITPase" activity and con- centrations of ITP in the cells (3). A methylene analog of ITP (IPCPOP) was chemically synthesized to explore ITP metabolism in erythrocyte lysates. This compound exhibited competitive inhibition for ITP with either partially purified human or rabbit enzyme with Ki values of 3.7 and 5.9 uM, respectively. The y phosphate of IPCPOP was attached to Sepharose by means of six carbon spacer molecule and the affinity column produced in this manner could be used to rapidly purify NTPH 200-fold from hemolysates by virtue of the affinity column's unique specificity for the enzyme. Preliminary data indicated that the cell-free accumulation of IDP and ITP in a three hour incubation was greatly enhanced by inosine and PRPP as opposed to IMP and PRPP. The role of inosine, IMP, PRPP, and NTPH in IDP/ITP accumulation was studied by the addition of various inhibitors, radioactive precursors, or intermediary metabolites to the incubation media. The proposed schemeikn~the synthesis of ITP and the role of NTPH which is suggested by these data is presented in Figure l. IMP (2) Inosine P. 1 Figure 1. Proposed Role of NTPH in ITP Metabolism in the Red Cell R5P ADP ITP (4) 1) ATP PPi IDP PRPP The enzymes involved in this cycle are the following: (1) NTPH, (2) 5'-nucleotidase, (3) proposed pyrophosphotransferase, (4) nucleoside diphosphokinase. PRPP and R5P refer to phosphoribosylpyrophosphate and D-ribose 5-phosphate. ad fo ox de' le the CHAPTER II LITERATURE REVIEW A. Transport and Turnover of Preformed Purines by the Red Cells Human erythrocytes lack three enzymes of purine metabolism usually found in other tissues, phosphoribosylpyrophosphate amidotransferase, adenylosuccinate synthetase and xanthine oxidase (6, 7, 8, 9). There- fore erythrocytes cannot synthesize purines gg_ngyg, AMP from IMP or oxidize hypoxanthine to xanthine or uric acid, and the red cells must depend on salvage and transport mechanisms to maintain their proper levels of nucleotides. Two reviews by Murray give a good background to these mechanisms by emphasizing the significance of the salvage path- ways and the regulatory controls of nucleotide biosynthesis from pre- formed purines (10, 11). The transport of purines across the red cell membrane does not seem to be a simple diffusion process. Although transportation of hypoxanthine, guanine and adenine has been shown to be very rapid (12, 13), Lassen has been able to distinguish a saturable from a nonsatura- ble transport system for one of the bases, hypoxanthine (13). Other investigators, deBruyn and Oei, have suggested that incorporation of Purine bases by intact red cells may be regulated by salvage enzymes associated with the cell membrane (14). The transport of nucleosides across the plasma membrane is also complex; Oliver and Paterson have described the transport as mediated by a "nonconcentrative, 7 'facilitated diffusion' mechanism of broad specificity" (15) . Furthermore, they have shown nucleoside transport to be independent of the base or sugar transport mechanisms (16) . Agarwal and Parks suggest that the metabolic fate of adenosine may be determined at the membrane surface by adenosine deaminase (l7) . Nucleotides have not been shown to be transported across membranes, probably due to the highly charged phospate moieties on the ribose. The transport processes described above not only supply the purine requirements of the erythrocytes but also allow the red cells to func- tion as distributors of purines synthesized by the liver to other tissues not capable ofgengvg biosynthesis (l8, l9). Prichardg_t_a_1_l_. have recent— ly shown that rat or rabbit liver clears a perfusate of the hydroxylated purines, hypoxanthine, xanthine and uric acid, but releases adenosine into the hepatic circulation (20). They postulate therefore that adeno- sineis the transported form of the purines which supply the requirements of tissues lacking gem biosynthesis. Free purines or nucleosides normally are not foundin the cell but exist in the form of nucleotides. Anion exchange chromatography of acid extracts of fresh erythrocytes indicates that adenine is present as AMP (13— 60 1M), ADP (100-270 (M) or ADP (0.85-l.7 pM) while guanine exists as GDP (20 11M) or GTP (10—35 11M) and hypoxanthine as IMP (0-50 (1M) and ITP (0-183 11M) (21 , 22, 23, 24, 25, 26). An interesting experiment by Magergi_:_a_l_. il- lustrates the metabolic turnover of these nucleotide pools 15mg (27). First red cells were incubatedjnyiiggto prelabel the nucleotide pools. Cells incubated with [MC]adenine incorporated label into AMP (4%),ADP (14%) and ATP (82%). Cells incubatedwith [14C]guanine incorporated label into IMP (10%), GDP (12%) and GTP (78%) while cells incubatedwith (”cihyp- Oxanthine incorporated [MC] label into GMP (27%) and IMP (73%). Prelabelled cells were then injected into individuals, and the half- lives of the purine nucleotides were determined. Their results indi- cated that adenine had a half-life of 8-9 days, guanine, 5-7 hours and hypoxanthine, 1 hour. Bartlett has also illustrated the turnover of the nucleotide pools by jn_vitrg labelling and subsequent analysis of the specific activities of these matebolites (23). In_vitrg_experiments with rabbit erythrocytes indicates that ade- nine is primarily used for adenine nucleotide synthesis while xanthine, guanine and guanosine are precursors of guanine nucleotides, and unlike human erythrocytes, hypoxanthine, inosine and adenosine can be converted to both guanine and adenine nucleotides (28). The only purines secreted from prelabelled cells are hypoxanthine, xanthine and xantho- sine regardless of which [14C] precursor is used (29). Combining the evidence derived from both rabbit and human erythrocytes and taking into account the enzyme deficiencies mentioned above, Figure 2 des- cribes the interconversions of purines in the human red blood cell. As may be seen in the figure, purines synthesized gg_ngvg_enter the scheme at the level of IMP (30). Thus biosynthesis and catabolism of purines is accomplished by controlled and defined pathways with IMP playing a key intermediary role. B. Enzymes Involved in Red Cell Purine Metabolism Several enzymes catalyzing purine conversions have been purified and characterized from human erythrocytes. A careful study of their kinetic parameters and susceptibility to regulation by other metabo- lites has added a great deal to the understanding of purine intercon- versions. This section will briefly list and describe some of these ATP = ADP :‘r:__——;. AMP*——— AR Adenylosuccinate “----'IDP i ITP___._,. V IMPZI-A .. /i \ gg_novo biosynthesis XMP::::::;;;-X GTP=_7—GDP‘_—__—_'—GMP/ XR/ UA 11 FIGURE 2. Interconversions of Purine Nucleotides in the Red Cell The following abbreviations are used in this figure: A, AR, AMP, ADP and ATP refer to adenine, adenosine, adenosine mono-, di-, and triphosphate, respectively; H, HR, IMP, IDP and ITP refer to hypoxan- thine, inosine, inosine mono-, di-, and triphosphate, respectively; UA, X, XR and XMP refer to uric acid, xanthine, xanthosine and xanthosine monophosphate, respectively; and G, GR, GMP, GDP and GTP refer to guanine, guanosine, guanosine mono-, di-, and triphosphate, respec- tively. enzymes, note any significant properties, and describe their role in metabolism. 1. Purine Nucleoside Phosphorylase (PNP) Purine Nucleoside Phosphorylase catalyzes the phosphorolysis of nucleosides: nucleoside + phosphate 1 base + ribose-l-phosphate. It has been purified from human erythrocytes and characterized in terms of 10 its physical and kinetic parameters (31). It shows specificity for hypoxanthine, xanthine and guanine and their nucleosides but will not react with adenosine. Zimmerman e_’_c__a_l_. have shown that the Km for ade- nine is much higher than that for hypoxanthine (32). Equilibrium favors the formation of the nucleoside but the enzyme is thought to function only in the catabolic sense jn_vivg. Formycin B, an analog of inosine with a C-C glycosidic bond, is a good competitive inhibitor of purified PNP (Ki = 100 uM) or PNP found in intact cells or hemolysates (33). 2. Phosphoribosyltransferase (PRT) Human erythrocytes have two enzymes which catalyze the general reaction: base + PRPP 2 nucleoside 5'-monophosphate + pyrophosphate (10, ll). Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is specific for hypoxanthine and guanine with little activity for xanthine while adenine phosphoribosyltransferase (APRT) is specific only for adenine. HGPRT has only recently been purified to homogeneity (34). Studies examining the properties and kinetics of red cell HGPRT have been hampered by the less than pure enzyme preparations used and by the fact that PRPP, one of its substrates, is rather unstable (35, 36). However, the evidence available indicates that HGPRT does not follow classical Michaelis-Menten kinetics but behaves as an allosteric enzyme (37). Red cell HGPRT is competitively inhibited by GMP and to a lesser extent by IMP for the binding of the substrate PRPP (38, 39). Develop- mental changes in both APRT and HGPRT activities have been noted (40). The significance 105°C) was dissolved in dry DMF and concentrated in a 50 ml round bottom flask containing a stir bar. The CDI was redissolved in 5 m1 dry DMF, and the IPCP-containing solu- tion was added in three aliquots over a 15 minute period with constant stirring.r The solution was stirred for 30 minutes more after which 2.5 nunfles (45 uliters) water were added. The solution was stirred for a final 30 minutes in the presence of this water to hydrolyze the remain- ing C01 and prevent the formation of branched structures in subsequent condensations with phosphate. A small aliquot of the reaction mixture was analyzed for the formation of the IPCP-imidazolide by ascending paper chromatography in solvent 1. The solution was concentrated twice with dry DMF as before and resuspended in 26 m1 DMF. Phosphoric acid (2.5 mmoles) was added to an equivalent amount of nethyl-tri-groctylammonium hydroxide. This phosphate syrup was concen- trated and resuspended in dry DMF three times. The phosphate solution was finally resuspended in 10 ml of dry DMF, and the IPCP-imidazolide was added to the phosphate with a pasteur pipet. The reaction mix was dried twice more with DMF and finally suspended in ca. 20 m1 dry DMF, and the mix was stored over phosphorus pentoxide for four days. To convert the IPCPOP to the ammonium salt, the reaction mix was concentrated to a syrup and dissolved in 50 ml ether followed by the addition of 50 mmoles (3.15 g) ammonium formate in 50 ml water. The aQueous phase containing the IPCPOP ammonium salt was collected with the aid of a separatory funnel and evaporated with the flash evaporator at 30°(2. The solid was dissolved in water and reconcentrated to remove the fininoni um formate . 49 A 2.8 x 25 cm DEAE-Sephadex column was prepared as before (see section III, A, 3) and washed with 0.4 M TEAB, pH 7.5. IPCPOP was resuspended in water and adjusted to the same pH and ionic strength as the TEAB buffer and loaded on the column in about 300 ml solution. The column was then washed with 400 ml 0.4 M buffer followed by a 3 liter linear gradient from 0.4 M to 0.8 M TEAB, pH 7.5. The effluent was monitored for UV absorption and conductivity. Fractions containing the major peak, IPCPOP, were combined, concentrated by the flash evaporator at 30°C, and the IPCPOP was converted to its sodium salt in a method similar to that already described for ITP (see section III, A, 3). The IPCPOP sodium salt was characterized by its UV, IR, and 13C-NMR absorp- tion spectra, and the purity of IPCPOP was determined by HPLC and the UV-absorption of a weighed sample. c. Synthesis of Sepharose-4B covalently attached to IPCPOP Preparation of an affinity resin coupling IPCPOP through the 7- phosphate to a six carbon spacer molecule covalently attached to Sepharose-4B was accompliShed by the method of Barker et_al, (168, 169). The N-trif1ouroacetyl-grphosphoryl 6-amino-l-hexanol imidazolide was prepared and reacted with IPCP to form y-(N-triflouroacety1-6- aminohexy1)-IPCPOP which was then purified by column chromatography. After the triflouroacetyl blocking group had been removed, the result- ing y-(6-aminohexyl)-IPCPOP was reacted with cyanogen bromide-activated Sepharose-4B (170). N-triflouroacetyl-6-amino-l-hexanol phosphate (TFA-HAP) was puri- fied by dissolving it in water and passing it through a 2.2 x 10 cm Dowex 50 x 8 (20-50 mesh, H+) column. The acidic effluent was collected and concentrated by flash evaporation at 35°C. The compound was 50 estimated to be pure by ascending paper chromatographyirisolvent II. TFA-HAP was dried twice with dry DMF using the flash evaporator at 40°C attached to a high vacuum pump. 234 mg (m0.75 mmoles) of the syrup were dissolved in DMF, dried with DMF as above and resuspended in 2 m1 dry DMF. C01 (3 mmoles, 487 mg) was dissolved and stirred in 5 ml dry DMF to which the TFA-HAP prepared above was added in two aliquots 10 minutes apart. After 30 minutes 3 mmoles (54 uliters) water were added, and the solution stirred for 20 minutes more. The formation of the imidazolide was demonstrated by ascending paper chromatography in solvent III. The compound was concentrated, dried twice with DMF as before and redis- solved in ca. 3 ml distilled hexamethylphosphoramide (HMPA). IPCP (0.5 mmoles) was converted to its methyl-tri-gfoctylammonium form and dried with DMF as described (see section III, C, 2b). The IPCP was then dissolved in ca. 3 m1 HMPA and combined with the TFA-HAP imidazolide. The reaction mixture was placed ig_yaggg_ over phosphorus pentoxide for 3 days. A 1.3 x 45 cm Dowex 1x2 (200-400 mesh, Cl') was prepared in 50% ethanol. The HMPA containing the product was diluted 4-fold with 50% ethanol and loaded on the column. The column was washed with 250 ml 50% ethanol followed by a 1 liter linear gradient from 0.01 N HCl to 0.01 N HCl containing 1.0 M LiCl. The effluent was monitored with an ISCO UA5 UV monitor at 254 nm. and collected in 15 m1 fractions. The tubes containing the major peak were combined and the solution was neu- tralized with LiOH and concentrated to ca. 30 ml. To remove the TFA blocking group, the solution was adjusted to ca. pH 14 with LiOH and stirred for 2-3 days. HPLC was used to monitor the 51 course of the reaction, and the final product, y-(6-aminohexyl)-IPCPOP was ninhydrin positive. A 3.4 x 40 cm Bio-Gel P2 column (200-400 mesh) was prepared and washed with 50 mM TEAB, pH 7.5 at a flow rate of 60 m1/hour. Thirty ml of the y-(6-aminohexyl)-IPCPOP solution was carefully layered on the column and eluted with the 50 mM TEAB buffer. The effluent was moni- tored for UV-absorbing material and conductivity. The desalted frac- tions containing the UV peak were concentrated and dissolved in water to a final concentration of 10 mM y-(6-aminohexyl)-IPCPOP and cooled to 4°C. 