QUINOLINIC ACID. PHOSPHORIBOSYLTRANSFEMSE IN CASTOR BEANS Thesis for the Degree of Ph. D.‘ MICHIGAN STATE UNIVERSITY DAVID F; MANN 1972 » ... .— —_. “var. LIBRARY Michigan State University This is to certify that the thesis entitled Quinolinic Acid Phosphoribosyltransferase in Castor Beans presented by ~» David F. Mann has been accepted towards fulfillment of the requirements for Ph-D - degree in Jinnhemiatry Pt [1. 7.21% Major professor DateJmmhw 0-7639 .31: ‘ BINDING BY "In I I mm; 3 sons ‘BUGK swam mc. RARY Bl NDERS roar, Ilflllflj ISI’ a I 1' 09 t“(: I v: ' _ I I _ 7.7": ‘1‘, ,4 "urlbosy.:~ , , .‘ J"? '?aonue1tx~t5s . LE)“ SI-L‘. - ‘ ._ ”I Ricux J2 A , » - ~... , ,..- Vitaleculer weight :t'I fitZL'fl' -‘.' ..'. $7.; :1 01513. The E £103 bios}: :fiimlinic 3C catalyzes the S‘Pi-Csphoribc acid moflonucl. has purified SEEdlingS Of 1 included a he ~Ogra Phy, hyd; : n a <4 ABSTRACT QUINOLINIC ACID PHOSPHORIBOSYLTRANSFERASE IN CASTOR BEANS BY David F. Mann The purpose of this study was to purify quinolinic acid phosphoribosyltransferase and study its role in the fig gg!g_biosynthesis of NAD in the castor bean plant. Quinolinic acid phosphoribosyltransferase (E.C. 2.4.2.__.) catalyzes the following reaction: quinolinic acid + i 5-phosphoribosyl-1-pyrophosphate (PRPP) ———————9»nicotinic acid mononucleotide + pyrophosphate + C02. This enzyme , was purified SOO-fold from the endosperm of etiolated seedlings of Ricinus communis L. by a procedure which included a heat denaturation step, DEAE Sephadex chroma- tography, hydroxyapatite chromatography, and isoelectric focusing to give a 20% overall yield. The enzyme appeared VZ5~‘. homogeneous after SDS-disk gel electrophoresis, but the native enzyme gave three closely migrating bands on 5:5 analytical disk gels at pH 8.3. The three proteins were €Lo£cthe same molecular weight but differed slightly in charge, as determined by Ferguson plots of the Rf values a: different maze was 5: PC or -90°C " e ‘.- 3.2;:1: coma 1.‘ :31. In a: $2“ for act; 592+; however the enzyme e): divalent cat: at l x 10-3 l: rection. At 53% inhibitic and the emerge eqution was :e-ction from as ineffecti‘ ranging from “5 PH 6.1. mm; kid SuggESte; 3‘Cal'b0Xy1 gr aetivitYI bUt “as m . David F. Mann at different acrylamide concentrations. The purified enzyme was stable for several months when stored at 4°C or -90°C in 0.05 M potassium phosphate buffer (pH 7.0) which contained 50% sucrose (w/v) and 0.01 M_dithioerythri- , tol. In addition to the substrates, the enzyme required , Mg2+ for activity. Neither Ca2+ nor Ba2+ could replace ' 2+ 2+ 2+ Mg ; however, in the presence of Mn in place of Mg , the enzyme exhibited 28% of its normal activity. The 2+ 2+ 2+ 2 2+ divalent cations, Ni , Co , Cd , Fe +, and Pb , all at 1 x 10-3 M, nearly completely inhibited the enzyme reaction. At 1 x 10-4 M, Cu2+ and Zn2+ each effected 80% inhibition. The reaction had a 910 value of 2.6, and the energy of activation calculated from the Arrhenius equation was 17.5 Kcal. Quinolinic acid gave 50% pro- tection from heat denaturation of the enzyme, but PRPP was ineffective. The enzyme displayed a broad pH optimum, ranging from pH 6.5 to pH 7.7. Its isoelectric point t. was pH 6.1. Inhibition studies with analogues of quinolinic acid suggested that the enzyme needed both the 2- and 3-carboxyl groups on the pyridine ring for maximum activity, but that the carboxyl group at the 2 position was more important for effective competition. A series _ of reported and suspected inhibitors, nicotinic acid azalE‘JCir‘e’ ‘ fc: 15% inhiL meal plOts '3 tat the enz" 3:131 and FREE graiient cent tively. Elec iezo..strated i :zits with a r The 11 ransferase w the etiolated been plant. ' .18 (Le M 133 with a rapid 5 ms of Nico‘ \ :ev .e also shoe NV \0 Pathwa «'d L tOuacco ma A ‘ W ”he Tee w... David F. Mann azaleucine, had no effect on the enzyme activity except for 15% inhibition by NAD at l x 10--3 M. Double recip- rocal plots of the initial velocity studies indicated that the enzyme formed a ternary complex with quinolinic acid and PRPP. The molecular weight of the enzyme was estimated by Sephadex G-200 chromatography and sucrose density gradient centrifugation to be 68,000 and 72,000 respec- tively. Electrophoresis on SDS-polyacrylamide gels demonstrated that the enzyme was composed of two sub- units with a molecular weight of 35,000 each. The level of quinolinic acid phosphoribosyl- transferase was found to be loo-fold or more higher in the etiolated seedling than in the adult green castor bean plant. This large increase in enzyme activity in the dg 2912 pathway for NAD biosynthesis correlated well with a rapid surge in ricinine biosynthesis. Regenerated roots of Nicotiana rustica, which synthesizes nicotine, were also shown to have elevated levels of quinolinic acid phosphoribosyltransferase. The elevation of the Q2 2922 pathway for NAD biosynthesis in castor beans and tobacco may represent a universal manner in which plants that synthesize pyridine alkaloids compensate for the loss of the pyridine nucleus. '77" I I U... " -1 in Pa: ‘ WINOLINIC ACID PHOSPHORIBOSYLTRANSFERASE IN CASTOR BEANS By m“ 9. David 15‘:‘ Mann A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 -Otion. .3. fi’ . a_ Paul - IDIDICATED no I, ions andmy parents, Clyde and Jessie V and my wife, Dotty - .; ially: ".13. t . ilt' . Ln." éf “Ce-)2": I . . H Pellard, Paul 5’333estions a 1' esneciany encouragement Studl’ togethe. this thesis. ACKNOWLEDGMENTS "€C”"'NI wish to express my appreciation to Dr. Richard ‘3yeigyerrum for his counsel and guidance during this ‘ .3,‘ L.- E£€qestigation. I would also like to thank Drs. Clifford i.T W a "Iuw' » APaul Kindel and N. Edward Talbert for their '§§;--yestions and corrections they made in this manuscript. . L-l . J ‘ {fifiwgspecially, want to thank my wife, Dotty, for her ”.fihnguragement, tolerance, and help during our graduate 17.;filiyft69ether and for her many suggestions in writing ‘. \\-‘.l'\.’ {4. .4. I . - ,vfiv§“thesis. The financ1al assistance from the National & ’.\""p?. .ffi-‘Egtgtes of Health is acknowledged. , 4 . ' ‘65".1" . 0 .. (“’1 3'wtvclya:4. ‘ .‘IqHQIa‘t'rM‘ .IQIOLVia lvy‘C1h' n. _, iii 9:! v" h‘~ . ‘ Y‘T "h k, .‘iufl‘OfluLkG. iLo‘ m at? 01“ TABLE 115'? OF FISH? LIST OF ABBRL ZITEFATL'.£ Rz' UV"? “:6. :5me All; Chenicals Plants . Stbstrates Enzyme Ass: Paper Chro: Scintillat; reparatior omatc ryla: ”Olecular V FOtEin De: 1p' “lame De POIYac TABLE OF CONTENTS Page I I I I I I I I I I I I iii ACKNOWLEDGMENTS . LI ST OF TABLE S . O O O Q I I Q . C I I 0 v i f . LIST OF FIGURES I I I I I I I I I I I I I Viii |‘~ '. ' LIST OF ABBREVIATIONS . . . . . . . . . . . xi k. ' LITERATURE REVIEW . . . . . . . . . . . . 1 ”TERIALS MD METHODS O O O I O D O I D O O 2 1 ‘ :1 chmical s I O O I I O O I O 0 0 O I O 21 ' I l" , Plants I I I I I I I I I I I I I I I 23 i - 7. substrates I I I I I I I I I I I I I I 24 5 Enzyme Assays. . . . . . . . . . . . 28 - f. Paper Chromatography . . . . . . . . . . 32 ' Scintillation Fluids . . . . . . . 32 ¢ .- Preparation of Materials for Ion Exchange :‘ ."_. I. - ' V Chrmatography I I I I I I I I I I 3 3 ‘.» Polyacrylamide Gel Electrophoresis. . . . . . 34 ' 3' Molecular weight Estimation . . . . . . . . 35 Protein Determination . . . . . . . . . . 36 Ricinine Determination. . . . . . . . . . 36 ’{RESULTS. . . . . . . . . . . . . . . . 38 Purification of Quinolinic Acid Phosphoribosyl- .‘transferase . . . . . . . . . . . . 38 “gaflnzyme Isolation. . . . . . 38 Discussion of the Purification Scheme. . . . 45 Enzyme Stability. . . . . . . . . . . 49 Enzyme Purity. . . . . . . . . . . . 50 _ I ABSay Characteristics of Quinolinic Acid Phos- 1‘7;- phoribosyltransferase . . . . . . . . . 60 7 Metal Requirements . . . . . . . . . . 64 [{Product Identification. . . . . . . . . 67 iv pH Opt; Terpera Substra vat; Effect Effect Effect Acii Kinetic Physical C Holecul G-ZC Sucrose Electrai PhYSiOlog; Ricinin NiCOtin: Assa- Nicotin The Lew Acid' Devel PlanJ p'a‘rity hochemical Mich I pH Optimum and Isoelectric Point . Temperature Activation . . vation. . . . . Effect of Divalent Cations . . Effect of Quinolinic Acid Analogues Acid Phosphoribosyltransferase. Kinetics . . . . . . . . . i Physical Characteristics. . . . . } G- -200 Column. . . . i Physiological Studies. . . . . . I Ricinine Biosynthesis. . Assay . . . . . . . . . Nicotinamidase . . Developing and Mature Castor Bean Plants. . . . . . . . . DISCUSSION . . . . . . . . . . Purity . . . . . . . . Biochemical Properties . . . . . pH Optimum . . Protection Against Heat Inactivation. Inhibitor Studies . . . . . . . Kinetics . . . . . . . I Physical Characteristics. . . . . Physiological Characteristics . . . REFERENCES 0 O O I I C I O O O Substrate Protection Against Heat Inacti- Sucrose Density Gradient Centrifugation. Electrophoresis in SDS- Polyacrylamide Gels. Nicotinic Acid Phosphoribosyltransferase The Level of Quinolinic Acid and Nicotinic I Acid Phosphoribosyltransferases in Effect of Potential Inhibitors on Quinolinic Molecular Weight Estimation Using a Sephadex Page 68 74 74 80 84 84 89 89 95 98 98 104 105 113 126 128 131 132 132 133 135 135 136 142 LIST OF TABLES Page " 1. Purification of Quinolinic Acid Phosphoribosyl- transferase from Castor Bean Endosperm . . . 48 =e 2. Requirements for Quinolinic Acid Phosphori- ~ bosyltransferase Activity Purified from Castor Bean Endosperm . . . . . . . . 63 3. Quinolinic Acid Phosphoribosyltransferase Product Identification . . . . . . . . 71 4. Substrate Protection Against Heat Inactivation . 77 fi 5. The Effect of Various Cations on Quinolinic ‘ Acid Phosphoribosyltransferase Activity. . . 79 31 _ 6. The Effect of Substrate Analogues on the Rate A; of Nicotinic Acid Mononucleotide Formation. . 81 v3 7. The Effect of possible Inhibitors of Quinolinic m Acid Phosphoribosyltransferase. . . . . . 85 8. Kinetic Data for Quinolinic Acid Phosphoribosyl- {-. transferase as Determined from Initial - fr Velocity Studies in the Absence of Product. . 92 9. Requirements for Nicotinic Acid Phosphoribosyl- transferase Activity in Castor Bean Extracts . 106 10. Recrystallization of Nicotinic Acid to Constant Specific Activity . . . . . . . . . . 112 f.t9 11. Physiological Development of the Etiolated '- ,Castor Bean Plant Grown in Moist Vermiculite at 30°C I O D O O 0 C I . O I O C 116 '; 12. The Specific Activity of Quinolinic Acid Phos- ' phoribosyltransferase, Nicotinic Acid Phosphoribosyltransferase, and Nicotinamidase in Green Castor Bean Plants. . . . . . . 120 71 Oil ‘4 .‘l. @‘.=9~ t The S; L’ she: phc: 1n 5* The 58¢ phor Phcs dasc Specific Activity of Quinolinic Acid Phos- 'phcribosyltranaferase, Nicotinic Acid Phos— phoribosyltransferase, and Nicotinamidase manybeans and pracco . . . . . . . fl; .; gf§‘-:§hn Specific Activity of Quinolinic Acid Phos- .“ phoribosyltransferase, Nicotinic Acid TPhosphoribosyltransferase, and Nicotinami- Mdaae _in Leaves and Roots of Tobacco Plants. ’- Syntncs;s Fr 1:. . Slut LC "’I 1‘55? .’ ESQSETi 61" '1‘. Eiurion ' v i . (H. w). 1 . ‘21..“ Blutiru him]; ( ‘ 1; Poem. 'u 'POIYACrv.-uv . linik; ML“ . '. 1,. ’ .tl’w 120).. ". - 3? Densitomwtv: 7 Elect: pi":-= _ _ .Jf-by '. ‘.Z.‘:.'.' .r Minolta»: 1k. :3 2'.'. 511.3‘ " Pictivit‘, Eu): 1 a». L" (”T fiz' Ly": ." I \ .irL‘LL ”11.; '1'. g' “lyacrylar 1'. -".3n:.-= ...~ k_iwotein (Q; g;, en; g!r 1.1 a . '7 ,Iwelec t1. Focusum .-'-‘:' _ \ion of Nicotinic PC _d x.p'wn H.nn WWIJOJC‘ Acid PF: 052)} t.’ 1“."“112. 3 L: s \ nicotinamide adenine dinucleotide. This sequence was also found to be operative in plants (14), bacteria (15), fungi (16), and avian tissue (17) (see Figure 1). 159:. Th. 11...- i4 n43”. r, we.“ 9. I“ .w s e.” U k: .p 0.; a 1r: . y... ;: ,_ . m . :1: :1. 9.2.3.5; coda...“ waexx. t nutmeg 4. Cflomu...‘ .41.er r... .w , 53...». f vice“. 30mg. 32w; U, 35m. ch11»- .ukvmeah Rug...» 1...... .3... elm; my... .mtnumn. no“ puck}. can. 21053 man. 3. 2:14.551. .1. issuer awnwamxémwwum mac mnwubai .mha ”5.3.3... .3822? . :65. q. 3mm .mEmummm mmusu Ham on COEEOO ma >m3£uwm umavcmmlmmwoum may mmmnm£3 .mamfiflcm cw haze Uwumuumcoamv cmmn mm: mmznumm nowuumwa mna .mam>wuowmmmu munmwm can .mwumuomn .mHMEHcm cw canon :mmn m>mn U can .m .d mmmznumm o>oc we was .mwmmsuGMmOHn aéz mo mmmacumm mmnnp m£BII.H musmflm ocduoaauncun acacovz aUhAOUHahouxau + vwo< uwcfiuouaz tau: uwuuumn< oufiuooHuscun oc«:un< ocafiacwuoo«z ouuauom ¢ouquou< “no. unquoo~uaconox uufiuooauacoco: : cough-05m wnfiENCNuou«z taut uacfiuouwz.llllll.ku< vacaaocwso . 1‘ A 1,: + a m Eu: flunk: y<328<3E8oz uln 9322332 cunnoum>uh vau< owcwuouaz The: anthems . (U lc‘ a}. (18) 'r liver, were ziie mononuc and PRPP. '1 Capared to “F a ‘ 7.8;55 am H pnoribosyltr :etection Ur: $.- “rst had to an endogenou located in t cw “e“ylfltran mg (20), There are two other pathways involved in NAD bio— synthesis. The second route was found in 1965 by Dietrich 23 El (18) who, using an enzyme preparation from rat liver, were able to obtain the formation of 94¢] nicotina- mide mononucleotide from l}4q] nicotinamide, ATP, Mg2+, and PRPP. The Km for nicotinamide was 3.0 x 10-6 g as compared to a Km of 1.0 x 10-1 fl originally reported by Preiss and Handler in 1957 (19). The nicotinamide phos— phoribosyltransferase activity with the low Km had eluded detection until it was realized that enzyme extracts first had to be treated with protamine sulfate to remove an endogenous inhibitor. All the enzymatic activity was located in the cytoplasm. It is interesting that the other enzyme in this pathway, nicotinamide mononucleotide adenylyltransferase, was first reported in 1950 by Korn- berg (20). Hogeboom and Schneider (21) followed up the work on the Kornberg enzyme and showed by means of dif— ferential centrifugation that nearly all of the NMN adenylyltransferase activity was located in the nucleus. It has been just within the last few years that biochemists have found a possible role for NAD in the nucleus. Besides the normal oxidation-reduction reactions, NAD has been implicated in the control of DNA synthesis. Haines gt 31 (6) found that the activity of nuclear NAD glycohydrolase and its associated poly(ADP)ribose poly- merase was lowest in rat nuclei actively synthesizing DNA. this enzyme grateins was in rat live: The: the Phk'siolc i-‘a‘l the Diet Cr niCOtinar. 33"» nicotir. F‘In " . ‘ hwmnamme I~-tluated I he 41's and t1 0f EN This enzyme system cleaved NAD to form nicotinamide and ADP-ribose, which was then polymerized into chains of ADP-ribose, 25 residues or less long (7). Burzio and Koide (8, 9) showed that ADP-ribosylation of nuclear proteins was related to the inhibition of DNA synthesis in rat liver nuclei or chromatin preincubated with NAD. There is some confusion in the literature as to the physiological role in mammals of the Preiss-Handler and the Dietrich pathways, starting with nicotinic acid or nicotinamide respectively. Hayaishi gt gt (23) feel that nicotinic acid is a better precursor of NAD than nicotinamide as determined by injection of equimolar quantities (78 nmoles) of the EAT] labeled compounds into the portal vein of mice and measurement of the appearance of 94¢] labeled NAD in the liver. However, they found that large doses (82 to 164 nmoles) of nico- tinamide resulted in a higher incorporation into NAD than equal doses of nicotinic acid. A large per- centage of the injected nicotinic acid was excreted in the urine, whereas the nicotinamide was believed to be deamidated by intestinal bacteria over a period of several hours and the resulting nicotinic acid used as a source of NAD in the liver (24). On the other hand, Kaplan gt gt (25) and more recently Chaykin gt gt (26) demon- strated that nicotinamide rather than nicotinic acid was the more efficient precursor of NAD. As stated an”; refore, the the vitamin 7.26) 'njecte either nice: the normal ( veg'ing all 2 they were a‘; precursor cf 3.3313,, 111 mg I thesis of m. Vf‘v vv—v' before, the