'Fines' were removed from Sepharose-4B, and 10 ml of the resin were combined with 20 ml 2.5 M potassium phosphate, pH 12.0. The Sepharose-4B was cooled to 3°C and stirred while cyanogen bromide (1 g/ml acetonitrile) was rapidly dispersed into the solution with a syringe. The reaction was allowed to proceed exactly 8 minutes, after which the mix was filtered rapidly through a scintered glass funnel. The resin was washed immediately with 200 ml cold water (within 1.5 minutes total), and the cake was added to the cold y-(6-aminohexyl)- IPCPOP and adjusted to pH 9.5 with concentrated NaOH. The Sepharose-4B was stirred overnight, after which it was stored in the reaction mix without stirring for two days. The UV absorbance of the supernatant was determined to calculate by difference the amount of y-(6-amino- hexyl)-IPCPOP coupled to the resin. 3. General Procedures a. Preparation of hemolysates For routine analysis of NTPH whole blood was centrifuged at 4000 9 fiar 5 minutes at 4°C to sediment the cells. The plasma and buffy 52 coat were removed by aspiration, and the red cells washed once or twice with 3-5 volumes 0.9% saline and collected by centrifugation. The washings were discarded and the packed cells lysed with 9 volumes cold 1 or 2 mM DTT. The debris was removed by centrifugation at 30,000 g for lO-15 minutes and the top half of the supernatant was used for NTPH and hemoglobin analyses. In some special cases the lysate was prepared more concentrated or dilute than that described here. For analysis of the cell-free synthesis of ITP, the procedure was modified in that the packed cells were lysed with an equal volume of cold water for 10 minutes. In some experiments, lysates prepared in this manner were given one to three treatments of Norit A (10 mg/ml) for 5 minutes to remove endogenous nucleotides. The charcoal was removed by centrifugation at 30,000 g for 10 minutes at 4°C. b. Blood cell separations and sonication procedures Cell separations methods have been reviewed by various authors (171-174). A combination of a few of these methods was used to separ- ate blood components for the comparison of NTPH activity in platelets, granulocytes, lymphocytes and erythrocytes. Only carefully cleaned silanized glassware (see III, B, 2) or plastic materials were used in these experiments. Twenty m1 of blood was drawn fresh daily by venipuncture using EDTA-containing vacutainers. TWo m1 of 3.8% sodium citrate, pH 7.4, was mixed with 10 ml of whole blood, and platelet rich plasma (PRP) was obtained by centrifugation of the blood at 740 gav in a swinging bucket rotor (IEC Model HN centri- f119(9) for 5 minutes at room temperature. The PRP was removed with a PiPet and the remaining cells were washed twice with a buffered salt soltrtion (355) containing .01% glucose, 5 uM CaClz, 98 uM MgCl 0.54 2’ 53 mM KCl, 1 mM sodium EDTA, 5 mM B-mercaptoethanol, 14.5 mM Tris and 126 mM NaCl at pH 7.4. The cells were centrifuged as before, and the two washings were combined with the PRP to obtain the platelet fraction of the blood. After the number of platelets was determined in this solu- tion (see III, C, lb), the platelets were cooled to 4°C for 2 hours and then centrifuged at 35,000 g for 15 minutes. The supernatant was care- fully removed by aspiration; and the pellet was stored at 4°C until it could be sonicated. White cells were separated from erythrocytes by dextran sedimenta- tion (172). Following the removal of the platelets, the blood cells were adjusted to 18 ml with BSS and mixed with 3.6 ml 6% dextran pre- pared in BSS. With the tube placed in a vertical position, the red cells were allowed to settle for 45 minutes at room temperature leaving a leukocyte-rich supernatant. The white cells were collected by cen- trifuging this supernatant at 740 gav for 5 minutes at room temperature and resuspended gently with the aid of a pasteur pipet in 5 ml fresh BSS. An aliquot of erythrocytes from the bottom of the tube was washed three times with BSS. The cells were collected each time by centrifugation at 740 gav for 5 minutes at room temperature. An appropriate dilution of the cells were counted and the remainder were centrifuged as above, the supernatant was removed and the red cells were placed at 4°C. The lymphocytes were separated from the granulocytes by the ISOpaque-Ficoll method of Boyum (174). Two ml of Ficoll-paque were placed in each of two silanized glass ignition tubes. Each of these was carefully layered with 2.5 ml of the suspended white cells and centrifuged at 400 gav for 30 minutes at room temperature in a swinging 54 bucket rotor. The interphase between the BSS and Ficoll-paque was collected with a pasteur pipet and washed three times with at least 3 volumes BSS. The cells were collected by centrifugation at 740 gav each time for 5 minutes at room temperature. These cells found at the inter- phase were characterized by a staining technique (see III, C, 1b) and suspended in 1 ml BSS for counting. The cells, predominately lympho- cytes and monocytes, were again centrifuged as above, the supernatant was carefully removed and the pellet placed at 4°C prior to sonication. The red and white cells collected at the bottom of the Ficoll— paque were washed three times with 0.87% NH4C1 to lyse the remaining erythrocytes. Each time the cells were collected by centrifugation at 740 gav for 5 minutes at room temperature. The last pellet was charac- terized by a staining procedure (see III, C, lb) and suspended in 2.0 ml BSS for counting. The cells, predominately granulocytes, were centrifuged as before, the supernatant was removed and the pellet was placed at 4°C. The various fractions were sonicated to insure that all the cells were lysed completely. The erythrocyte pellet was suspended in ca. 9 volumes cold 2 mM DTT. The granulocyte and lymphocyte pellets were each suspended in 1 m1 cold 2 mM DTT and the platelet pellet was sus- pended in 2 or 4 ml cold 2 mM DTT. These four suspensions were soni~ cated at 4°C with two 15 second bursts delivered with a microprobe powered by a Biosonik sonicator at 50% capacity (Model BIO III, Bron- will Scientific). The debris was removed by centrifugation at 30,000 g for 20 minutes at 4°C. The volumes of the granulocyte, lymphocyte and Platelet debris were considered negligible but the volume of the ery- throcyte lysate was measured since considerable debris was present. 55 All NTPH analyses were performed on the same day that the blood was drawn. Both 0.5 mM ITP and 0.5 mM ATP were used in all NTPH analy- ses to determine the enzyme activity Specific for the hydrolysis of ITP. NTPH and protein analyses have been described previously (see III, C, la). c. Henderson's methods for the accumulation of [14CJITP in intact erythrocytes Henderson's procedures for the analysis of [14C]ITP in intact erythrocytes have been described in detail (5, 72). Briefly his proce- dure involves the following methods. Erythrocytes, 2% suspensions in modified Fisher's medium, were incubated at 37°C for 2 hours in the presence of 100 uM [14C]hypoxanthine. Neutralized perchloric acid extracts were prepared and separated by one-dimensional, polyethyleni- mine-cellulose, thin-layer chromatography. Areas containing IMP, IDP 14C]ITP accumula- and ITP were cut out and the radioactivity analyzed. [ tion was reported to be linear for two hours with negligible radioac- tivity accumulating in IDP. d. Analysis of nucleotides in blood or lysates Various procedures for the extraction of free nucleotides from cells have been reviewed by Mandel (175). P. R. Brown has compared some of these techniques for use with HPLC (176-178). Perchloric acid extraction followed by neutralization with potassium hyroxide was used successfully to determine the ITP concentration in fresh blood. Tri- chloroacetic acid extraction followed by neutralization with Tris was most convenient for the determination of the cell-free synthesis of ITP in hemolysates. Neutralized extracts containing nucleotides are Stable for months at -20°C (176, 179). 56 Twenty ml of blood were drawn by venipuncture into a heparinized vacutainer. The hematocrit was determined by filling a small hematocrit capillary, plugging its end with a seal, centrifuging it in an IEC MB centrifuge for 5 minutes and measuring the percentage of packed cells to total volume. Fifteen ml of whole blood measured in a marked silanized tube were mixed with 50 pliters of a [3H]ITP internal standard (see III, A, 4c) and combined with 30 ml 0.6 N perchloric acid in a 50 m1 centri- fuge tube. The precipitated protein was stirred vigorously at 4°C for 5 minutes and removed by centrifugation at 35,000 g for 10 minutes at 4°C. The supernatant was filtered through Whatman 1 paper and neutra- lized with ca. 9 ml 10% KOH. The potassium perchlorate salt was removed by centrifugation at 20,000 g for 10 minutes at 4°C. The pH was adjusted to ca. 7.5 and the extract was lyophilized to dryness. The solid was taken up in 10 m1 cold water, centrifuged and lyophilized a second time. If necessary this process was repeated to obtain a pre- cipitation free solution with a total volume of ca. 1 ml. The exact volume was noted and 10 uliter aliquots were analyzed by HPLC, and the effluent of the HPLC in the ITP region was monitored for radioactivity (see III, C, 1e). The neutralized extract was stored at -20°C with very little decomposition in a month. Reaction mixtures for synthesis of ITP in cell-free hemolysates were extracted with two volumes ice cold 12% tricholoacetic acid (TCA); usually 0.1 ml of the reaction mix was added to 0.2 ml 12% TCA. The precipitate was removed by centrifugation at 20,000 g for 5 minutes at 4°C. A 100 uliter aliquot of the supernatant was neutralized with 7 mg Tris and stored at -20°C until it was analyzed by HPLC (see III, C, 1e). 57 e. Determination of Km__a__n_d_l_(,i for NTPH Methods for the determination of enzyme kinetic constants have been reviewed by Segel (180). For Km or Ki determinations, either ly- sates or partially purified enzymes were used at a concentration which gave a maximum NTPH activity of 10 to 20 units. The method of Lee and Wilson was used to compensate for the significant utilization of substrate at low substrate concentrations (181). Thus the average sub- strate concentrations were determined by averaging each initial and final concentration (S), and the values so obtained were substituted for the initial substrate concentrations in the determination of the Km by Lineweaver-Burk plots. The Km for a set of S and "initial" velocity pairs was determined by a computer program using the method of Wilkin- son (167, see also III, C, lg) and plotted in the form, l/S vs.-$—. i Similarly Lineweaver-Burk plots were obtained at four different inhibi- tor concentrations. The apparent Km values from each of these analyses were plotted against the inhibitor concentration of each, and the Ki was determined as the negative of the intercept on the inhibitor axis. This method of analysis was valid since the inhibition was shown to be competitive. f. Affinity chromatography of NTPH The techniques for the practical application of affinity chromato- graphy have been reviewed by Lowe and Dean (182). The following pro- cedure provided the best combination of conditions tested forthe purification of NTPH by means of the IPCPOP-Sepharose affinity resin (see III, C, 2c). All procedures were conducted at 4°C. The NTPH- containing solution (15 m1 of a 1:4 lysate) was dialyzed against 1500 MT containing 50 mM Tris-Cl, pH 7.4, 1 mM MgCl2 and 1 mM GSH. A 0.9 x 58 6.5 cm affinity column was prepared with this buffer, and the sample was loaded at ca. 60 ml/hour. Following elution of the major peak of pro- tein, an additional 40 ml buffer were passed through the column. The NTPH was eluted by allowing the resin to equilibrate with 1.5 ml buffer containing 10 mM MgCl 10 mM ITP, 1 mM GSH and 50 mM Tris-Cl, pH 7.4, 2. for 30 minutes followed by washing the column with ca. 40 m1 of the initial buffer. The protein solutions were concentrated and separated from ITP by pressure dialysis using an Amicon Model 52 apparatus with a 43 mm PM 10 ultrafiltration membrane. The concentrated solution was resuspended in initial buffer and reconcentrated two additional times to remove the ITP. The NTPH and protein were assayed by procedures described before (see III, C, la). 