dietary as well as the circulating form of the vitamin is usually nicotinamide (22). Chaykin gt gt (26) injected mice interperitoneally with 900 nmoles of either nicotinic acid or nicotinamide. This is considered the normal daily intake of the vitamin in mice. By sur- veying all the body tissues rather than just the liver, they were able to show that nicotinamide was the only precursor of NAD in the spleen, skeletal muscle, kidney, ovary, lung, heart, and brain tissue and that this syn- thesis of NAD was via the Dietrich pathway. In the liver and intestine, however, nicotinic acid was rapidly con— verted to NAD and then to nicotinamide by the Preiss- Handler pathway. The nicotinamide thus formed from nicotinic acid could be used by the peripheral tissue as a precursor of NAD. These findings are supported by the earlier tt gtttg work of Greenbaum and Pinder (27), who showed that the Dietrich pathway was the only operable pathway for NAD biosynthesis in rat mammary gland. The first enzyme in both the Preiss-Handler path- way (nicotinic acid phosphoribosyltransferase) and the Dietrich pathway (nicotinamide phosphoribosyltransferase) as well as the microbial and plant nicotinamidases and the mammalian NAD glycohydrolase are subject to a variety of control mechanisms. Nicotinic acid phosphoribosyl- transferase from various organisms spans the gamut from dependency on ATP to independency of ATP. The enzyme €;?.~. A .l g» “oz- Asta 5 1e .6 “ vhile the e: PEEP (32, 3 e:.zy:e was with the co £3053 8. sub ltsande (3;; the ATP was oh» I Vauanisms . V 'wv from Astasia tgtgg was completely independent of ATP while the enzyme purified from bovine liver was activated by ATP and had a 10-fold lower K.m for nicotinic acid and PRPP (32, 33). The ATP used to activate the bovine liver enzyme was apparently stoichiometrically cleaved to ADP with the concomitant formation of nicotinic acid mono- nucleotide. Nicotinic acid phosphoribosyltransferase from t. subtilis and yeast required ATP for activity; Imsande (34) and Gholson gt gt (35) demonstrated that the ATP was stoichiometrically cleaved to ADP in both organisms. Dietrich and his co—workers (18) studied nico- tinamide phosphoribosyltransferase from rat liver and found that ATP was a positive allosteric effector of the reaction. Their enzyme synthesized NMN in the absence of ATP but only at elevated levels of PRPP and Mg2+. The Km for nicotinamide decreased from 1 x 10.1' g in the absence 6 of ATP to 3.0 x 10- g in the presence of ATP. Nicotina- mide phosphoribosyltransferase was inhibited 50% by 5 x 10-4 fl NAD. It would therefore seem that this enzyme is an important control point in the biosynthesis of NAD in mammals. Microbial nicotinamidase was also inhibited by NAD; however, the nicotinamidase from rabbit liver was not (70, 31). Nicotinamide itself had an inhibitory effect on mammalian nuclear NAD glycohydrolase and a ‘ }. «MALE r1- v- 1 ‘ I "‘D g‘. :‘reribosyltr first step i The Isre Physiol fmgi Which 34’ (28, 29’ 10- act as a niCOtinamidE 1“ ' .60 blos"nt rail" Wherea poly(ADP)ribose polymerase as well as rat liver micro- somal NAD glycohydrolase (37). It seems therefore that the pyridine nucleotide cycle is no exception to the phenomenon that the end product of a specific metabolic sequence (NAD) inhibits the enzyme (nicotinamide phos- phoribosyltransferase or nicotinamidase) catalyzing the first step in its formation. The Preiss-Handler pathway is probably of much more physiological importance in plants, bacteria, and fungi which have an active nicotinamidase sensitive to NAD (28, 29, 30). The nicotinamidase allows the cycle to act as a true salvage pathway which can convert the nicotinamide formed from the breakdown of NAD to nicotinic acid. The nicotinamidase in mammals had a very high Km for nicotinamide (1 x 10-2 g) (31) and probably is not very significant. From this and ta ytzg feeding experi- ments with nicotinamide (26), it would seem that the Dietrich pathway is of more physiological significance in mammals. Also, only two moles of ATP were needed for NAD biosynthesis from nicotinamide via the Dietrich path- way, whereas three moles of ATP were needed when starting from nicotinic acid via the Preiss-Handler pathway except in the case of Astasia tgtgg (32), where nicotinic acid phosphoribosyltransferase was ATP independent. The third route for NAD biosynthesis is the 9g novo pathway. As stated earlier, tryptophan was able to c .r“ fEC.&C€ n1- acid format pfan (39). Hayaishi (3 . E (LL) L C ( linic acid tr‘.~'§‘~0phan r mononucleot; nation, 5 10 replace nicotinic acid in the diet of certain mammals (11), but investigators were unable to detect any nicotinic acid formation in cell-free extracts incubated with trypto- phan (39). It was not until 1963 that Nishizuka and Hayaishi (39) using a rat liver homogenate found that (UL) PAC] 3-hydroxyanthranilic acid or (UL) [}4C] quino- linic acid (previously thought to be an end product of tryptophan metabolism) could form [}4C] nicotinic acid mononucleotide and 14 2 C02 in a PRPP and Mg + dependent reaction. This finding established nicotinic acid mononucleotide rather than nicotinic acid as the com- pound made from tryptophan. Nishizuka and Hayaishi (40) demonstrated that nicotinic acid was not an intermediate in the reaction and that the decarboxylation of the a-carboxyl group of quinolinic acid was probably a concerted reaction involving the formation of a positive charge on the quaternary nitrogen adjacent to the depart- ing carboxyl group. Soon after quinolinic acid was established as an intermediate in the conversion of tryptophan to nicotinic acid mononucleotide in mammals, quinolinic acid phosphoribosyltransferase was also found in plants and bacteria (41, 42). Earlier work by Henderson, Byerrum, and co-workers (43) had established that tryptophan could not serve as a precursor of the pyridine ring in plants. Further tg vivo work with tobacco plants by Byerrum gt gt .45 (lair-Or‘s'“a carbon coupon? 3.;tosphate. C free pIEPal'atL Lndicated two genesis in ‘ precursor to ‘ case from clihilI g3 (45) anaerobically formation of tetrahydrofol ration of the (64, 45). Pa anaerobe, Cl< acetyl CoA, . 131$ stud that it cool glycerol and C .or the bios Rene 0f the °f quinolin- Identif it at Qui is the one 11 (44) demonstrated that aspartic acid and some three carbon compound, probably glyceraldehyde or glyceraldehyde— L 3-phosphate, condensed to form the pyridine ring. Cell- free preparations from aerobic and anaerobic bacteria indicated two different pathways for quinolinic acid bio- synthesis in these organisms. Aspartic acid was a common precursor to both systems, but the other three carbons came from dihydroxyacetone phosphate in aerobically grown E. ggtt (45) and glycerol and N-formyl-L-aspartate in anaerobically grown t. ggtt (63, 64). The anaerobic formation of nicotinic acid in E. ggtt appears to be a tetrahydrofolate dependent reaction while aerobic for- mation of the pyridine ring is an FAD dependent reaction (64, 45). Partially purified extracts from the obligate anaerobe, Clostridigg butylicug, utilized aspartic acid, acetyl CoA, and formate to form quinolinic acid (46). tg'ytgg studies with yeast showed that it was unique in that it could use tryptophan under aerobic conditions and glycerol and aspartic acid under anaerobic conditions i for the biosynthesis of quinolinic acid (65). As yet none of the intermediates in quinolinic acid biosynthesis has been established. This work is hindered by low yields of quinolinic acid and a tedious assay procedure for the identification of quinolinic acid. Quinolinic acid phosphoribosyltransferase, which is the one enzyme common to the various gg novo pathways fa: RAD biosy: :3: d'fferent l is sole cartI 233‘ mutant (4 —. 1a"; a nolecul: centrifugatio. ———— inactive sub; 3C1. Further decarboxylati nucleotide wa- thistle to der. P?1v 'HStalline e Some cating the ex muting t2 bosyltransf e: integrated w ShSEpjed an "T *r] ”Sing ti" "iQEr 31:}, 2.5 x ' 12 for NAD biosynthesis, was purified from bovine liver by two different groups (40, 47) as well as from a pseudomo- nad mutant (48, 49, 50) which used quinolinic acid as its sole carbon source. The crystalline bacterial enzyme had a molecular weight of 165,000 as determined by ultra- centrifugation and could be irreversibly dissociated into inactive subunits of 54,000 M. Wt. using 4.8 M quanidine- HCl. Further proof of a concerted mechanism for the decarboxylation and formation of nicotinic acid mono— nucleotide was obtained when Packman and Jakoby (47) were unable to demonstrate any exchange reactions with their crystalline enzyme preparation. Some tentative evidence has been presented indi- cating the existence of possible control mechanisms modulating the activity of quinolinic acid phosphori- bosyltransferase, whereby the gg 2222 pathway would be integrated with the salvage pathway. Gholson gt gt (47) observed an 88% inhibition of activity with 1 x 10-3 M ATP using their purified bovine liver enzyme, and Had- wiger gt gt (41) observed a 77% inhibition of activity with 2.5 x 10'2 M ATP using crude preparations from castor bean cotyledons. Kahn and Blum (32) showed that nicotinic acid at 1 x 10.3 M inhibited quinolinic acid phosphoribosyltransferase by 33% in crude enzyme prepar- ations from Astasia longa. However, no inhibition with nicotinic acid was observed with the bovine liver enzyme g: the caste-r afore, nicct for activ 1:31 nechanis As ca ' o o rosyntne 51 s 133 ther€forq Ii‘jElY Sprea; 5.33 been £011. attire Cycle :i:e (26) , 13 g1. castor bean cotyledon preparation. As stated #2 ;:;;e, nicotinic acid phosphoribosyltransferase needed ‘fiégP for activity (33); therefore one could invoke a con- _fi;'trol mechanism for the two pathways based on ATP levels. j. As can be seen from the three pathways for NAD biosynthesis briefly outlined here and pictured in Pigure 1, it is a very complex process. To date the Dietrich pathway has been established only in mammals and therefore awaits further investigation to see how widely spread it actually is. The Preiss-Handler pathway has been found in most organisms studied, although the entire cycle is limited to the liver and intestine in mice (26). The gg gggg route uses tryptophan in mammals and is limited to the liver and kidney (40). In plants and bacteria, however, the 9g gggg route for NAD bio- gynthesis uses aspartic acid and various other three- catbon precursors to form quinolinic acid, which is then ”.1- 3 Vi. iggggele_but involves some loss of the pyridine nucleus. 55: ' ‘w‘giqqtinamide and nicotinic acid are excreted in man, 5;. .. ’ Plants also fc ni niootinazn: tailed trigom :‘ze much more £29, 52). Little gyridine nucl. :icotinanide fife atle to b Picnic and ac 14 .,,5¥:{; é also form N-methyl derivatives of nicotinic acid -’52§§d“nicotinamide; however, N-methyl nicotinic acid, also F called trigonelline, predominates, probably because of " the much more active nicotinamidase present in plants (29, 52). Little is known of the complete metabolism of the pyridine nucleus in plants as they cannot excrete excess nicotinamide as is the case in animals. Various bacteria are able to break the ring down to compounds like pro- pionic and acetic acid (72) or malic acid (73), which can then easily enter general metabolism again. Certain plants, however, use pyridine compounds in additional reactions to form pyridine alkaloids such as ricinine . ; in the castor bean plant (1 mg ricinine/gm fresh weight), . nicotine in the tobacco plant (5% of the dry weight of gicotiana rustica) and anabasine, also in the tobacco ‘2: plant. Free pyridine was found in the rayless goldenrod, _§plopappus hartwggi (Gray) Blake, where it made up 2.04% Qf;the dry weight (53). The reader should be aware, how- 21 ever, that alkaloids are not end products of plant 'nytnwmabolism. To the contrary recent work showed that :‘administered [}46] ring labeled ricinine was actively 14 K}: ,L qugfitabolized to CO2 in the castor bean plant (71). i "h. ; ji- 1&3 n... Two recent papers (29, 52) on NAD biosynthesis eotide cycle in plants. Using physiological amounts .iLL] 'r ‘ .2 L L lah'e‘ rerois that i ..: ' ‘ ' an. tnat .m ‘ \" '5 ' and —') NR1“ HR is that when the plan pyridine nucl tantrast to l were injecti anrease in t 'v‘C‘dd argue ‘ iosyntnesis the met ac: . d: the 16 Stan 15 30f [?4Q] labeled compounds, they established by tg ytgg methods that the Preiss-Handler pathway was operative and that NAD and NADP were broken down by way of NAD ————+ NMN -———+ Nicotinamide riboside ——-—> Nico- tinamide. Another important concept established in this work is that NAD and NADP levels remain constant even when the plant is given high nonphysiological doses of pyridine nucleotide precursors (52). This is in direct contrast to long-accepted results with rats and mice where injections of nicotinamide lead to a several fold increase in hepatic NAD concentrations (60). This finding would argue for the existence of tighter controls on the biosynthesis of NAD in plants than in animals. Constant pyridine nucleotide levels were also established for E. ggtt. Using an t. ggtt mutant lacking quinolinic acid phosphoribosyltransferase and therefore requiring nicotinic acid, Lundquist and Olivera (61) showed that when the media contained a large excess of 14C nicotinic acid, the level of the pyridine nucleotides remained con— stant. These experiments were, of course, performed at a certain stage of development or growth and should not be misconstrued to mean that the pyridine nucleotide pool is constant throughout development, for in fact it -is not. Yamamoto (62) showed that there were variations in the pool levels of NAD/NADH and NADP/NADPH during :ezration, g: inflower, cor: The co: 3; when coupl. acid, nicotine. :ne trip throu i‘fie and Scot tale? leaves. mation of E feeding 5 nmol “Wt of tisg acid was Co—fLI ”“1011 0f ml five 93% COR'I Czelline' whil fresh weight 360:) (52) Obt Eatever, “1823' Titles, of e: tide per 9r an incorporatio‘,‘ * I “1" n . .icotinic ac< is not; the“ 59 | an EXCellE I v—V'_ 16 germination, growth, and subsequent senescence in beans, sunflower, corn, watermelon, rice, and wheat. The constant NAD pool becomes even more interest- ing when coupled with the fact that the excess nicotinic acid, nicotinamide, and quinolinic acid (supposedly after one trip through the cycle) are stored as trigonelline. Ryrie and Scott (29) demonstrated this in their work with barley leaves. Normally they obtained a 4.9% incor- poration of E4C] nicotinamide into trigonelline when feeding 5 nmoles of this precursor per gram of fresh weight of tissue. However, when nonradioactive nicotinic acid was co-fed with the E46] nicotinamide a larger pro- portion of the label was funnelled into trigonelline. Five nmoles of nicotinic acid per gram of fresh weight gave 9.3% conversion of the PAS] nicotinamide to trig- onelline, while 25 nmoles of nicotinic acid per gram of fresh weight lead to 31.0% conversion. Godavari and Way- good (52) obtained similar results using wheat leaves. However, they routinely used a rather high dose, 55 nmoles, of either ENC] nicotinic acid or [14C] nicotina- mide per gram of fresh weight. This led to an average incorporation of approximately 80% of the labeled pre- cursor into trigonelline. These workers stated that nicotinic acid is toxic to plants while trigonelline is not; therefore trigonelline biosynthesis appears to ' be an excellent detoxification mechanism (62). Mothes 174) also 5qu 2:;ound less The i: :iie cycle am as first sugc posed that NP; gyridine alka ea‘idence rega nucleotide in even thongh a 1i3'95 themse‘. llCQtinamide) and niCOtine Ci labeled 33’cle (1.9. r adenine dinuc tide) ShOWed ricinine at 2 11a; 1”Pisults were Obtaine some WeStic Since it is have been tr inm (55 F Seve ricinine big 17 (74) also suggested that methylation generally makes a compound less