9. Cell-free synthesis of ITP Hemolysates were prepared as described (see III, C, 3a). Incuba- tions were carried out in 10 x 75 mm culture tubes and included 0.2 ml of the lysate, 50 mM sodium phosphate buffer, pH 7.0, and other sub- stances indicated. When Na4 PRPP was substituted for MgzPRPP, an addi- tional 5 mM MgCl2 was added to the mixes. The reactions were carried out for 3 hours with aliquots removed and extracted (see III, C, 3d) at indicated time points. HPLC analysis of the nucleotides in these extracts has previously been described (see III, C, 1e). CHAPTER IV RESULTS A. Genetic Variability of NTPH 1. Population Distribution The population study presented in Figure 3 was performed by S. A. Fuller, a graduate student in genetics. A random survey of NTPH speci- fic activity in the erythrocytes of a Caucasian population (262 indivi- duals) in the mid-Michigan area was conducted over a two month period. At least two different populations of individuals are readily apparent from this distribution. The specific activity of the low NTPH group, 18% of the population, ranges from undetectable levels to 27.5 nmoles ITP cleaved/mg hemoglobin. The high NTPH group, 82% of the population, includes specific activities that range from 27.5 to 125 nmoles ITP cleaved/mg hemoglobin. Other subgroups are suggested by these data, but Whether or not they are indeed distinct classes must await confirming EVidence by an independent method of analysis. These results are comparable with the overall characteristics of the papulation survey for erythrocyte "ITPase" conducted by Vanderheiden (3). His population data was also resolved into two classes with the 1dwer, 19% of the population, ranging from undetectable to 180 nmoles Pi released/hour/g hemoglobin and the upper, 81% of the population, hanging from 180 to 720 nmoles Pi released/hour/g hemoglobin. Since Varlderheiden also presented some evidence that the "ITPase" was 59 60 .mcowuwucou xcmmm ucmucmum smug: oanm pm mmpzcwe om cw aHH apes: P mmN>Fosu>5 cues: mez~cm mg“ eo “cacao was“ me umcwewu me sue>wuum map: to pee: < .mumcumnzm msu mm ahH gov: mcowuwccoo ammmo usmucmum sous: mpmowpawsu cw um~>Pmcm mm: Penn iw>wucw sumo do mummxp Fqu ems a mo xuw>euum Inez 8;» cowpmpaaoa :mwmmozmo a mo mppou vmm mzu c_ 3.5.52 3:83 1&2 .8 8:3 585 < .m 83.; 61 coepmpzaom cowmmuaou m eo mp_mu umm may cw apw>wpu< uwwwomam Inez mo xm>s=m Eoccmm < .m weaned Aeeaopmoeax me\moweav seeseoo< oec_ooam Iaez om, o__ cop oa ow om om cm as om om op o I. _ , l V L r r.. [I , I m I L l N— .m F1 ”w T. 1 mp .m I .IJ 1 ON W3 n e U ill, 0. .. F - a... m I. .rIL 1 mm rlur|l_ I 1 mm 62 inherited as a codominant trait in human erythrocytes, we further pur- sued the analysis of NTPH in related individuals. 2. Inheritance of NTPH in Two Families Two interesting family studies were conducted early in the inves- tigation of the inherited variability of NTPH in red cells (Figure 4). The first is a family (0d) with two parents who have relatively high specific activities. The children in this family also have high NTPH levels. The second family study (Gr) demonstrates parents who have very low to undetectable levels of NTPH. Four of the progeny also have very low levels of NTPH while the other has a comparatively high NTPH level. However, this latter person was receiving blood transfusions at the time of the analyses; thus the relatively high NTPH level determined for red cells drawn from him may not be characteristic of red cells produced by his own marrow. To verify the NTPH level of the members of the Gr family, the analyses were repeated by S. A. Fuller; these results are also shown in Figure 4. Blood from more families is currently being collected to define more clearly the inheritance of the red cell NTPH specific activity. 3. Mixing Study with Hemolysates of Red Cells of Selected Individuals To determine whether intracellular inhibitors or activators of NTPH are present in the red cell, equal volumes of different hemolysates were mixed to observe if the NTPH activities were additive. As can be seen in Table 1, three of the four experiments gave additive results, while the other experiment indicated a significant difference between the observed and expected result with the observed 26% less than the expected. Since experiment 3 was essentially repeated in experiment 4 63 0d. Family e @ .73. [8F @— $4 Gr. Family (we {a (0) (2) 85* 'i‘ o (L, (ll) (2) (2) (1) (2) Figure 4. NTPH Specific Activity in the Red Cells of Members of Two Families. The numerical values represent the NTPH activity expressed as nmoles ITP cleaved/mg hemoglobin under standard conditions. The parentheti- cal values are the results of an independent NTPH analysis. *This individual was receiving periodic blood transfusions at the time his blood was drawn for this NTPH analysis. 64 Table 1. Study of NTPH Activity in Mixed Hemolysates* NTPH Activity Experiment Lysate Source Expected Observed Student t Test 1 VLV + MG 298 335 N.S. 2 SF + MG 484 354 N.S. 3 AJM + MG 777 575 .05 > p' > .01 4 J00 + MG 942 873 N.S. * Hemolysates (1:10) of blood of five individuals were prepared, ana- lyzed for NTPH activity separately and analyzed in equal volume mixtures as indicated in the table. Each analysis was done in triplicate and all experiments were performed in one day. The expected and observed activities are expressed in units/m1 lysate. NTPH specific activities (nmoles ITP cleaved/mg hemoglobin) of the subjects studied were as follows: VLV, 13; SF, 31; AJM, 69; J00, 64 and MG, 6. with negative results, the search for intracellular effectors of NTPH by this method was not continued. Neither were effectors detected by studies in which partially purified NTPH was mixed with lysates of blood cells of selected indivi- duals (5). 4. The Michaelis Constant for ITP of NTPH in Hemolysates of Selected Individuals A study was undertaken to determine if the Km of NTPH was the same in NTPH-containing lysates of red cells of selected individuals with greatly different NTPH specific activities. The data presented in Table 2 indicates that no correlation can be observed between the NTPH specific activities and the respective Km values among these indivi- duals. The variability of the Km that is reflected in the high stan- dard deviations is probably due to the technical difficulty of getting good kinetic data at low enzyme activity in hemolysates. 65 Table 2. The Michaelis Constant (Km) of NTPH of Human Red Cell Lysates from Selected Individuals * NTPH Specific Activity Km i S.D. (Units/mg Hemoglobin) (x105 M) 12 3.1 i 0.63 17 1.5 i 0.80 18 2.4 t 0.26 28 2.5 i 0.33 36 4.4 t 0.49 46 1.2 i 0.42 49 2.5 i 0.26 70 3.7 i 0.32 84 2.5 i 0.52 106 5.1 i 0.56 124 2.0 i 0.34 * The NTPH specific activity for the red cells of each individual was determined in triplicate under the standard assay conditions. The ini- tial velocity of NTPH in lysates was determined for 12 to 14 ITP con- centrations over the range, 10-500 uM. Km values were determined by using average substrate concentrations and a computer program of the method of Wilkinson (see Methods). 5. A Comparative Study of NTPH Activity Found in Erythrocytes, Granu- locytes, Lymphocytes and Platelets Since NTPH activity had previously been found in all tissues examined of the rabbit and rat (2,144), it was of special interest to determine whether the genetic variability expressed in human red cells was reflected in other human tissues. Thus procedures were developed to separate blood cells into defined populations and to analyze each type for NTPH activity. 66 The complete characterization of cell populations and NTPH analy- ses of erythrocytes, granulocytes, lymphocytes and platelets from selected individuals are listed in Tables 3, 4, 5 and 6, respectively. The mean cell hemoglobin concentration in the 19 erythrocyte analyses was 2.90 s 0.071 (3.15.) mg hemoglobin/108 cells, and the mean red cell protein concentration as determined by the Lowry method in 17 of these samples was 3.87 i 0.082 (S.E.). These results indicate good internal consistency in the methodology over the two month period that these analyses were performed. One can see that individuals were selected to represent an NTPH range of 3 to 97 nmoles ITP cleaved/mg hemoglobin. The units of activity/107 cells were adjusted for nonspecific ITP hydrolysis (0-27%) measured by substituting ATP for ITP in the reaction mix - The granulocyte populations were composed of 92-99% granulocytes and 1-8% lymphocytes and contained an average of 5.10 i 0.35 (S.E., N=17) mg protein/108 cells. The units/107 cells were adjusted for activity due to platelet contamination (O-ll%) and for nonspecific hydrolysis of ITP (0-39%). The lymphocyte populations were composed of 84-95% lymphocytes, 2-14% monocytes and l-12% granulocytes. The cells had an average 6.5 i 0.37 (S.E., N=l6) mg protein/108 cells. The units/107 cells were ad.l'usted for activity due to platelet contamination (0-5%) and for non- specific hydrolysis of ITP (0-29%). Platelet preparations were contaminated with activity, 2-10%, and Pmtein, l-8%, due to large cells. The average amount of protein per 9911 was calculated as 0.174 t 0.0078 (S.E., N=l7) mg protein/108 C811- 67 Table 3. Characterization and NTPH Analyses of PopulationscfliErythro- cytes --- NTngSpecific Activity Protein/Cell . SW“ 3.3%? if)? 321i: 1‘7—‘1‘N6t 321‘? 33532135 T'é‘g—n-o c2” 5 THFTTT—mo "2“? VLV 14 0.34 3.7 9.8 2.94 3.77 AJM 76 0.71 20.6 53 2.80 3.90 DL 92 0 19.8 71 2.16 2.77 CV 33 0.18 11.1 25 3.39 4.46 SF 32 0.02 8.2 24 2.60 3.40 VLV l6 0 4.2 12 2.69 3.48 AJM 64 0.43 16.3 45 2.60 3.60 SF 28 0.08 7.3 20 2.68 3.70 DL 97 0.86 21.0 70 2.25 3.01 CV 35 0 11.2 25 3.19 4.46 SF 36 0.10 8.6 - 2.38 - VLV 15 0.08 5.2 - 3.61 - BH 18 0.37 3.5 11 2.10 3.06 CV 53 0 15.3 36 2.88 4.24 SF 43 0.16 13.0 30 3.07 4.32 DY 3 0.26 0.71 1.8 3.03 3.92 SF 44 0.25 12.3 32 2.86 3.84 CV 37 0.02 10.6 30 2.89 3.55 AJM 76 O 21.2 59 2.80 3.59 Activity was determined in triplicate for each sample under standard NTPH assay conditions and was calculated as (a) nmoles ITP hydrolyzed, (b) nmoles ATP hydrolyzed and (c) nmoles ITP hydrolyzed - nmoles ATP hydrolyzed. Protein was determined in triplicate for each sample by the cyanmethemoglobin or Lowry methods (see Methods). Aliquots of cells to be analyzed were diluted and counted with the aid of a count- ing chamber. The individuals are listed in the chronological order that their cells were analyzed. 68 Table 4. Characterization and NTPH Analyses of Populations of Granulo- cytes NTPH Specific Activity Characterization Subject ATP Unitsa Net UnitsP Net UnitsP mg Protein Gran. Lym. Plat.C 107 Cells 107'Cells mg Protein 108 Cells % % % VLV 0 56 162 3.45 92 8 11 AJM 14 97 257 3.79 95 5 2 DL 5 163 580 2.80 97 3 2 CV 0 51 88 5.76 97 3 4 SF 0 101 160 6.32 95 5 1 VLV O 60 138 4.33 - - 2 AJM 13 112 364 3.10 98 2 2 SF 0 150 211 7.10 98 2 0 DL O 180 236 7.61 99 1 1 CV 2 132 210 6.28 99 1 0 SF 0 101 - - 98 2 - VLV O 46 - - 97 3 - BH 11 65 126 5.15 97 3 2 CV O 117 181 6.47 99 1 0 SF 15 128 192 6.70 95 5 1 DY 11 17 41 4.19 99 1 0 SF 27 139 280 4.96 97 3 0 DV 12 87 176 4.96 99 1 1 AJM 6 201 543 3.69 99 1 O Activity was determined in triplicate for each sample under standard NTPH assay conditions and was calculated as (a) nmoles ATP hydrolyzed and (b) nmoles ITP hydrolyzed - nmoles ATP hydrolyzed. Protein was determined in triplicate by the Lowry method (156). Aliquots of cells to be analyzed were diluted and counted with the aid of a counting chamber and a representative aliquot of each population was stained with Wright and Geimsa stains. The types of cells were classified by their morphology (see Methods for further details). The platelet con- tamination (c) is expressed as the percent of the total NTPH activity which was contributed by the platelets. The individuals are listed in the chronological order that their cells were analyzed and correspond to the individuals listed in Table 3. 69 .m mPamP cP cumPP umauP>PocP mcm om vcoammssou mam mmePmcm mam: mPPmm stzm Pmcp smuco PmumoPocossu mgp :P omamPP msm umanP>Pch ch .umPmmea mcp An mmuznPsucoo mm: gngz APP>Puum :aPz Pmmou mg» Po ucmucmn mcu mm ummmmsaxm mP Puv :oPPchEmpcou PumPmPa mzP .PmPPmumo smgmszP soc muozpmz mmmv amoPozasoe stgu Po umPPPmmmPu mam; mPPmu Po mmaxu ch .chum mmEPmu cam usmPsz cpPz nchmum mm: :oPmeaaoa comm Po PoscPPm m>PPmpcmmmsams m mam smnamsu chucaom m Po oPm mgp cuPz umpcaom mam cmuaPPm msmz nmNPPmcm ma om mPPmu Po mpoacPP< .PmmPv mogume Pegs; mg» »n mumoPPaPcu cP ucheLumn mm: chPoLa .umNPPosna; aP< mmPoE: inmNPPoscx; QPP mmPoE: Pav mam mmNPPosmzc QP< mmPoE: va mm mumPaquo mm; mcm mcoPqu icou Pmmmm :aPz msmncmum smug: mPaEmm zoom soP umuPPaPsP :P uchELumu mm: qu>PPo< mmpaoogaexd Po mcoPPszaoa Po mmmaPmc< :aPz mcm coPPmNPsmPumsmso .m manP 7O o m. .m P N m mm Na.c mNm PNN mm Pm N e P am mm.m mma PmN NN mm P N m mm 8P.e NaP Pm Pm Po m.o N N as me.P Nme oPm NN Pm m.o a N mm PP.m mas Poe aN >8 a NP oP NP 08.8 NNN PPN ON IN - m m cm - - PNP aP >d> - N m cm - - er P mm P NP P am oP.m PNN NNN Pm Pm P a 8 cm PP.e mam mom PN do - - - - - - - - mm m.o a m Pm oo.m PNP emm ma 28< N - - - ma.m oaN PON o PAP o m P om NN.N mNN aNN ON am P m PP em ma.P aNN P8P 0 >0 P P m om Po.m P8P _ mmm PP do m.o N a mm aP.a mNP PNN mm zo< m N aP em mm.m NPP ooP mN >o> P P ., . P w P mPPom oP erPoca me . - . : . . .a a . pr o.PePa .eesm .oeoz .2»; ePoPosa e .maPPe=.Poz. oaPPez Poz maPPea aP< Poonosm eoPPaNPcoPoasaem . PuPsPPo< oPPPooam IaPz.. ll 89,805.53 Po 203238 PO mmmemPE :aPz new 2053238ng .m 2&2. 71 .m mPooP :P omomPP mPoooP>PooP moo op ocoommssoo oco omePocm msmz mPPmo stco Pogo smoso PooPmoPocosgo moo :P omomPP mso mPoaoP>PocP ch .mPPmo mmmzo mo ompzoPsocoo chuoso Pomosmo so aoP>Pooo ocmosmo moo smcoPm mo ommmmsoxm mP Pov :oPPocPEopcoo PPmo mmsoP ch .smoEoco moPocooo o Po oPo moo con omocaoo oco omoaPPo msmz omePocm mo op mPPmo Po mooooPPo m>Pooocmmmsomm .PomPv oocome Pszoo one on mpooPPoPsP cP omoPesmomo mos :Pmooso .omNzPosoag oP< mmPoE: tomNAPosoP; oPP mmPoEc Pay oco omePosoP; oP< mmPoE: Pov mo omooPooPoo mo; oco mcoPoPo icoo sommo zon osooooum smog: mPoEom zoom soP mpmoPPoPso cP omoPEsmomo mo: zoP>Puo< umPmPoPo Po mcoPoonooo Po mmmeoc< :on coo coPpoNPsmpoosozu .o mPomP 72 N ,a NP.o . NNP . o.o NN.P zo< N N NP.o NNN N.P PP.o Po N N PP.P NNN P.N NN.P am a N PP.o. No P.P aP.o Po e N NN.P NNN N.N NN.P mm a P NP.o NON P.P am.o Po P N NN.P NPP N.P No.0 IN - - - - - - PNP - - - - - - PN N N PP.o NNN N.P PP.P Po 8 N PN.o aNN N.N NN.P PP P NP NP.o NNN N.N NN.o PN N N NP.o NNN o.N NN.o PNN a N NN.P NNP N.N NN.P _ PNP N NP NP.o NNN N.P NN.P Pm N N NP.o eNN P.a NN.P Po N N NP.o NNN N.P NN.P on P N NP.o NNP a.N NN.P .PNN P N NN.P NNP N.N PP.P P4P PPP ePaPosa. ._ PNP.PPPPPPo< aPPoo.NoP . V ePoPosa Ne .. . NPPao PNP W. .NPPoo PNP oPPoo dosed ePoPosa Ne oMPPeP Poz omPPeP,Poz (NNPPeP PPN PooPoPN eoPPeNPsoPoesaeo . 1 .NPPPPPoN oPPPooaw IaPz ‘ (inuuuununnuuuuuu aPoPoPePa Po aeoPPaPsaoa Po moaPPae<_Nst aea eoPPaNPcoPoesNeo .N oPeNP 73 The units/107 cells were adjusted for nonspecific hydrolysis of ITP (9-40%). The variation of NTPH activity in human red cells is correlated with the activity in human granulocytes, lymphocytes and platelets in Figures 5, 6 and 7, respectively. In each case there is a significant positive correlation (p<.01) of activities indicating that the variation is reflected in each of these cell types. It should also be noted that the activity per cell and the range of the variation in each type is quite different. In the individuals tested in this study the erythro- cytes ranged from 0.7 to 21 units/107 cells, the granulocytes from 17 to 201 units/107 cells, the lymphocytes from 91 to 462 units/107 cells and the platelets from 1.1 to 7.2 units/107 cells. These data provide evidence that the inherited variability of NTPH activity of the red cell demonstrated by the population survey and family studies in Figures 3 and 4 is also expressed in other blood cell types. 6. NTPH Activity in the Red Cells of 13 Patients with Muscular Dystro- phy Blood from 13 patients with various forms of muscular dystrophy became available through the laboratory of Dr. C. Suelter, Michigan State University. The NTPH activities of these people, listed in Table 7. exhibited little deviation from the activity found in the general Population (Figure 3). 3- Jielationship Between NTPH and the Accumulation of [14C]ITP in Agatact Erythrocytes It came to our attention that Dr. J. F. Henderson of the Univer- sity of Alberta was interested in the basis for the variation of 74 '01 All C) 200 ' '1 ‘ 160 120 80 7 Granulocyte NTPH (Units/10 Cells) 4O l l 1 l 5 10 15 20 Erythrocyte NTPH (Units/107 Cells) Figure 5. Correlation of NTPH Activities in Granulocytes and Erythro- cytes The NTPH analyses for each data point were conducted in triplicate on a single day, and the activity for each cell type was adjusted for nonspecific hydrolysis of ITP and activity due to platelet contamina- tion (see Methods). The seven individuals involved in the study are represented by the following symbols: DY (A), VLV (0), BH (A), SF (x), CV (I), AJM (o) and DL (0). 75 I 1 l l r = 0.84 p < .01 500 )- .1 I) 33 '8 9’ 400 l\ 0 Z .3 E; 300 3 I 0.. f— 2 33 200 >, u o .: cs E >, -J 100 - c, . l L l 1 5 10 15 20 Erythrocyte NTPH (Units/107 Cells) Figure 6. Correlation of NTPH Activities in Lymphocytes and Erythro- cytes The NTPH analyses for each data point were conducted in triplicate on a Single day, and the activity for each cell type was adjusted for nonspe- Cific h drolysis 0f ITP and activity due to platelet contamination (see Methods). The seven individuals involved in the study are represented By the following symbols: DY (A), VLV (0), BH (A), SF (x), CV (I), AJM (o) and DL (0). 76 8.0 ' Platelet NTPH (Units/107' Cells) l l l 5 10 15 20 Erythrocyte NTPH (Units/107 Cells) 1 Figure 7. Correlation of NTPH Activities in Platelets and Erythrocytes The NTPH analyses for each data point were conducted in triplicate on a single day, and the activity for each cell type was adjusted for non- specific hydrolysis of ITP (see Methods). The seven individuals involved in the study are represented by the following symbols: DY (A), VLV (0), BH (A), SF (x), CV (I), AJM (o) and DL (0). 77 Table 7. NTPH Activity in the Red Cells of Patients with Muscular Dystr0phy* Pat'°"' S°x (yggis) ”1°9"°S‘S (051t37fiéiilfiogi§liliy 1 F 19 limb girdle 47 2 M 10 Duchenne 55 3 M 13 Duchenne 65 4 M 15 Duchenne 67 5 M 17 Duchenne 64 6 M 18 Duchenne 65 7 M 15 Duchenne 72 8 M 17 Duchenne 71 9 M 18 Duchenne 64 10 M 18 Duchenne 76 11 F 37 Myotonia 60 12 M 21 Duchenne 53 13 M 19 Duchenne 37 *Blood was obtained from these patients by Dr. C. Suelter, Department of Biochemistry, Michigan State University. The NTPH activity in the washed red cells of each individual was determined in triplicate under standard assay conditions. [14C]ITP accumulation from [14C]hypoxanthine in intact erythrocytes from various individuals (72). We undertook a cooperative study with his laboratory in which the red cells of 93 individuals were analyzed for [14C]ITP accumulation and NTPH specific activity. Since some of the blood was stored at 4°C and -20°C prior to NTPH analysis, the stability 0f NTPH was tested under these conditions. Storage of blood at 4°C for 5 weeks had little effect on the ac- tivi ty of NTPH (Figure 8); however, storage under these conditions did increase the endogenous phosphate level in the red cells and hence the absorbance values obtained in the no substrate controls of the NTPH 78 ‘T T’ r T T 80 ‘ -—4l 50 P -—4.-—— P 40 F A NTPH (Units/mg Hemoglobin) Time (Weeks) Figure 8. NTPH Activity in Red Cells Stored in Heparin for 5 Weeks Three samples of whole blood were stored in heparinized vacutainers for 5 weeks. At each time point indicated, aliquots from each sample were analyzed in triplicate for NTPH activity under standard condi- tions. Blood from the following individuals was used in this study: AJM (O), WGC (X) and VLV (O). 79 assay. Other evidence indicated that NTPH was stable for months during the storage of frozen packed red cells. Figure 9 indicates that [14C]ITP accumulation and NTPH specific activity in blood cells of selected individuals may be described by an inverse hyperbolic relationship. A theoretical curve is fitted to the data which describes a substrate-enzyme relationship between % [14C]ITP accumulation and NTPH, predicted by assuming no variation in the synthe- sis of ITP among individuals, assuming steady state conditions and assuming Michaelis—Menten kinetics (see Appendix A). This relationship corroborates and expands the data of Vanderhei- den that suggest that [14C]ITP accumulation from [14C]inosine is limited by an "ITPase" (3). Whereas Vanderheiden suggested three classes of individuals that accumulated different amounts of ['4c11TP, Figure 9 indicates that a continuous spectrum of individuals are scattered along a hyperbolic relationship between % [14C]ITP accumula- tion and NTPH specific activity. C. Analysis of Endogenous Levels of ITP in Whole Blood Since the theoretical consideration of the data in Figure 9 sug- gests that ITP may be a normal constituent of red cells (see Appendix A) and since Vanderheiden had previously reported the presence of ITP in fresh extracts of human erythrocytes, experiments were designed to quantitate the ITP extracted from blood of selected individuals. Fresh blood without removal of serum or leukocytes was used in these experi- ments to minimize any change in the pool size of endogenous nucleotides. 80 E'rTllITl 35 r i - a: . C .2 q 4.) f0 '3 E 3 u 1 U <1: 0. t: C? q. o " ‘6 at T . P o :‘i; c». ' o .4 . .012 3. 20 4O 60 80 NTPH (Units/mg hemoglobin) Figure 9. The Relationship Between [14C]ITP Accumulation in Intact Red Cells and NTPH Specific Activity [14C]ITP accumulation in red cells of selected individuals was analyzed by Dr. J. F. Henderson's laboratory at the University of Alberta and expressed as a percent defined as ([14C]ITP/[14C]ITP + [14C]IMP) x 100. NTPH activity was determined in triplicate for each sample under stan- dard assay conditions. 81 1. Preparation of the Internal Standard, [3H]ITP To monitor the recovery of ITP by acid extraction of blood, [3H]ITP of high specific activity was prepared by deamination of [2-3H]ATP (27 Ci/mmole). DEAE-Sephadex purification of the [3H]ITP is shown in Figure 10. The major peak was pooled, concentrated and ana- lyzed by HPLC (Figure 11). The standard was 90% pure with major conta- minants, [3H]ATP (5%) and [3H]nucleoside mono- and diphosphates (5%). Reanalysis of this standard following storage in ethanol and ammonium hydroxide at -20°C indicated less than 1% loss of the radiochemical purity of [3H]ITP. HPLC analysis of this standard on the most sensitive UV scale failed to detect any UV-absorbing material in the ITP region of the chromatogram (Figure 12). This analysis also served to quantitate the [3H]ITP in an aliquot of the standard solution of [3H]ITP. 2. Resolution of Nucleotides by HPLC In order to demonstrate the resolution of nucleoside 5'-triphos- phates and to identify various peaks in the HPLC chromatograms of biolo- gical extracts, the retention times were determined for various standards (Table 8). XTP is the nucleotide least resolved from ITP. 3. Analysis of Endogenous ITP Levels in the Blood The blood of three individuals with different NTPH specific acti- vities was analyzed for the presence of endogenous ITP pools by HPLC. Figure 13 is a chromatogram of an extract of blood of an individual with very low NTPH activity, 3 nmoles ITP cleaved/mg hemoglobin. One can observe the presence of a small UV-absorbing peak at a retention time coinciding with the internal [3H]ITP. This HPLC analysis was repeated four times on the same extract of this individual with similar results. 82 40- 30F [I - 'I . Radioactivity (cpm) x 10.2 o d l o 10 1- . i " . O. ./\O i o d' 1’ o " /\{ \ o «d‘ 19' \ C . 3:00; .T. . 4 J 1 1".'1. 0 10 20 30 40 50 6O 7O 80 Fraction Number Figure 10. DEAE-Sephadex Purification of [3H]ITP [3H]ITP was purified following deamination of [3H]ATP by DEAE-Sephadex (fliromatography with a 200 m1 linear gradient from 0.4 M to 0.7 M TEAB, (WT 7.5, at a flow rate of 9 ml/hour. Fractions (2.5 ml each) were collected and aliquots were analyzed by liquid scintillation. Peak 'fractions were characterized by HPLC as described in the Methods as (A) a 3mixture of [3H]ADP and [ 3H]IDP and (B and C) a mixture of [3H]ATP and [ 3H]ITP. 83 '—_ll 1 T r l .025 _ I . 50 11 ~ '2 PE. .020 - ll 4 40 x C A s I 8 3 ‘ 8P 8 ° :2” 5 .015 ~ I - 30 .,>. D «p 8 | | 8 U1 0 '2 I 1 E A cc .010 P 1 - 20 ’l‘ v I . C | l .005 __ 1' g 10 I o, 0. 7.3. .0": .\. J , 0 5 10 15 20 25 Time (minutes) Figure 11. Radiochemical Purity of the Internal Standard, [3H]ITP The nucleotides were separated on a Perkin-Elmer 1250 HPLC equipped Twith a Partisil-lO SAX column in a buffer, 0.6 M KHZPO , pH 3.4, at a flow rate of 55 ml/hour. The [3H]ITP was injected si8ultaneously wiifl1 a standard ITP solution. Fractions (0.5 minute) were collected and analyzed by liquid scintillation. 