reactive or "metabolically stabilized." The interconnection between the pyridine nucleo- tide cycle and the biosynthesis of ricinine and nicotine was first suggested by Leete and Leitz (54), who pro- posed that NADH might serve as a possible precursor in pyridine alkaloid biosynthesis. To date most of the evidence regarding the actual incorporation of a pyridine nucleotide into one of the alkaloids is circumstantial, even though all the precursors of the pyridine nucleo- tides themselves (quinolinic acid, nicotinic acid, and nicotinamide) are readily incorporated into ricinine and nicotine (41, 57, 67, 68). Waller gt gt (14) using [14é] labeled intermediates of the pyridine nucleotide cycle (i.e. nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and nicotinamide adenine dinucleo- tide) showed that these compounds were incorporated into ricinine at about the same level as nicotinic acid. Sim— ilar results using 94C] labeled NAD in tobacco plants were obtained for nicotine biosynthesis (75). There is some question regarding the meaning of these results, since it is doubtful that the pyridine nucleotides could have been transported across the cellular membrane intact (55, 56). Several jumping off points have been proposed for ricinine biosynthesis. Yang and Waller (58) suggested :2: possible :j;i:.clinic ac Lie” nicoti adenine dinnc linic acid in second route YiCLLS data sh :Lie undergoe :itrile group Slggested an 18 two possible pathways, the first of which would involve quinolinic acid and the pyridine nucleotide intermediates (i.e., nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide) resulting from the entry of quino- linic acid into the pyridine nucleotide cycle. The second route would begin with nicotinamide because pre— vious data showed that the carboxyamide group of nicotina— mide undergoes intramolecular dehydration to form the nitrile group of ricinine (57). Hiles and Byerrum (59) suggested an alternative route for ricinine biosynthesis based on results from competitive feeding studies using [}4é] quinolinic acid and a ten-fold molar excess of non- radioactive NAD. The assumption made was that if NAD were an obligatory intermediate, the [}4C] quinolinic acid incorporated into ricinine should be diluted by the NAD. Their results showed, however, that when [}4S] quinolinic acid was co-fed with NAD, there was in fact a three-fold increase in incorporation of the quinolinic acid into ricinine. They therefore suggested that quinolinic acid need not go through the pyridine nucleo- tide cycle at all to be incorporated into ricinine. Waller gt gt (14) performed some experiments to assess the efficiencies of nicotinic acid and quinolinic acid as ricinine precursors. They found that quinolinic acid incorporation into ricinine was twiCe that of nicotinic acid or nicotinamide, all administered at the are level of 53:1: was f2 :ize-fold exce fed with four 340% decreasi minine, sug' the label fro: That also did IZIIEY‘SIX LI.“ I 5:3: .noles c iiis case the :jsinolinic acli "35 converteé When fed with that qUinolir acid; this i; 33:18}? and \t' tri‘SOIl‘EIline Plants, then “name to d i :‘lcn of the Title. Thes shorter time aimlinic d Yang 8‘ . sperm“ 1 19 same level of 600 nmoles per 7-day-old seedlings. This finding was followed with co-feeding studies. When a nine-fold excess of nonradioactive quinolinic acid was fed with four nmoles of [?49] nicotinic acid, there was a 40% decrease in nicotinic acid incorporation into ricinine, suggesting that the quinolinic acid diluted the label from the [?4S] nicotinic acid entering ricinine. They also did the converse experiment, that is of feeding thirty-six nmoles of nonradioactive nicotinic acid with four umoles of P49] quinolinic acid per plant. In this case they obtained 4.9% incorporation of PACJ quinolinic acid into ricinine and 5.4% incorporation when fed with the nicotinic acid. These results indicate that quinolinic acid is used in preference to nicotinic acid; this is not incongruous with the data from the barley and wheat experiments where excess nicotinic acid was converted to trigonelline. If this conversion to trigonelline had been occurring in the castor bean plants, then the co-fed nicotinic acid would have been unable to dilute the labeled quinolinic acid, since much of the nicotinic acid may never have entered the cycle. These experiments should be repeated using shorter time periods and physiological quantities of quinolinic acid and nicotinic acid. Yang gt gt (58) published a very interesting experiment in 1965 in which they showed that the t :e::entage 0. ' TI .1 § c] nicoti: increased. C aficient pre- —_—_— niicates tn; ticsynthesis . gated a poss azwo- or tn: :2 hnorpora Prel; "m quinoli: Wine the: be“ “er under; in E‘lants an 20 percentage of labeled ricinine decreased as the amount of ENC] nicotinic acid or [14C] quinolinic acid fed increased. Quinolinic acid again served as the more efficient precursor at all levels tested. This experiment indicates that there are definite controls on ricinine biosynthesis. Recent work by Johnson gt gt (69) sug- gested a possible feedback control. They found that a two- or three-fold excess of unlabeled ricinine decreased the incorporation of labeled quinolinic acid into ricinine. Preliminary evidence in our laboratory indicated that castor beans had considerably more quinolinic acid phosphoribosyltransferase than nicotinic acid phosphori— bosyltransferase. This might help explain the reason that quinolinic acid served as a better precursor of ricinine than did nicotinic acid. In order to obtain a better understanding of the pyridine nucleotide cycle in plants and of quinolinic acid incorporation into ricinine, the purification and characterization of quinolinic acid phosphoribosyltransferase were under- taken. It was also decided to establish the level of several of the key enzymes in the pyridine nucleotide cycle to determine their influence in channeling pre- cursors into ricinine. We a; nicotinic ac adenine dim following CCj ”“195 With Me Me .ent gra part the pu Chemicals . adEnine dinucleotide. Dr. Hiles also 5 following compounds which were used in Methyl 3-carboxypyridine-2-ca 3-Cyanopicolinic acid 3-Amidopicolinic acid 4-Hydroxyguinolinic acid 2-Hydroxynicotinic acid 3-Cyanopyridine-2-carboxylate ‘4 vIntertwine purest available. : 13h Chemical Com-an L2,4 -Pyridinedicarboxylic acid “.3fé-Pyridinedicarboxylic acid asfi-Pyridinedicarboxylic acid 5-Pyridinedicarboxylic acid igfiydroxy quinoline 21 MATERIALS AND METHODS We are indebted to Dr. Richard Hiles who made the nicotinic acid mononucleotide and the nicotinic acid ynthesized the the-inhibitory .4 studies with quinolinic acid phosphoribosyltransferase. rboxylate Methyl 3-amidopyridine-Z-carboxylate Methyl 3-cyanopyridine-Z-carboxylate L“ The other chemicals used in this research were reagent grade. Co-factors and enzymes were for the most 'Nicotinic acid Nicotinamide Trigonelline Picolinic acid war/Sea. ..... a.