84 .coPPNPPPP -cPoN oPooPP an omNNPNcN ooo coPmms APP mg» cP omPomPPoo msmz m:=wPEN m. ov NcoPooosP .soocxPe No Po moms 3oPP N Po .o.m com xx 2 o. o smPPPo o 2P cEaPoo x2 .NP msomPP 85 (———-o -——) Radioactivity (cpm) x 10'2 OPPPINP .OsaOeaPN POOsOPOP oOP OP NoPPPsOOeP OOPOsONOO-PO .NP osOOPs PNmPOOPEV mEPP ON OO ON ON OP O u i Poo. OP r a NOO. LO P- if MN 1 moo. P om f i ooo. (-—-——) Absorbance (254 nm) 86 .mmsOomooso NPNPPOOO oom :oPPoosoxm ch OP OPP PO POOPooos NNO e OooaoPOOP .OPPPINP .OsOOOOPN PeesmPeP oOP .OOPPOPPPPOPON OPOOPP PO OOPOOOOO was POPPPPOOOPONP oco coPmms oPP moo :P omPomPPoo msmz mmuwcPw m.ov NooPPoosP .sOOOPPe NO PO does zOPP a PO .O.N I . Os Pg 2 0.0 .sOPPOO 8 OP OeOPoo NON OP-PPNPPPOO a OPP: OPP: ONNP smePN-OPssoa a co omNaPoco No: PoPooPmoem; me\om~>PosoP: OPP NmPoEO m "PuP>PPom oPPPooaa NOPZP OOOPO PO Po Poesexo Nos a PO POOOPPO POPPPP OP O oooPm Pa Po PoOstm 0 Po Poospxm o> Po PoOstu o> Po ooosoxm looms Pom O omoooPocP .oPPP: o .osoocoom PocsmPcP mOP .coPo -OPPPooPoN oPooPP Po omoomomommoz PPPPPooOoPoNs oco :oPmms oPP moo cP omoomPPoo msoz PmooxPE m.ov NooPPoosP .sOo;\PE No PO does POPP O PO .O.N IO . OONIO z O.O .POPPOO O OP OeO_OO xPosoao oPP mmPoEc mm "PPPPPooo oPP -POOON IOPZV OOOPO PN PO POOPPNO oz ooo oooP oomP oooP oooP oooP ooom oomm ooom oomm d — 4 q d u i— - ‘ \I q I 8 P. . ,P i O.P s P P P/ _” i P r P P N” P. 1 m.o Po. P 7 P P i P, P “ .P.P " P T P P P _ _ \ NO P. P P P _ — — — - _ — o —— J P _ _ .P N.o a!» PP M P). _ D 2 Pk .— g as p~ — —<- a — _ I f ‘ s —.:- >7“ — 1 Foo (PP P P.P...r P \ all .}o\s T L. P P P P P omuiiirsiii-1\_ P P LPo Absorbance 104 Figure 22. The 15.08-MH2, Decoupled 13c-NMR Spectra of (A) ITP, (B) IPCPOP, and (C) the Expanded Methylene Region of IPCPOP These 13C-NMR spectra were obtained as described in the Methods. The spectrum for A was accumulated with 15, 368 scans having a l6 u second pulse with no recycle time and was analyzed with the line broadening set at 1.00 Hz. The spectrum for B was accumulated with l4, 512 scans having a 12 u second pulse with a 3 second recycle time and was ana- lyzed with the line broadening set at 1.00 Hz. Spectrum C was accumu- lated under the same conditions as B but analyzed with the line broadening set at 4 Hz. The arrow indicates an impurity in the IPCPOP sodium salt. Chemical shifts are expressed in parts per million downfield from tetramethyl- silane. 105 I l l l I 200 160 120 WTWMWWWWWWW The l5.08-MHz, Decoupled 13C-NMR Spectra of (A) ITP, (B) Figure 22. IPCPOP, and (C) the Expanded Methylene Region of IPCPOP. 106 the spectrum for IPCPOP (Figure 228), the C-5 peak is clearly visible. Thus the missing peak in the ITP spectrum may be explained by the experimental conditions during data collection. The IPCPOP spectrum also shows an impurity at l72 ppm which is very likely sodium bicarbon- ate since the bicarbonate carbon appears in the region of l62 ppm and was present during the purification of IPCPOP. The following features distinguish the IPCPOP spectrum from the ITP spectrum. The C-S' doublet is shifted upfield by 1.5 ppm and the coupling constant, ZJP-C’ is decreased from 6.6 in the ITP spectrum to 5.2 Hz in the IPCPOP spectrum. The methylene itself appears upfield at 28.8 ppm as a triplet in Figure 228 which is partially resolved into a quartet in Figure 22C (IJP-C = 124-l3l Hz). Thus the coupling constants for the two P-C bonds are almost identical. The chemical shifts and coupling constants for the carbons of ITP and IPCPOP are listed in Table ll. Peak assignments were made by comparison of these data with the spectrum for IMP published by Koto- wycz et al. (185). 3. Synthesis of IPCPOP Covalently Attached to Separose-4B An affinity resin was prepared by coupling the y-phosphate of IPCPOP by means of a 6-carbon spacer molecule to Sepharose-4B. This was accomplished by the method of Barker gt_al, (168, l69). N-triflouracetyl-6-aminohexane-l-phosphate (TFA-HAP) was purified by cation exchange chromatography and analyzed by paper chromatography (Figure 23). The imidazole of TFA-HAP was prepared by reacting TFA-HAP with l,l'-carbodiimidazole and the product was characterized by paper 107 Table 11. The 15.08 MHz, Decoupled 13C-NMR Chemical Shifts and Coupling Constants of ITP and IPCPOP in Aqueous Solution* ITP IPCPOP Carbon Chemical Coupling Chemical Coupling Shift Constant Shift Constant (ppm) (Hz) (ppm) (H2) Impurity 172.2 C-6 159.7 159.8 C-4 149.8 149.7 C-2 147.4 147.5 C-8 140.8 140.9 C-5 N.D. 124.6 C-1' 88.4 88.5 . 3 _ 3 - C-4 85.1 JP_c—8.8 85.0 JP_c-8.l C-2' 75.7 75.6 C-3' 71.3 71.2 c-5' 66.1 23 =6.6 64.6 2a =5.2 P'C 1 - P-c Methylene - 28.8 JP_C=124-131 13 * The peak assignments were made from the C-NMR IMP spectrum published by Kotowycz et al. (185). The chemical shifts are expressed in parts per million dBWfifield from the resonance of tetramethylsilane. The impurity in the IPCPOP is likely sodium bicarbonate (see Text). chromatography in solvent III as shown in Figure 24 below. However, the free imidazole and product were not separated by this system. The methyl-tri-groctylammonium form of IPCP was reacted with TFA- HAP-imidazolide under anhydrous conditions. HPLC indicated the appear- ance of a product with a retention time greater than that of IPCP. This product was purified by Dowex 1 column chromatography as shown in Figure 25 and characterized by HPLC, Figure 26A. The impurity in the product coincided with the RT of IPCP but may also be y-(6-aminohexy1)- IPCPOP as shown in Figure 268. Figure 26 further demonstrates that 83% (If the TFA-blocking group was hydrolyzed during a 3 day treatment with 108 A 0.63 0.63 .34 O .34 0 O .12 I l l J J l a ‘b c a V6 c Characterization of N-triflouracety1-6-aminohexane-l- Figure 23. phosphate by Paper Chromatography Paper chromatography of (a) P., (b) TFA-HAP and (c) HAP was carried out in solvent II. Chromatogrdm A was developed with an ammonium molyb- date spray for the detection of phosphate, and chromatogram B was developed by spraying with 1.0 N NaOH and drying to hydrolyze the tri- flouracetyl blocking group followed by spraying with ninhydrin for the detection of primary amines. The numerical values indicate the Rf at the center of each spot. A B 0.74 0.74 0.28 l I L 1 a b a b Figure 24. Characterization of the Imidazolide of N-triflouracety1-6- aminohexane-l-phosphate by Paper Chromatography Paper chromatography of (a) TFA-HAP and (b) TFA-HAP-Imidazolide was performed in solvent III. Chromatogram A was developed by spraying witfil'1.0 N NaOH and drying followed by spraying with ninhydrin. Chro- matogram B was developed by the use of an imidazole-specific spray. The numerical values indicate the Rf at the center of each spot. 109 WW I T 1 2.0 r- . ow») 11.00 E S 1.5 - -o.75 e, 1 P a. , 5 8 ‘ .— C‘U U {3 1 '5 o A § 1.0 - 1. fi0.50 1 . v 0 V l 10.25 '\. 00. U. g 0..'. .C .32.___“0 , 0 1 0 20 30 40 50 Fraction Number Figure 25. Purification of y-(N-triflouracety1-6-aminohexy1)-IPCPOP by Dowex Column Chromatography .A column (1.3 x 45 cm) containing Dowex 1 x 2 (ZOO—400 mesh, C1 form) was loaded with the sample and eluted with 250 m1 ethanol followed by a 1 liter linear gradient from 0.01 N HCl to 0.01 N HCl containing 1.0 M LiCl. Fractions, 15 ml each, were collected and monitored at 254 nm. 110 A 1 I l B 1 l I .035 - P .030 __ .J E? .025 - P C <1" m 21 8 .020- P C (U .Q S. O .8 < .015- J .010 F' P .005 P P l J 1 m l 0 5 10 15 0 5 10 15 Time (minutes) Figure 26. HPLC Analysis of (A) y-(N-triflouracetyl-6-aminohexyl)- IPCPOP and (B) y-(6-aminohexyl)-IPCPOP Analyses were performed on a Perkin-Elmer 1250 HPLC with a Partisil-lO SAX column using a phosphate buffer, 0.75 M KH2P04, pH 3.4. Chromato- gram 8 is the analysis of a solution of y-(N-triflouracetyl-6- aminohexyl)-IPCPOP treated 3 days with 1.0 N LiOH. 111 1.0 N LiOH. Finally the ninhydrin-positive y-(6-aminohexyl)-IPCPOP was desalted with Bio-Gel P2 column chromatography (Figure 27). About 24% of the IPCP starting material was recovered as y-(6-aminohexyl)-IPCPOP. A sketch of this product is shown in Figure 28 below. This ligand was then coupled through its primary amine to 10 ml CNBr-activated Sepharose-4B. Final analysis (see III, C, 2c) indicated a concentration of 7.3 pmoles ligand/m1 Sepharose. E. Application of a,B-Methylene-ITP Analogs to the Study of NTPH 1. Inhibition of NTPH with IPCPOP To be sure that the coupling enzyme, yeast pyrophosphatase, was not being affected by IPCPOP, a range of concentrations of IPCPOP and PPi were treated under the standard NTPH assay conditions at a reduced concentration of yeast pyrophosphatase (0.01 unit/m1 reaction mixture). The IPCPOP/PP, ratio was varied from 0 to 7.5 with no effect on the activity of the pyrophosphatase. Figure 29 clearly shows the competitive inhibition of NTPH with IPCPOP. The Vmax remains the same while the apparent Km for ITP increases with increasing concentrations of the inhibitor. A replot of the apparent Km, determined by the method described in Figure 29, versus the concentration of IPCPOP is shown in Figure 30. The Ki was calculated from the x-intercept of the replot as 5.9 uM for partially purified human NTPH and 3.7 uM forpartially purified rabbit NTPH. Kinetic analysis of an NTPH containing lysate also indicated competi- tive inhibition by IPCPOP, but the data did not fit a straight line in the replot of apparent 5n versus IPCPOP concentration (Figure 30). This nonlinearity could not be accounted for by the hydrolysis of IPCPOP during the experiment, and no alternative explanation has been tested. 112 1 l I l l 2.0 - QIVV--O> CI: 450 15 \ I D E on 1 76‘ g I :1 ~40 2 E: 1.5 _ 73 E; 8 9 .3» C D .g g D” -30 .5 1: 8 a D g 2% 1 o - o / 2 P 8 1 420 If ' 0.. I .’ ‘. ° ° '11 0 ‘~10 " \ 1:1 . \ o 1 l3 --CL-1J--lJ-thJ--t3-t3I :5 diacl -- -—- I ,PQIZIIPPJPPPPJ 20 40 60 80 100 Fraction Number Figure 27. Bio-Gel P2 Column Chromatography of y-(6-aminohexyl)-IPCPOP A column (3.4 x 40 cm) containing Bio-Gel P2 (2004400 mesh) was loaded with the sample and eluted with 50 mM TEAB, pH 7.5. Fractions, 5 ml each, were collected and monitored for absorbance and conductivity. 113 ”(in o H u H HZN-(CH2)6-P-0-P-CH2-P-0-CH 0 0' 0‘ o“ ou 0H Figure 28. Structure of y-(6-aminohexyl)-IPCPOP Purified ITP (96% by HPLC analysis) was used in these studies to lower the phosphate background in the NTPH assay controls which lacked enzyme and thus increase the reproducibility of the enzyme assay. 2. Affinity Chromatography of NTPH Since affinity chromatography of an enzyme may be affected by temperature, concentration of the ligand, pH, ionic strength of the buffer, concentration of the protein as well as other factors (182), the effect of these variables on the purification of NTPH from hemoly- sates was tested extensively by S. A. Fuller and the best results are presented here. Experiment 1 in Table 12 shows the purification of NTPH from a lysate of red cells. Greater than 76% of the activity loaded on the column was recovered in the effluent. About 53% of the activity was recovered following an ITP elution and indicated a 200- fold purification of NTPH from the initial lysate. Experiment 2 in Table 12 shows the results of the affinity chro- matography of a partially purified preparation of NTPH from human red cells. Thus 123% of the activity loaded on the column was recovered in 114 T T l T r l a 280 - H A! 240 1' P A0 £3 5 160 P P P4::' 120 - A D 'i o o Figure 29. Competitive Inhibition of Partially Purified Human NTPH with IPCPOP NTPH (specific activity = 3500 units/mg protein) was determined under standard conditions at various concentrations of purified ITP (96%). The velocity is expressed as the net absorbance at 700 nm in the Rath- bun and Betlach assay for phosphate (see Methods). Substrate concen- trations were recalculated by the method of Lee and Wilson (181) and lines were fit to the data by a computer program using the method of Wilkinson (167). Each point plotted here represents an average of two determinations. The following IPCPOP concentrations were used: 0 mM (x), 0.025 mM (0), 0.075 mM 03) and 0.15 mM (A). 115 l T 1 0.50f P I 0.40.. P E E E 0.30 4 :4 P I C OJ 0 $- § 0.20 D P P I O 0.10 3 J J l 1 0 0.04 0.08 0.12 0.16 IPCPOP (mM) Figure 30. Effect of IPCPOP on the Apparent Km of NTPH The apparent Km was calculated at each IPCPOP concentration as des- cribed in Figure 29. The two straight lines were fit to the data by the method of least squares. Legend: (I) partially purified rabbit NTPH, specific activity = 247,000 units/mg, (:0 partially purified human NTPH, specific activity = 3500 units/mg, (o) NTPH-containing lysate, specific activity = 15 units/mg. 116 Table 12. Purification of NTPH by Affinity Chromatography* Recovery Specific Activity (%) (Units/mg) Purif1cat1on Experiment 1 NTPH-containing lysate 100 30 Buffer elution 21 Buffer + ITP elution 55 6080 200 Experiment 2 Partially purified NTPH 100 1140 Buffer elution l7 Buffer + ITP elution 106 4990 4.4 * Affinity chromatography was performed with a 0.9 x 6.5 cm column con- taining IPCPOP-Sepharose-4B at 4°C. The column was eluted with buffer at pH 7.4 containing 1.0 mM MgCl followed by a buffer containing ITP and 10 mM MgCl as described in Ehe Methods. NTPH-containing lysate was prepared b; a 1:4 hypotonic lysis of red cells, and partially purified NTPH was prepared by the method of Morris (142). The protein was determined by the method of Lowry et al. (156). the effluent with 106% appearing during the ITP elution at a greater than 4-fold purification. F. Kinetic Differences Between Human and Rabbit NTPH Table 13 is a comparison of the kinetic constants of human and rabbit NTPH. The Km for ITP, Km for GTP and Ki for IPCPOP was in each case less for the rabbit enzyme than the human. The Lineweaver-Burk plot for the determination of the Km for GTP in Figure 31 indicates that GTP at high concentrations inhibits the rabbit enzyme in agreement with results reported earlier (153). Furthermore,high ITP concentra- tions have also been reported to inhibit the rabbit NTPH (1). In con- trast to these results neither GTP (up to 1.0 mM, Figure 31) or ITP (up to 2 mM, data not shown) was observed to inhibit the human NTPH. J. 117 1 1 1 1 1 50 ,. IIL .. I - 40 II E) AC) 0 (N 30 1— . q =?' . 20 F ‘ .- . I 10 " I. . ' ‘ I I I 1 1 l l l 2.0 4.0 6.0 8.0 10 12 1 GTP (mM) Lineweaver-Burk Plot for the Determination of the Km for Figure 31. GTP of Rabbit and Human NTPH NTPH was determined under standard conditions with varying concentra- tions of GTP. The velocity is expressed as the net absorbance at 700 nm in the Rathbun and Betlach assay for phosphate (see Methods). Lines were fit by a computer program using the method of Wilki1nson 1167). Legend: (I) partially purified rabbit NTPH (specific activity= 247,000 units/mg), (0) partially purified human NTPH (specific activity = 3500 units/mg). d951' 118 Table 13. Kinetic Constants for Human and Rabbit NTPH* Human NTPH Rabbit NTPH Km for ITP 23.5 i 2.2 uM 16.0 i 1.0 uM Km for GTP 3.82 i 0.46 mM 2.74 t 0.71 mM Ki for IPCPOP 5.9 pH 3.7 pM * Human and rabbit NTPH were partially purified by the method of Morris (142). The specific activities of human and rabbit NTPH were 3500 units/mg and 247,000 units/mg, respectively. The K for ITP and GTP was determined as described in Figure 29 and 31, regpectively, and the K was determined as described in Figure 30. The standard deviation is g1ven for the Km values. Whether these kinetic differences indicate a molecular difference in the NTPH from these two sources is not known. G. Cell-free Synthesis of ITP Zachara reported that red cells incubated in the IPP media (10 mM inosine:10 mM pyruvate:50 mM phosphate) of Duhm, Deuticke and Gerlach (186) accumulated 98 nmoles ITP per 100 ml cells during a 2 hour period at 37°C (119). Although Zachara used Dowex l-x8 to analyze the ITP accumulation in these cells, studies in our laboratory indicated that nucleotides including ITP could readily be separated and quantitated by HPLC (see Figures 32 and 33). Suspension of intact red cells (NTPH activity = 15 nmoles ITP hydrolyzed/mg hemoglobin) in the IPP media at 37°C for 2 hours followed by analysis of the nucleotides by HPLC indicated an accumulation of 64 nmoles ITP/100 ml red cells, a result similar to that reported by Zachara. Experiments were then designed to analyze cell-free synthesis of ITP. 119 Figure 32. HPLC Analysis of Hypoxanthine, Inosine, IMP, GMP, IDP, ITP, and ATP TCA extracts of lysates were analyzed on a Perkin-Elmer 1250 HPLC with a Partisil-lO SAX column eluted with( (A) 0.05 M KH P04, pH 3.4, (B) 0.2 M KH P0 , pH 3.4, and (C) 0. 5 M KHZPO, pH 3. i at a flow rate of 55 m1/houg. The retention time of standar s are marked as follows: (a) inosine, (b) hypoxanthine, (c) IMP, (d) GMP, (e) ADP, (f) IDP, (9) GDP, (h) ATP, (i) ITP and (j) GTP. 120 ab c d e f 9 A I B I I r I 1111 A.002~ 1 1 1- .30 1- - g $ 81 g .001 - - f5 .25 - '1 e O (D 2 ’\ .21 1 1 L E .20 . P 0 10 20 g Time (minutes) " h i j 8 I I I I I g C 1 1 1 S .15 ' ‘ .003 ' ‘ U" .Q < A E C $ .10 - - g .002.— P G) U C B L O .05 - A é’ .001- P . ELL L 1 I 1 0 5 10 0 10 20 Time (minutes) Time (minutes) Figure 32. HPLC Analysis of Hypoxanthine, Inosine, IMP, GMP, IDP, ITP, and ATP HPi an GMP 0b31 that the S 121 I I I T 25. /.d E20? . at +a ’/,/’//’ fi x101- / «I 1U 83 51- . -1 /l l J 1 100 200 300 400 ITP (pmoles) Figure 33. Standard Curve for the Quantitation of ITP by HPLC HPLC analysis of ITP was performed as described in Figure 32C. The peak height of ITP is plotted against the amount of ITP in the 10 uliters injected on the column. ITP concentrations were determined by the UV absorption of ITP at 248.5 nm (extinction coefficient = 12.2 mM). 1. Stimulation of Cell-free Synthesis with PRPP In agreement with Hershko gtggl, (29), we were not able to find an IMP kinase activity in hemolysates by the substitution of IMP for GMP in the guanylate kinase assay (187). Furthermore, in an attempt to observe ITP synthesis in hemolysates either by sonicating the cells in the IPP medium described above or lysing the cells with water followed by addition of the components of the IPP medium, we could detect very little ITP accumulation by HPLC even if the hemolysates were prepared from blood of individuals with relatively low NTPH activity and IPCPOP was included during the incubation to inhibit the remaining NTPH. Thus a search was initiated to find a metabolite or set of conditions which might enhance the synthesis of ITP. The results in Table 14 indicate that the combination of PRPP and inosine gave a 4-fold stimulation of the synthesis of IDP and ITP. 0n the other hand, this stimulation was 122 Table 14. Stimulation of Cell-free ITP Synthesis with PRPP All incubations in these experiments were performed at 37°C for 3 hours. The reaction mixtures (0.3 ml each) included 0.2 ml of a lysate (packed cells-water, 1:1) of VLV red cells (NTPH activity, 15 nmoles ITP hydro- lyzed/mg hemoglobin), 50 mM sodium phosphate buffer, pH 7.0, and the “ metabolites listed. RlP and REP refer to a,D-ribose 1-phosphate and D-ribose 5-phosphate, respectively. Mg PRPP was purified as described in the Methods. TCA extracts of the mixtures were neutralized and analyzed by a Perkin-Elmer 1250 HPLC equipped with a Partisil-lO SAX column (see Figure 32). Nucleotides were quantitated by peak height (see Figure 33). Experiment 1 Incubation Mixture 123 Nucleotides Analyzed by HPLC Metabolites (mM) (nmoles/ml mix) Ino IMP IDP MgZPRPP G1ucose ATP IDP ITP IDP+ITP 0 0 0 0 5 175 O 0 0 5 0 0 0 5 151 9 24 33 5 0 0 5 5 119 69 76 145 0 5 0 0 5 131 25 19 44 0 5 0 5 5 163 l3 19 32 O 0 5 O 5 96 - 874 - Experiment 2 Incubation Mixture Nucleotides Analyzed by HPLC Metabolites (mM) (nmoles/m1 mix) Ino Mg2PRPP RlP PP: ATP IDP ITP IDP+ITP 0 5 5 0 119 - 0 - 5 5 5 0 103 21 59 80 5 0 5 0 95 9 0 9 5 0 0 5 131 - 0 - Experiment 3 Incubation Mixture Nucleotides Analyzed Metabolites (mM) (nmoles/m1 mix) Ino MgzPRPP Glucose M92C12 RSP ATP ATP ITP 0 0 5 0 0 0 235 0 0 5 5 0 0 0 262 0 5 0 5 0 0 0 168 17 5 5 5 0 0 0 176 70 5 5 5 0 0 0 172 58 5 0 5 5 0 0 163 21 5 0 5 0 O 2 434 12 5 0 5 0 5 2 319 12 124 not observed when any of the metabolites, a,D-ribose 1—phosphate, PPi, MgClz, D-ribose 5-phosphate or ATP was substituted for PRPP. Furthermore, the stimulation of the synthesis of IDP/ITP by PRPP was independent of the presence of glucose. 2. Correlation of IDP/ITP Synthesis with the Presence of Inosine and Hypoxanthine The results in Table 14 further indicate that inosine cannot be substituted by IMP. Thus inosine and PRPP gave 4-fold more synthesis of IDP/ITP than the same concentrations of IMP and PRPP. This suggests that the biosynthesis of IDP/ITP may not occur through IMP as an inter- mediate. Table 15, Experiments 1 and 2, presents additional evidence that cell-free IDP/ITP synthesis is best correlated with elevated con- centration of hypoxanthine and inosine rather than the concentrations of IMP in the reaction mix. It may also be noted from this table that inhibition of HGPRT with 5 mM IMP, 0.5 mM GMP, or 5 mM GMP (39) elimi- nates the requirement for PRPP, perhaps by increasing the availability of endogenous PRPP or the PRPP synthesized from inosine (43). IMP, and perhaps GMP, may also inhibit NTPH (1) and thus enhance the accumu- lation of IDP/ITP. 3. Incorporation of [14C]inosine and [3H]hypoxanthine into IDP/ITP In contrast to the accumulation of IDP/ITP from high concentra- tions of unlabeled inosine, accumulation of [14C]ITP from [14C]inosine not only requires PRPP but also requires the additional presence of IMP (Table 16, Experiment 1). It seems likely that at low concentrations, [14C]inosine in the presence of PRPP is rapidly incorporated into IMP through the combined action of purine nucleoside phosphorylase and HGPRT. A high concentration of IMP would then slow this incorporation 125 Table 15. Correlation of Cell-free IDP/ITP Synthesis with Elevated Con- centrations of Hypoxanthine and Inosine The VLV lysate used in Experiment 1 and the DY lysate used in Experiment 2 (NTPH activities of 15 and 3 nmoles ITP hydrolyzed/mg hemoglobin, respectively) were treated with 10 mg charcoal/ml lysate for 5 minutes and the charcoal was removed by centrifugation. A11 reaction mixtures included 5 mM MgC12 and 50 mM sodium phosphate, pH 7.0. R5P refers to D-ribose 5-phosphate. Metabolite analyses were performed as described in Table 14. 126 Experiment 1 Incubation Mixture Metabolites Analyzed by HPLC Time Metabolites (mM) (nmoles/ml mix) (nmoles/m1 mix) (hrs.) Ino IMP Na4PRPP Hyp Ino IMP ATP IDP ITP IDP+ITP 3 5 0 O 3.1 .029 1.6 110 9 19 28 3 0 5 0 0.25~ .010 6.5 81 0 0 0 3 0 0 5 .009 .010 0.48 192 0 0 0 3 5 5 0 5.9 .086 3.0 156 27 118 145 3 5 0 5 1.2 .057 7.0 150 25 57 82 3 0 5 5 0.8 .048 7.2 132 15 43 58 0 5 5 5 3.2 6.23 0.05 90 - 0 - 3 5 5 5 4.4 .238 7.7 122 19 99 118 Experiment 2 Incubation Mixture Metabolites Analyzed by HPLC Time Metabolites (mM) (nmoles/m1 mix) (nmoles/m1 mix) (hrs.) Ino IMP GMP RSP Na4PRPR Hyp Ino IMP GMP ATP IDP ITP IDP+ITP 3 5 0 0 0 0 3.5 .057 1.2 - 90 19 57 76 3 0 5 O 0 0 0.31 .009 6.4 - 111 0 l9 l9 3 0 0 0 0 5 .009 .009 0.43 - 35 0 0 0 0 5 5 0 0 0 1.9 3.1 6.8 - 63 0 0 0 3 5 5 0 0 0 3.5 .076 7.6 - 177 49 133 182 3 5 0 0 0 5 1.1 .038 6.1 - 192 37 142 179 3 O 5 0 0 5 .073 .009 7.2 - 78 9 38 47 3 5 5 0 0 5 1.6 .048 11.7 - 189 43 13 176 3 0 5 0 5 0 .093 .019 7.3 - 132 15 24 39 3 0 5 0 .5 0 .009 .009 6.7 - 108 9 19 28 3 5 0 5 0 0 3.1 .038 1.8 7.3 165 37 90 127 3 5 0 5 O 0 3.5 .048 1.8 1.3 144 55 123 178 127 Table 16. Cell-free Incorporation of [14C]Inosine and [3H]Hypoxanthine into IDP/ITP Each reaction mixture in Experiments 1 and 2 included DY lysate (NTPH activity, 3 nmoles ITP hydrolyzed/mg hemoglobin), 5 mM MgC12, and 50 mM sodium phosphate, pH 7.0. The specific activity of the [14C]inosine used was 52 mCi/mmole. Each reaction mixture in Experiments 3 and 4 included VLV lysate (NTPH activity, 15 nmoles ITP hydrolyzed/mg hemoglobin) and 50 mM sodium phosphate, pH 7.0. The MgzPRPP was purified as described in the Methods. RlP refers to a,D-ribose l-phosphate. The [3H]hypoxanthine used in these experiments had a specific activity of 1.8 Ci/mmole. All incubations were carried out for 3 hours at 37°C and the metabo- lites were analyzed as described in Table 14. Radioactivity in each region was collected and analyzed as described in the Methods. NM 051119 1% 19 Experiment 1 Incubation Mixture 128 Metabolites Analyzed by HPLC Metabolites (mM) (cpm) [14c11no Na4PRPP IMP Hyp+In0 IMP ITP 0.13 5 1317 39,727 20 0.13 5 1337 41,700 216 Experiment 2 Incubation Mixture Metabolites Analyzed by HPLC Time (hrs.) 14 Metabolites (mM) (cpm) [ C]Ino Ino IMP Na4PRPP Hyp+Ino IMP ITP 0.5 0.13 5 5 5 27,635 6,909 N.D. 1.5 0.13 5 5 5 13,506 21,107 31 3.0 0.13 5 5 5 3,801 32,033 194 Experiment 3 Incubation Mixture Metabolites Analyzed by HPLC Metabolites (mM) (cpm) Ino MgzPRPP Glucose [3H]Hyp Hyp+Ino IMP ITP 5 5 5 .033 23,119 51,415 959 0 5 5 .033 3,979 80,353 35 Experiment 4 Incubation Mixture Metabolites Analyzed by HPLC Metabolites (mM) (cpm) Ino MgzPRPP R1P [3H]Hyp Hyp+Ino+IMP (IDP ITP 5 5 5 .033 78,643 212 767 5 0 5 .033 80,114 68 169 0 5 5 .033 74,559 - 0 0 0 5 .033 71,852 - 0 129 of radioactivity into IMP by its mass action effect on the HGPRT catalyzed reaction as well as the actual inhibition of HGPRT (39) and thus conserve [14C]inosine for synthesis of IDP/ITP. Further evidence that the concentration of inosine during the incubation may be directly correlated with the incorporation of [14C1inosine into ITP is presented in Table 16, Experiment 2. Unla- belled inosine in this experiment was increased by 5 mM over that in Experiment 1. Comparison of the results of these two experiments indicates that 5 mM [14C]inosine at a specific activity of 1.3 mCi/ mmole was incorporated to the same extent as 0.13 mM inosine at a spe- cific activity of 52 mCi/mmole. It may be observed that following 3 hours of incubation, the radioactivity in the hypoxanthine and inosine region of the chromatogram was 3.4-fold higher in the case with high initial inosine concentration (Experiment 2) than in the case with low initial inosine concentration (Experiment 1). Thus it seems that the incorporation of [14C]inosine into IDP/ITP as well as the incorpora- tion of unlabelled inosine into IDP/ITP may be correlated with condi- tions which prevent or slow the inosine incorporation into IMP. Table 16, Experiments 3 and 4, demonstrates that at low concen- trations [3H]hypoxanthine is readily incorporated into IMP but not IDP or ITP. However a high concentration of unlabelled inosine with the same amount of [3H]hypoxanthine yielded more than 1% incorporation of the label into IDP/ITP. In this case it seems that inosine itself may have been labelled by the [3H]hypoxanthine by means of the reversable purine nucleoside phosphorylase reaction (188) and the high concentration of inosine prevented all the radioactivity from being incorporated into IMP during the 3 hour incubation (see Experiment 3). a,D-ribose an. UT o 110 "Br 130 1-phosphate (dicyclohexylammonium salt) alone could not stimulate the incorporation of [3H]hypoxanthine into ITP (see Experiment 4), but this may simply be due to the low concentration of hypoxanthine in this experiment. 