“ Amersham[Searle Corporation E-14C1Quinol inic ac id New England Nuclear K & K Laboratories, Inc. E-I4C]Nicotinic acid DL-4-aza1eucine di HCl fi-14C1Nicotinamide 14 Kontes Glass Compagy L [ C] Aniline-HCl 4 Rubber septa P CJBaCO3 Center well (for CO2 94d Toluene (standard) collection) ggckard Instrument Company! Inc. Hydroxide of Hyamine lO-X, p-(diisobutylcresoxyetho— xyethyl) dimethylbenzylammonium hydroxide POPOP, 1,4-bis-2(4-methyl-5—phenyloxazolyl)-benzene (scintillation grade) PPO, 2,5-diphenyloxazole (scintillation grade) BBOT, 2,5-bis [2-(5-tert-buty1benzoxazolyl)]-thiophene (scintillation grade) P-L Bioctgmicals, Inc. Adenosine 5'—triphosphate Nicotinamide-adenine dinucleotide Nicotinamide-adenine dinucleotide phosphate S-phosphoribose 1-pyrophosphate (magnesium salt) Canalco gtgrmacia Fine Chemicals, I_ne- Acrylamide N,N'-methylene-bis-acrylamide Sephadex G-200 TEMED, N,N,N',N'-tetramethy1 DEAE Sephadex A-50 ethylenediamine Blue Dextran 2000 Ammonium persulfate Mallinckrodt Chggical Works Clarkson Chemical Company Sodium dodecyl sulfate 95% Hydroxyapatite C Toluene Sigga Chemical Company Bovine serum albumin Ovalbumin Dithioerythritol a-chymotrypsinogen Sire Chenic Quinolir Alcohol Catalase HES, Z-I fonic 'EPES, l zine-l Bicine, glycir Patheson Col \ Naphthal Sand (5: 23 Sigma Chemical Company (cont.) Quinolinic acid Diaphorase (pig Alcohol dehydrogenase heart) Catalase Acid phosphatase MES, 2-(N-morpholino) ethanesul- Snake venom phos- fonic acid phatase HEPES, N—2-hydroxyethylpipera- zine-N'Z-ethanesulfonic acid Bicine, N,N-bis(2-hydroxyethyl) glycine Eastman Kodak Company p-Dioxane gathgson Coleman & Bell Naphthalene Sand (Standard Ottawa) Plants Ricinus communis L., variety Hale, was selected as the biological material used in most of this thesis. These seeds were very graciously supplied by Mr. Walter Domingo, Director of the Oil Seeds Production Division of the Baker Castor Oil Company, La Mesa, California 92041. The seeds were treated with a 10% solution of .‘. Orthocide (50% N-[(trichloromethyl)thio]-4-cyclohexene-l, 2-dicarboximide, "captan") and germinated in moist ver— miculite for four or five days at 30°C. The flats were tightly covered with aluminum foil. Etiolated seedlings of approximately the same physiological age were used throughout (i.e., the endosperm was firm and the secondary root structure was well developed). The t the roots we: jusr prior t: regenerate ac. :11 other pl: unless other Late sized by the the Skraup s densed with exidiZed to raflganate 01-' “Te added j 24 The tobacco plants used were Nicotiana rustica L. The roots were removed from plants grown in the greenhouse just prior to flowering and new roots were allowed to regenerate according to the method of Byerrum et 31 (43). All other plants were grown under standard conditions unless otherwise stated in the text. Substrates Labeled [2,3,7,8-14C] quinolinic acid was synthe- sized by the method of Fleeker and Byerrum (79). Using the Skraup synthesis (80), (UL) E4C] aniline was con- densed with glycerol to form quinoline, which was then oxidized to quinolinic acid by means of an alkaline per— manganate oxidation (see Figure 2). The following reagents were added in order: 87 mg of pulverized FeSO4-7H20, 0.84 ml of anhydrous glycerol, 227 mg redistilled aniline, 3.23 mg (UL) [14c] aniline (0.25 mCi), 0.18 ml redistilled nitrobenzene, and 0.48 ml concentrated H2504. The flask, equipped with an air-cooled reflux condenser, was placed in a 140°C Wood's metal bath for 6 hr. The reaction mixture was then steam distilled to remove all the nitro- benzene. After the addition of 9 ml of 40% KOH, the reaction mixture was steam distilled again. This time the distillate, which contained the 94C] quinoline, was collected in 0.1 g NaOH and 150 ml of 5% KMnO4 were added dropwise. The flask with the KMnO4 solution was 25 I ¢<‘< (It‘d: I Eoum pwom owcflaocwdv HUv H Im.b.m.NH (( C .mcwawcm mu mo mwmwnucw HH AADV can Houmomam mnu.~ magmas 26 3.82 -0: u... 204 25.83 2:230 2:24 .285 28.8052 .. N .. z .. z - .N .8... * _ r $ um i AAVI .. Al -. .. zoom. .25 . 6%: .. .. . zoom: “:3 in a steam ty filtrati excess mm in; the sol 118 (so-10: 27 fitted with a water cooled reflux condenser and placed in a steam bath for 16 hr. The MnO2 formed was removed by filtration; absolute ethanol was used to convert excess KMnO4 to MnOZ, which was again removed by filter- ing the solution. The solution was applied to a Dowex 1X8 (50-100 mesh) formate column (2.7 cm i.d. x 30 cm) and the quinolinic acid was eluted with a 1000 m1 linear formic acid gradient (0.0 to 0.4 g). The quinolinic acid fraction was evaporated to dryness on a rotary evaporator and recrystallized giving a 31% yield. The [2,3,7,8-14C] quinolinic acid contained 117 dpm per nmole. The synthesized product was 100% radiochemically pure as determined by autoradiography and co-chromatography with standard quinolinic acid in three solvent systems (A,B,D). The a-carboxyl group contained one-quarter of the total radioactivity as determined by decarbox- ylation technique of Scott gt 31 (81). The other radioactive substrates, [6-14C] quino- linic acid, [7-14C] nicotinic acid and [7-14C] nicotina- mide, were obtained commercially and checked for purity by co-chromatography on Whatman No. 1 paper in two dif- ferent solvent systems (A and B). The magnesium salt of PRPP was obtained from P-L Biochemicals and used in the characterization studies whereas the sodium salt of PRPP (90% purity) was obtained from Sigma Chemical th Company (for 1/26 the cost of the magnesium salt) and used in cation. End :ave 10% nor 1 Quii aczivity was! 14 502 relea: nicotinic a: Cixture for M ml, the Si‘ren: 0.5 PhCSPhate ( fiinclinic reaction fj aPO‘iYethy hlldrox ide 28 and used in the assay procedures throughout the purifi- cation. Under comparable conditions the magnesium salt gave 10% more activity than the sodium salt. Enzyme Assays Quinolinic acid phosphoribosyltransferase activity was determined either by quantitating the 14CO2 released or by measuring the formation of E-14C] nicotinic acid mononucleotide. The standard assay mixture for the enzyme contained, in a total volume of 0.8 ml, the following reagents at the final concentrations given: 0.5 to 5 milliunits of enzyme;* 115 m§_potassium phosphate (pH 7.0); 12.5 MMgCl 0.187 m_M [2,3,7,8-14c] 2. quinolinic acid and 0.375 mg PRPP. The 25 ml erlenmeyer reaction flasks were equipped with a rubber septum and a polyethylene hanging well which contained 0.2 m1 of hydroxide of hyamine lO-X and a folded paper wick. The flasks, minus the PRPP, were equilibrated for 5 min with shaking in a 30°C water bath; the reaction was started by the addition of PRPP. After 20 min, the reaction was stopped by the addition of 0.3 m1 of 10% perchloric acid through the rubber septum. To insure complete 14 absorption of the CO2 by the hydroxide of hyamine, * A unit of enzyme activity is defined as that amount of enzyme needed to catalyze the formation of l Lunole of product per min under the given assay con- ditions . yell was t the scinti sgectrcrnet four or fi :rometer. has initia results in with a wic 29 the flasks were allowed to stand for one hr. The entire well was then placed in a counting vial with 6 m1 of the scintillation fluid A and placed in a scintillation spectrometer. The vials were counted after a period of four or five hr equilibration in the scintillation spec- trometer. This assay procedure using hydroxide of hyamine was initially checked using standard [F4C] BaCO The 3. results indicated that 0.2 m1 of hydroxide of hyamine with a wick was sufficient to quantify up to 200 nmoles of C02. The results also indicated that one hr was ample time to allow for complete absorption of the released 14C02. The second, more sensitive, assay procedure measured the formation of nicotinic acid mononucleotide using paper chromotography. The assay mixture.contained the following reagents at the final concentrations given in a total volume of 0.2 ml: 115 m§_potassium phosphate (pH 7.0), 5 m§_dithioerythritol, 12.5 m§_MgC12, 0.375 3%. PRPP, and 10 to 30 microunits of enzyme. The reaction was started by adding 0.3 mfl_[§-14C] quinolinic acid (4.15 uCi/umole) and it was allowed to proceed for 2 hr at 30°C. The reaction was stopped by heating the flask in a boiling water bath for one min. Unlabeled quinolinic acid (1.6 nmoles) was added to each assay to serve as a chromatographic standard and the protein was removed by centrifugation in a clinical centrifuge for 33in. Appr spotted on :tromatogra‘ 13,.36) was the [14C] 1 (R, .14), a by scanning DatOgram Sca COIIespOndi: Cit and {313 'I'ith Sc inti quinolinic was detemj 310nm, as: if Cpm (pr: Since the acid uSed ZOHOnuCleC \Mtr 30 3 min. Approximately 10 ul of the assay mixture were spotted on Whatman No. 1 paper and developed by descending chromatography with solvent system A. Quinolinic acid (Rf.36) was detected under a mineralight uvs-ll