4. Effect of NTPH Activity on IDP/ITP Accumulation As might be expected, the activity of NTPH in the lysates affected the rate of ITP accumulation in the three hour incubations. As demon- strated in Table 17, the highest accumulation of ITP was obtained with the lysates with lowest NTPH activity. Table 17. Effect of NTPH Activity on the Cell-free Synthesis of IDP/ITP* Incubation Mixture Nucleotides Analyzed Lysate . Metabolites (mM) (nmoles/ml mix) Subject NTPH (3318:) Inc IMP GMP Na4PRPP IDP ITP IDP+ITP DY 5 5 0 0 49 133 182 DY 5 5 0 5 43 133 176 DY 5 0 0.5 0 55 123 178 VLV 15 5 5 0 0 27 118 145 AJM 65 5 0 0.5 5 15 33 48 * All reaction mixes included lysates, which had been treated once with charcoal (10 mg/ml lysate); 5 mM MgC12 and 50 mM sodium phosphate, pH 7.0. The 3 hour incubation procedures for the lysate of the red cells of each subject were performed on different days. The nucleotide analyses were performed as described in Table 14. 5. Other Conditions Affecting Cell-free IDP/ITP Synthesis To uncouple IDP from ITP synthesis by removing ATP and to test whether IDP synthesis was ATP dependent, the endogenous nucleotides were removed by treatment of the lysates with charcoal. As can be seen 131 in Table 18, extensive charcoal treatment reduced both the ATP concen— tration and capability of these lysates to synthesize IDP/ITP. Exper- iment 2 in Table 18 indicates that very little IDP/ITP synthesis was recovered even when ATP or PRPP was added back to these lysates. Thus the experiments so far have not demonstrated an uncoupling of IDP from ITP synthesis or shown that IDP synthesis is completely independent of ATP concentrations. Sonification of crude hemolysates seemed to decrease endogenous levels of ATP, perhaps by release of a phosphatase associated with the membrane, however these lysates were no longer capable of cell-free synthesis of IDP or ITP. When stored for months at -20°C, the washed packed red cells from one individual (DY) retained the ability to accumulate ITP under cell- free conditions. 132 Table 18. Effect of Extensive Charcoal Treatment of Hemolysates on Cell-free IDP/ITP Synthesis Experiment 1 Incubation Mixture Metabolites Analyzed by HPLC Lysate Metabolites (mM) (nmoles/m1 mix) (nmoles/ml mix) Charcoal Treatment Ino IMP Hyp Ino IMP ATP IDP ITP IDP+ITP 1 X 5 5 3.5 .076 7.6 177 49 133 182 2 X 5 ' 5 3.8 .048 6.1 18 0 17 17 3 X 5 5 - - - 30 9 24 33 Experiment 2 Incubation Mixture Metabolites Analyzed by HPLC Lysate Metabolites (mM) (nmoles/ml mix) (nmoles/ml mix) Charcoal Treatment Ino GMP Na4PRPP ATP Hyp Ino IMP ATP IDP ITP IDP+ITP 2 X 5 0.5 0 0 - - - - 0 - - 2 X 5 0.5 0 1 - - - - 5 - - 2 X 5 0.5 5 0 1.01 .057 6.6 - 8 - - 2 X 5 0.5 5 1 1.03 .076 6.7 123 6 10 16 The reaction mixtures in Experiment 1 contain DY lysate (NTPH activity, 3 nmoles ITP hydrolyzed/mg hemoglobin) and the mixes in Experiment 2 contain VLV lysate (NTPH activity, 15 nmoles ITP hydrolyzed/mg hemoglo- bin). Prior to the 3 hr incubation these lysates were treated with charcoal, 10 mg/ml lysate, for 5 min and the charcoal was removed by centrifugation. This process was repeated where indicated. All mix— tures also include 5 mM MgCl and 50 mM sodium phosphate, pH 7.0. Metabolite analyses were per ormed as described in Table 14. CHAPTER V DISCUSSION A. The Genetic Variability of NTPH At the time of the purification and characterization of NTPH from rabbit reticulocytes by Chern, MacDonald and Morris (1), it was observed that the NTPH activity in human red cells varied more than lOO-fold from one individual to another, yet the specific activity was constant in the red cells of any one individual over a period of years. Concurrent with these observations Vanderheiden published a population distribution and 15 family studies of an "ITPase" activity in the red cells, data which he interpreted as evidence for the codominant trans- mission of this activity (3). Vanderheiden was inadvertantly measuring a coupled reaction between NTPH and endogenous red cell pyrophosphatase and thus either variation in NTPH or red cell pyrophosphatase activity could have accounted for his observed distribution of "ITPase" activity in the human population. To ascertain whether this distribution reflected the variation of NTPH activity and to establish a framework for the study of the inheritance of NTPH as well as for a study of the metabolic implications of this activity, S. A. Fuller conducted a popu- lation survey of the specific activity of NTPH in red cells. The survey clearly demonstrates the variation of NTPH activity among indivi- duals and furthermore suggests that individuals may be classified into at least two groups, those with low or high activity based on the 133 134 specific activity of their red cells (Figure 3). This distribution corresponds well with the population survey presented by Vanderheiden indicating.that the variation of the "ITPase" which he observed was primarily due to variation of NTPH. Vanderheiden however suggested yet a third classification of individuals, those with very low activity. Whether this third classification is indeed a distinct group which represents a unique genotype cannot be established by evidence presented by Vanderheiden or supported by data presented in this dissertation. However as discussed below, the metabolic consequence of very low red ,5? cell NTPH activity (0—3 nmoles ITP hydrolyzed/mg hemoglobin) may be distinct from the metabolic consequence of low activity (3-27 nmoles ITP cleaved/mg hemoglobin). Thus it seems that the specific activity of NTPH in the red cells is an inherited trait by virtue of the simi- larity of the two population distributions described here and the fact that NTPH activity remains unchanged in any individual over a period of years. Other data presented in this dissertation also support this con- clusion. First, the two family studies, particularly the Gr family (Figure 4), indicate that the NTPH activity of the progeny reflect the activity of their parents. S. A. Fuller is currently expanding these studies to include other families with the hope of defining the pattern of inheritance. Second, the results of the survey of NTPH activity in granulocytes, lymphocytes and platelets emphasize that the variation of NTPH activity is not limited to the erythroid cells but is reflected in other tissues as well. Thus any explanation of the molecular basis of this diversity cannot be limited to red cell development or metabo- lism. 135 Consistent with the results of the tissue survey of the rabbit (2), the NTPH activity per cell was lowest in the erythrocyte (see Figure 5-7). Although the NTPH per platelet was similar to the red cell activity, the platelet activity probably reflects the NTPH activity of a small portion of the cytoplasm of a megakaryocyte, the platelet- producing cell. 1 Possible explanations for the molecular basis of the NTPH varia- tion may be classified into two groups. First, the actual amount of NTPH protein may vary due to alterations in the synthesis or degrada- 1 tion of the enzyme in the cells or second, variation may be caused by NTPH molecules with altered kinetic parameters for the substrate ITP which may be caused by the presence of isozymes or intracellular effec- tors in different proportions or combinations in the cells of different individuals. A number of these possibilities has been tested. Pre- vious data from this laboratory as well as the data offered by Harris and H0pkinson suggest that the variation cannot be attributed to elec- trophoretic isozymes. With NTPH from rabbit liver, rabbit red cells and human red cells, Wang and Morris could observe only one band on disc gel electrophoresis specific for the hydrolysis of ITP (2). Although Harris and H0pkinson report that by their method NTPH migrates as 2 or 3e1ectrophoretic:bands, they could not observe qualitative differences of NTPHelectrophoreticnmufility while screening a "large number of red cell lysates" (145, see also Literature Review). Table 2 indicates that the Km for ITP is the same for NTPH in lysates of selected individuals which exhibit a wide range of NTPH specific acti- vities. The mixing study in Table 1 and data reported earlier (5) give no indication of intracellular inhibitors or activators. Current 136 studies in this laboratory are designed to eliminate or test other possible molecular causes of this variation. 8. The Metabolic Consequences of the Genetic Variation of NTPH A number of hypotheses have been offered over the past ten years concerning the physiological significance of NTPH. Hershko, gt_gl, sug- gested that NTPH may be involved in the control of the intracellular concentration of GTP, a substrate of NTPH hydrolyzed at 10% the rate of ITP (121); Wang and Morris suggested that NTPH is necessary to prevent incorporation of ITP into RNA or dITP into DNA (2) and Vanderheiden postulated that NTPH is one of the enzymes of an ITP-IMP cycle which controls intracellular concentrations of ATP (144). To support the assumption inherent in the last two hypotheses that ITP is the physio- logical substrate of NTPH, it is important to establish that the substrate-enzyme relationship exists between ITP and NTPH. Vanderhei- den had suggested as early as 1965 that the presence of ITP in the red cells of some individuals could be attributed to the lack of an I'ITPase" and furthermore that the variation in the accumulation of [14C]ITP from [14C]inosine by intact cells could be attributed to variation in this same "ITPase" (26, 3). The data in Figure 9 as discussed in Appendix A support the conclusion that the rate of [14C]ITP accumulation by intact red cells in the presence of [14C]hypo- xanthine is related to the NTPH specific activity of these cells in a manner predicted by Michaelis-Menten kinetics. Furthermore direct analysis of nucleotides by HPLC indicated that a UV-absorbing peak coinciding with ITP was present in extracts of blood which had a very low red cell NTPH activity, 3 nmoles ITP cleaved/mg hemoglobin (see Figure 13). On the other hand concentrations of GTP (Table 8 and 137 Figures 13-15) were quite similar for blood of individuals with a 10- fold range of red cell NTPH activity. One can conclude therefore that NTPH activity has a physiologically significant enzyme-substrate relationship with ITP in the red cell both in artificial incubation media and jp.vivg, Since ca. 1% of the population may have undetectable NTPH acti- vity in their red cells, the question may be asked whether other tis- sues of these individuals have undetectable activity as well and if so, whether the ITP concentration is unusually high in these tissues. Although we were not able to analyze tissues other than the red cells of individuals with undetectable NTPH activity, it is interesting that the one individual with very low red cell NTPH has greater than 5-fold more granulocyte NTPH activity and greater than 30-fold more lymphocyte NTPH activities compared to the red cell level of that enzyme (Tables 3-5). Thus tissue differences with regard to NTPH activity may prevent endogenous accumulation of ITP in cells other than the red cell, a cell which is not capable of DNA or RNA synthesis. 0n the other hand tissues may differ in the ability to synthesize ITP, a question which has not yet been studied. C. The Physiological Role of ITP and NTPH If we proceed with the hypothesis that NTPH is present to pre- vent incorporation of ITP or dITP into RNA or DNA, we may ask why ITP is synthesized at all. Since the red cell and perhaps other tissues clearly can synthesize ITP, it is possible that either ITP or interme- diates of its synthesis or an ITP-IMP cycle may have some physiological role. Since the biosynthesis of ITP has not been defined or studied 138 in red cell lysates, we worked to establish a cell-free system which had the capability to accumulate ITP. The substrate-enzyme relationship between ITP and NTPH in intact cells suggested that to observe any ITP synthesis it would be necessary to use the red cells of an individual with very low NTPH activity or find an inhibitor which is highly specific for NTPH in order to pre- vent degradation of ITP as soon as it was synthesized. Since the availability of individuals with very low NTPH activity is limited, we found it more practical to prepare an inhibitor. The work of Yount (183) with methylene analogs of ATP suggested that substitution of the oxygen with a methylene in the 6,8-position of ITP might yield a very specific competitive inhibitor of NTPH. IPCPOP was successfully prepared by two methods, deamination of commercial APCPOP and synthesis from adenosine and methylene diphos- phonic acid followed by deamination and phosphorylation of the result- ing APCP. IPCPOP was a very good competitive inhibitor of partially purified human NTPH (Ki = 5.9 0M) and NTPH present in lysates (Figure 29). IPCPOP was also coupled to Sepharose-4B to yield an affinity resin which was used to purify NTPH from hemolysates or partially purified preparation (Table 12). Preliminary cell-free experiments designed to observe accumulation of ITP by lysates incubated with the inosine, pyruvate and phosphate media used by Zachara (119) were unsuccessful even when lysate with low NTPH activity, 15 nmoles ITP cleaved/mg hemoglobin, was used in combination with the IPCPOP inhibi- tor. These studies indicated that the cell-free accumulation of ITP was not limited by NTPH alone but probably required a cofactor or set of conditions not being supplied by the incubation conditions. 