lamp; the PACJ labelled nicotinic acid mononucleotide (Rf .14), as well as the quinolinic acid, were located by scanning 1.5 inch wide strips on a Packard Radiochro- matogram Scanner. Three-inch sections of the strips corresponding to the two radioactive peaks, were cut out and placed in counting vials. The vials were filled with scintillation fluid B and counted. The percent quinolinic acid converted to nicotinic acid mononucleotide was determined by dividing the cpm, corrected for back- ground, associated with the product by the total number of Cpm (product and quinolinic acid) on the paper strip. Since the specific activity of the labeled quinolinic acid used and the percent conversion to nicotinic acid mononucleotide were known the amount of product could be calculated. Nicotinic acid phosphoribosyltransferase was assayed by measuring the formation of [}4C] nicotinic acid mononucleotide from [7-14C] nicotinic acid. The assay mixture contained the following reagents at the final concentrations given in a total volume of 0.2 ml: 25 mg potassium phosphate (pH 7.0), 4 m§_dithioerythritol, 20 mM.tris-potassium phosphate (pH 8.0), 60 m§_ATP, 1.24 r11 PRI‘ reaction we with shakin by heating Standard ni were added removed by Product in in order tc Bubnucleot were treat Cf quinolir Nit in? the fol niconnami: ing reaQEn Vclm’e of 7.3 V . L dit, 0‘ ‘ ehZYfie, 0'7311'4 [j allOwed t»] N] 30°C. 31 1.24 mg PRPP, and 10 to 30 microunits of enzyme. The reaction was started by adding 0.148 mfl_[7-14C] nicotinic acid (6.8 uCi/umole) and was allowed to proceed for 2 hr with shaking in a 30°C water bath. The assay was stopped by heating the flask in a boiling water bath for one min. Standard nicotinic acid and trigonelline (4 nmoles each) were added to the assay mixture before the protein was removed by centrifugation. Trigonelline was a side product in this reaction and had to be accounted for in order to determine the percentage of nicotinic acid mononucleotide formed. From this point on, the assays were treated in the same way as the chromatographic assay of quinolinic acid phosphoribosyltransferase. Nicotinamidase activity was determined by measur- ing the formation of [7-14C] nicotinic acid from [7-14C] nicotinamide. The reaction mixture contained the follow- ing reagents at the final concentrations given in a total volume of 0.15 ml: 36 mg_potassium phosphate (pH 7.0), 7.3 m§_dithioerythritol, and approximately 100 microunits of enzyme. The assay was started by the addition of 0.73 m§_[}-14C] nicotinamide (5.13 uCi/umole) and was allowed to proceed for one hr in a shaking water bath at 30°C. The assays were stopped by heating the flasks in a boiling water bath for two min. Standard nicotinic acid and nicotinamide (4 umoles each) were added to the assay mixture and the protein was removed by centrifugat suture wa: 21 descend assay mixt‘ ninolinic De 'a'ith sever 95% (V/v) l'butan01- (5H . 4)ZSO4 . (1386.8), . 32 centrifugation. Approximately 10 ul of each assay mixture was spotted on Whatman No. 1 paper and developed by descending chromatography with solvent system C. The assay mixtures were then treated the same as those for quinolinic acid phosphoribosyltransferase. Paper Chromatography Descending paper chromatography was performed with several solvent systems: (A) l-butanol-acetic acid- water (4-1-2 v/v); (B) 1 g ammonium acetate (pH 7.0)- 95% (v/v) aq. ethanol (3-7 v/v); (C) upper phase of l-butanol-acetone-water (9-1-10 v/v); and (D) 600 gm (NH4)ZSO4 per liter of 0.1 g potassium phosphate (pH 6.8), plus 2% (v/v) l-propanol. Scintillation Fluids Three scintillation fluids were used in this work. A: This toluene-based scintillation fluid was used to quantitate 14 CO2 trapped in hydroxide of hyamine and was prepared by adding 1.265 gm of POPOP and 19.0 gm of PFC to 3.79 liters of toluene. B: This scintillation fluid was used to count paper strips and contained 4 gm of BBOT per liter of toluene. After counting, the paper strips were removed and the vials of fluid were recounted. If the fluid was not contaminated (i.e. if the radioactivity equalled the background) it was reused. C: Aqueous samples (0.5 ml) were counted in Bray's scin" aiiing 289. 3.7225 gm 0 samples wer s;ectronete Die size) was s N - The in two Volu 3to 10 min m Siphone the gel Was 0'05 :1 POta 2~L-lerca‘Ptoe t'n'iCe dail) Hyc Packed in ‘ Ethan 33 Bray's scintillation fluid (5.0 ml) which was made by adding 289.8 gm of naphthalene, 28.98 gm of PPO, and 0.7225 gm of POPOP to 3 kgm of p-dioxane. All the samples were counted in a Packard Tri-Carb scintillation spectrometer. Preparation of Materials for Ion Exchange Chromatography Diethylaminoethyl Sephadex (A-50, 40-120u particle size) was suspended in water and allowed to swell for 24 hr. The resin was "defined" by suspending the gel in two volumes of water and allowing it to settle for 8 to 10 min after which time the material still suspended was siphoned off. After repeating this process 10 times, the gel was allowed to equilibrate with 10 volumes of 0.05 M potassium phosphate (pH 7.0) containing 0.01 M 2-mercaptoethanol for two days. Fresh buffer was added twice daily. Hydroxyapatite C (modified calcium phosphate) packed in 0.001 M phosphate buffer was suspended in 10 volumes of 0.05 M_potassium phosphate (pH 7.0) containing 0.01 M_2-mercaptoethanol. The buffer with no 2-mercapto- ethanol was changed twice daily for three days to ensure complete equilibration. Dowex 1X8 (50-100 mesh) was converted from the chloride form to the formate form by washing the resin with 40 volumes of 3 M ammonium formate (or until no :‘aloricie u solution c use by was EXCESS am T3 :5- ...lowed 1 Prepared L A :J (D *1 34 chloride was eluted as determined with a 1% (w/v) solution of AgNO3). The resin was then made ready for use by washing it with deionized water to remove the excess ammonium formate. Polyacrylamide Gel Electrophoresis The method of Ornstein and Davis (88, 89) was followed for disk gel electrophoresis. The gels were prepared using four stock solutions made in the following manner: solution A consisted of 48 ml 1 §_HC1, 36.6 gm tris, and 0.23 ml TEMED; solution B consisted of 28 gm acrylamide and 0.735 gm N,N'-methylenebisacry1amide; solution C consisted of 0.14 gm ammonium persulfate; solution D consisted of 48 ml 1 §_HC1 and 5.98 gm tris. A standard 7% gel was made by mixing 2.5 ml solution A, 5.0 ml solution B, 10 ml solution C, and 2.5 ml water. The gel solution was transferred to glass tubes (9.0 x 0.5 cm) and a small amount of water (3 mm) was layered on top of each gel. Samples for electrophoresis contained 12 pl solution D, 20% glycerol, protein (10 to 50 pgm) and water to a final volume of 100 ml per gel. The elec- trode buffer, pH 8.3, contained 0.605 gm tris and 2.85 gm glycine per liter of water. The anode was at the bottom of the apparatus. Electrophoresis was carried out at 3 mA per tube using bromophenol blue 35 as the tracking dye. Gels were stained in amido schwartz (2 gm amido schwartz, 250 ml water, 250 ml 95% ethanol, and 10 ml glacial acetic acid) and destained in 7% acetic acid or they were stained in Coomassie blue (0.05% in 12.5% trichloroacetic acid) overnight and destained in 10% trichloroacetic acid. Gels were assayed for enzyme activity by sectioning them with a Canalco gel slicer into 1.4 to 1.6 mm sections or slicing by hand with a razor blade into 8 to 9 sections per cm. Slices were immediately dropped into a test tube containing the regular assay components for the CO2 assay for quinolinic acid phos- phoribosyltransferase. The reaction was started by the addition of PRPP and the assays were allowed to proceed for 45 min at room temperature. The isoelectric focusing experiments were done with a LKB 8100 electrofocusing column of 110 ml capacity. The carrier ampholytes were in the 3-10 or the 5-7 pH range. The procedure for running the column was that specifically recommended for the LKB apparatus (86). Molecular Weight Estimation The estimated molecular weight of quinolinic acid phosphoribosyltransferase was determined by chromatography on Sephadex G-200 by the method of Andrews (83). Another estimate of the molecular weight ‘u—sngb was determin the method 0 SSS-nolyac ry gracedure of Prot of Lonny E in more pur ‘Jle ahsorba 5P€Ctr0phot {77), Pro “0 or 20: Equipped wi exclusion l IQCOVered e solution 12'. 