139 Many of the obvious cofactors such as nucleoside triphosphates or conditions such as variation of the pH did not stimulate ITP synthe- sis. But the observation that PRPP enhanced IDP and ITP synthesis in the presence of inosine but not in the presencecHiIMP (Table 14) suggested that synthesis of ITP may involve a unique reaction in which the pyrophosphoryl moiety of PRPP is transferred to the 5'-ribose position of inosine to yield IDP (Model I of Figure 34) as opposed to synthesis of IDP by phosphorylation of IMP (Model II of Figure 34). Evidence for this pyrophosphotransferase reaction is discussed below. The direct involvement of PRPP in IDP/ITP synthesis was deduced from the following observations. First, the inosine-phosphate media used in these studies is conducive for the synthesis of PRPP in that inosine can be metabolized to R5P and combine with ATP to form PRPP, a reaction catalyzed by PRPP synthetase in the presence of high phos- phate (43). But the production of PRPP by this pathway sacrifices ino- sine which also appears to be necessary for the synthesis of IDP/ITP. Thus the addition of exogenous PRPP stimulates IDP/ITP synthesis, perhaps because in this case neither inosine or PRPP is rate-limiting. Second, to confirm that the stimulation by PRPP was not caused by one of its degradation products such as PPi, R5P or RlP, these metabolites were substituted for PRPP in the cell-free system but none could stimu- late the synthesis of IDP/ITP. Third, since R5P can potentially yield ATP in this system by entering glycolysis at the level of glyceralde- hyde-3-phosphate, exogenous ATP was tested and not found to be an effector of IDP/ITP synthesis. However the possibility that the endo- genous ATP provided by the lysates was sufficient to effect the IDP/ITP synthesis cannot be excluded by data described here. Fourth, conditions 140 (4) ATP IDP (3) PRPP flQDEl—J PRPP ADP , (5) 7) ATP Hyp + Hyp Ino + IDP ATP R1P IMP (21PPI P1 MODEL 11 Figure 34. Two Models for the Role of NTPH in the Metabolism of ITP The enzymes involved in these cycles are the following: (1) NTPH, (2) 5 -nuc1eotidase, (3) proposed pyrophosphotransferase, (4) NDP Kinase, (5) hypothetical IMP Kinase, (6) PNP, (7) HGPRT. 141 which prevented the utilization of PRPP in other reactions also stimu- lated IDP/ITP synthesis. Thus 0.5 mM GMP or 5 mM IMP, known competi- tive inhibitors for PRPP of HGPRT (Ki = .014 and .14 mM, respectively, 39), could stimulate synthesis even if exogenous PRPP was not provided. The direct involvement of inosine in the synthesis of IDP/ITP was deduced from the following data. First, inosine and PRPP yield 4-fold more IDP/ITP synthesis than either IMP alone or IMP and PRPP. Second, elevated concentrations of inosine and hypoxanthine as detected by FiPLC following 3 hours of incubation were consistently associated with 'increased synthesis of IDP/ITP during this incubation period. Third, cronditions which prevented the utilization of hypoxanthine by HGPRT ii 150 stimulated IDP/ITP synthesis. Thus either 0.5 mM GMP or 5 mM LIEWP, inhibitors of HGPRT as noted above, not only spares PRPP but also sslaares hypoxanthine and thus prevents complete degradation of inosine (inuring the three hour incubation (Table 15). Table 14 shows clearly that IDP is readily converted to ITP in ‘tzf1ee cell-free system. Attempts to uncouple IDP synthesis from ITP sPYrIthesis by removal of endogenous ATP by extensive charcoal treatment <1'f’ the lysates were not successful since the charcoal treatment also a 1 tered the ability of these lysates to synthesize IDP (Table 18). SSPISv'I'I‘thesis of IDP or ITP could not be recovered even when ATP was added bac k to the system. From these data alone and the observations that NTPH activity a: 1F"1=f'the PRPP synthetase reaction as -2.0 i 0.5 kcal/mole (42). He 'frurther suggests that if one uses -11 kcal/mole for the free energy of hydrolysis of ATP as calculated by Alberty (189), one can calculate the free energy of hydrolysis of the P—glycosidic bond of PRPP to be (:ia. -9 kcal/mole at pH 7.5. 'On the other hand, the free energy of the phosphate hydrolysis of glucose 6-phosphate at pH 7.0 is reported '1:<) be -3.3 kcal/mole (190). Thus if one assumes that hydrolysis of ‘tlltee P-O-C bond of IDP is on the same order as hydrolysis of the P-O-C bond of glucose 6-phosphate, the reaction between PRPP and inosine to 15"i'eeld IDP would be expected to have a negative free energy on the oV‘cler of 5 to 6 kcal/mole. Model II would seemingly provide a futile cycle for IMP and ITP ‘""i'1th the net result of a loss each cycle of two high energy pyrophos- phate bonds and a decrease in the adenylate energy charge. It make 1' ‘7 tztle sense physiologically that the cell reduce the energy charge 146 during the situation of elevated concentrations of purine catabolic waste products inosine and hypoxanthine, particularly the latter which is already being salvaged into nucleoside monophosphates. On the other hand, Model I is much more attractive since it represents the salvage of the metabolic waste product inosine to a useful metabolite IMP which has central importance in the biosynthesis of AMP and GMP (see Figure 2). If Model I is indeed the mechanism by which IDP/ITP is synthe- :sized, it raises several interesting predictions and questions. First, r~ed cells of patients with purine nucleoside phosphorylase deficiency aissociated with severe combined immunodeficiency have been found to c:ontain both elevated concentrations of inosine and PRPP (104). Model 12 predicts that these same cells would have increased concentrations of LI TP particularly if these red cells had low NTPH activity. One may eausk whether other tissues in humans with this disease might also have £2 levated inosine and ITP concentrations. For example, if ITP does £EJility in granulocytes, lymphocytes and platelets. 2. The variation of NTPH specific activity of red cells is inde- F>endent of the Km for its substrate ITP. Intracellular effectors of NTPH activity were not detected. 3. NTPH specific activity is inversely related to the accumula- ‘t:ion of [14C]ITP in erythrocytes incubated jp_vitro with [14C]hypoxan- 1:!1ine in a manner predicted by Michaelis-Menten kinetics for an enzyme «Ell1c1 its substrate. Endogenous ITP was detected in an extract of blood (Dif’ an individual with very low NTPH activity. This evidence is inter- preted to indicate that NTPH limits the concentration of ITP allowed 'tZCD exist in erythrocytes. 4. A methylene analog of ITP (IPCPOP) is a good competitive ‘i nhibitor for ITP with a K]. = 5.9 .111 for partially purified human '\"I‘F>H. Attachment of IPCPOP to Sepharose-4B by means of a six carbon 3 pacer molecule provides an affinity resin for the purification of NTPH. 148 si co ph C8 51 K1 149 5. A cell-free system was established to study the biosynthe- sis of ITP in red cell hemolysates. Manipulation of the incubation conditions indicated that IDP may be synthesized by a unique pyrophos- photransferase which transfers the pyrophosphate of PRPP to the 5'- carbon of inosine. 6. NTPH is proposed to complete a metabolic cycle in which ino- sine is salvaged by PRPP to form IDP which is converted to ITP by NDP Kinase. The ITP thus formed is hydrolyzed by NTPH to prevent interference of ITP in transcription of RNA and to produce IMP, a metabolite essential in the gg_novo biosynthesis of GMP and AMP. APPENDIX A Theoretical Relationship Between Endogenous ITP in Human Red Cells and NTPH Specific Activity APPENDIX A THEORETICAL RELATIONSHIP BETWEEN ENDOGENOUS ITP IN HUMAN RED CELLS AND NTPH SPECIFIC ACTIVITY By the Michaelis-Menten equation one can relate substrate con- centration to the initial velocity of a reaction as given in Equation [1], where Vi is the initial velocity of an enzyme reaction, Vmax is the maximum velocity, [S] is the substrate concentration and Km is the Michaelis constant. [1] vi = vmax [SI/[S] + Km Furthermore, in the case of an irreversible enzyme such as NTPH (153), the velocity of the enzyme reaction at a steady state substrate concen- tration could be substituted for the initial velocity in Equation [1]. If, for the purposes of developing a theoretical relationship between the specific activities of NTPH and %[14C]ITP accumulation in the red cells of two individuals, one assumes (a) that the rate of ITP synthesis is constant among the subjects studied and equal to the rate of ITP degradation, (b) that the Km value for ITP is a constant among the subjects studied, and (c) that the %[14C]ITP accumulation observed in these analyses is proportional to the steady-state ITP concentration in the human red cells, then one may substitute the specific activity value of NTPH for Vmax and relate percentage of [14C1ITP accumulation to [S] by using a proportionality constant in the following equation: 150 151 121 vmax] [Sh/([511 + Km) = vmaxZ1312/(1512 + Km)- NTPH is assayed under conditions of optimal activity (1) and hence closely approximates Vma The Km for ITP is independent of the x' specific activity of NTPH in lysates of red cells from selected indivi- duals with markedly different NTPH levels (Table 2). The pr0portiona- lity constant relating the steady state ITP concentrations with the observed %[14C]ITP accumulation was calculated as 5.1 x 10'7 5 M by use of an average Km value of 2.8 x 10' M (from Table 2) and the reference points 2.5% [‘4c11TP, specific activity 70, and 14% [14C]ITP, specific activity 15. A best-fit theoretical curve relating [14C]ITP accumula- tion and NTPH activity was then prepared as shown in Figure 9. The entire range of experimental values correlate rather closely with the relationship defined by this mathematical treatment. If one recalculates the theoretical relationship in terms of the steady state level of ITP predicted by the proportionality constant above, then one can define the expected steady state levels of ITP in the red cells of-individuals with different NTPH specific activities as shown in Figure 35. From this curve one would predict ITP levels to be greater than 5 uM for any individual with an NTPH specific activity less than 20 units/mg. Moreover individuals with very low NTPH activity may be expected to have 50 uM or more ITP present in their red cells if no other mechanism limits the physiological level. In constrast to these predictions the HPLC analysis of endogenous ITP in the blood of an individual with an NTPH activity of 3 units/mg hemoglobin indicated that only 5 uM ITP was detectable (Figure 13). No ITP could be detected in the blood of two individuals with NTPH acti- vities, 15 and 30 units/mg hemoglobin (Figures 14 and 15). Special care 152 was taken in these experiments to minimize the amount of time between venipuncture and extraction of nucleotides (ca. 5 minutes). A [3H]ITP internal standard was added as the cells were extracted to monitor recovery throughout the extraction and analytical procedures. It may be however that the ITP present in intracellular pools did not com- pletely mix with the added standard during the extraction, in which case the HPLC analysis may be an underestimate of the endogenous pool sizes. An alternative explanation for the discrepancy between the theore- tical prediction of endogenous ITP levels and the HPLC analysis may be that the theoretical curve is based on a faulty assumption. In the mathematical treatment, it is assumed that Henderson's method for labelling the ITP pools does not significantly alter the pool size. This may not necessarily be true since Henderson eggpl, have shown in one case that prior to incubation of red cells with [14C]hypoxanthine no ITP could be detected by HPLC whereas after incubation, ITP was observed (72). Thus the analysis of the endogenous concentration of ITP in red cells is a problem which is unresolved, but it does seem that NTPH activity has a role in determining the intracellular steady state concentration. 7 l l 153 1 1 ‘1 ‘T 1 1 I 1 l 50 ' ‘ 1 i . 1 1H 5% 30 - ‘ 1 o. f F- i *‘ r 4 1 20 ' d 1 NTPH (Units/mg Hemoglobin) Figure 35. Theoretical Relationship Between Endogenous ITP in Human Red Cells and NTPH Specific Activity ‘This figure is the result of the mathematical treatment described in 10- ‘1 1 I 1 1 J I l L l in 20 4O 6O 80 100 gfippendix A on the data presented in Figure 9. REFERENCES i 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES Chern, C. J., MacDonald, A. B., and Morris, A. J. (1969) J. Biol. Chem. 2314, 5489-5495. Wang, J. K., and Morris, A. J. (1974) Arch. Biochem. Biophys. 161, 118-124. Vanderheiden, B. S. (1969) Biochem. Genet. 3, 289-297. (1970) Biochim. Biophys. Acta 215, 555-558. Soder, C., Henderson, J. F., Zombor, G., McCoy, E. E., Verhoef, V., and Morris, A. J. 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