36 was determined by sucrose gradient centrifugation using the method of Martin and Ames (84). Electrophoresis in SDS-polyacrylamide gels was performed according to the procedure of Shapiro 23 a1 (85). Protein Determination Protein determinations were done by the method of Lowry gt_31 (76) on crude fractions while the protein in more purified preparations was determined by measuring the absorbance values at 280 and 260 nm with a Hitachi spectrophotometer by the method of Warburg and Christian (77). Protein solutions were concentrated using either a 10 or 200 m1 capacity Amicon Ultrafiltration cell equipped with a PM-lO filter (10,000 molecular weight exclusion limit). At least 98% of the activity was recovered each time after concentration of the protein solution by this method. Ricinine Determination Ricinine was extracted from castor beans by homogenizing the entire plant in a Sorvall Omni-mixer with 5 ml of hot water (: 80°C) per gram of tissue for two min at top speed. The homogenate was then filtered and the residue was re-extracted. The combined filtrates were extracted with 1/4 volume of ether at least three times, or until no more lipid could be removed. The aqaeous pha rotary evap in either a (5CD ul) wa 8.5 in. , an i:- solvent '~.'isual ultr Leno and th 1" R of st f SPin-thimbl The eluted 101111 and t 260 “m with ricinine wa Standard r1. isolate the COCpQunds V F01 nu: leotide Of Ames an. OICinol (8 was deterrl 37 aqueous phase was concentrated almost to dryness on a rotary evaporator and then diluted to volume with water in either a 10 or 25 ml volumetric flask. The material (500 ul) was streaked on Whatman No. 1 paper, 22.5 x 8.5 in., and the chromatogram was developed overnight in solvent system A. The chromatograms were examined by visual ultra violet quenching with a Mineralight UVS-ll lamp and the portion exhibiting a fluorescent band at an Rf of standard ricinine was eluted with water using spin-thimbles (Reeve Angel Company, New York, N.Y.). The eluted ricinine was adjusted to a final volume of 10 m1 and the absorbance of an aliquot was measured at 260 nm with matched quartz cuvettes. The amount of ricinine was determined from a Beer's Law plot of standard ricinine. By using paper chromatography to isolate the ricinine, interfering ultraviolet absorbing compounds were eliminated. For the identification of nicotinic acid mono- nucleotide, phosphate was determined by the micro method of Ames and Dubin (81) and ribose was measured with orcinol (82). The NAD content of the plant tissue was determined by the method of Yamamoto (62). ions were Castor bea bucket. T distilled CEdures We ground wit RESULTS Purification of Quinolinic Acid Phosphoribosyltransferase Enzyme Isolation Endosperm tissue (120 gm) from which the cotyle- dons were removed was harvested from etiolated 5-day-old castor bean seedlings and immediately placed in an ice bucket. The endosperm tissue was weighed, rinsed with distilled water, and blotted dry. The remaining pro- cedures were carried out at 4°C. The tissue was first ground with sand in a cold mortar using two volumes of 0.05 M_potassium phosphate (pH 7.0) containing 0.01.M 2-mercaptoethanol. The crude extract was strained through four layers of cheese cloth and then centrifuged at 27,000xg for 20 min. The supernatant fraction, which contained the enzyme activity was adjusted to pH 7.0 with 0.40 M_NaOH-glycine (pH 10.0). The enzyme extract was then made 1 mM_in quino- linic acid with 0.08 M quinolinic acid and allowed to equilibrate for 10 min in the ice bucket. The enzyme, 100 m1 at a time, was heated in a 1 liter flask which was swirled in an 85°C water bath until the solution 38 reached 60 a EC°C wat ss'irlin was remove and the 5;; against fo remove the T2:- retoved by $221719 pre Sephadex ( quinOlinic r53 'v'ed by 2‘;ffer_ T 1:19 fracti 39 reached 60°C. The flask was immediately transferred to a 60°C water bath, incubated for one min, and then cooled by swirling in an ice bath. The precipitated protein was removed by centrifugation at 27,000xg for 15 min and the supernatant was dialyzed overnight at 40°C against four 4 liter portions of grinding buffer to remove the quinolinic acid. The precipitate which formed during dialysis was removed by centrifugation at 27,000xg for 15 min. This enzyme preparation (280 ml) was then placed on a DEAE Sephadex (A-SO) column (2.7 cm i.d. x 16.0 cm) and the quinolinic acid phosphoribosyltransferase activity was removed by eluting with approximately 800 m1 of grinding buffer. The elution pattern may be seen in Figure 3. The fractions from the column were assayed for quinolinic acid phosphoribosyltransferase activity as described in the methods section and protein was determined by 280/260 nm readings (77). Those tubes with the highest specific activity were pooled and concentrated to 30 ml using an Amicon Ultrafiltration apparatus equipped with a 62 mm PM-lO membrane. The concentrated enzyme was then placed on a hydroxyapatite C column (1.7 cm i.d. x 16.5 cm), washed with grinding buffer and eluted from the column with a 900 m1 linear gradient of 0.05 to 0.225 M_potassium phosphate (pH 7.0). The flow rate was approximately 4O —| - 1.1.x): :(HH.-C AOIIIIOV muw>fiuom oEMNcm LIV cwououm .poumnucmocoo pom pmaoom mums >uw>wuum owmwomdm swan m can scans Awninmmv mGOfluomum omega .pwuomaaoo mums mcofiuomnm HE Ha can an Hod HE vv mm3 mumu 30am one .Ahnv cmflumauno can museumz mo ponuos on» ma chAuMGwEumump samuoum £0fl3 pmumwnmusfl scans anemones sodas» pwcflmusoo ow Monaco 0» uoflum mcowuomum one .coHuoon come no muooowam HE ~.o msfims coauomm moonume on» GM ponwuommp mo pmmmmmm mm3 wfihncm one .sEsHoo Romnmv xopmnmmm mdma Housman m Scum wmmuommcmuu lawmonwuonmmonm owns UflsHHOCHsv mo mHfiMOHm coausamnl.m ousmwm 41 .3832 c0782... om cm 9 om 8 CV Om ON 0. I _ _ _ i H mo noon w. m. U m, o._ .. i009 / w. B I 00m. 51 :82, 5:5 in oE>~cmi (de) um: 02/300,, After ass. :hcribosy as before :Vgnb muions dialyzed . solution amired . T utilized CClumn Ca a PH grad was PIEpa with 50111 COStained CCntained filmine S was $leDst middle th we . 42 30 ml per hr and 8 ml fractions were collected. Figure 4 shows the elution pattern from this column. After assaying the fractions for quinolinic acid phos- phoribosyltransferase activity and protein concentration as before, those fractions (#55-68) having the highest specific activity were pooled. An Amicon Ultrafiltration apparatus was again used to concentrate the pooled fractions. The concentrated enzyme (6 ml) was then dialyzed overnight against 600 volumes of 1% glycine solution (pH 7.0) to remove excess potassium phosphate acquired during elution from the hydroxyapatite column. The last step in the purification procedure utilized an isoelectric focusing column. For this column carrier ampholytes (LKB) were used to establish a pH gradient from 5-7. The stepwise sucrose gradient was prepared by dilution of 24 aliquots of solution A with solution B to a final volume of 4.6 ml. Solution A contained 47% sucrose and 1.25% ampholyte and solution B contained 0.4% ampholyte. The enzyme (6 ml) in 1% glycine solution (pH 7.0) from the hydroxyapatite column was substituted for an equal amount of solution B in the middle three fractions. The voltage was increased step- wise over a three-hour period to 700 volts and was held there until the current had stabilized around 0.50 mA (44 hours). Then the column was slowly_drained and 2 ml fractions were collected and assayed for quinolinic acid 43 AOIIOV eufifiuoo ofihusm LIV aeouonm .onos csonm omonu ou HoHeEeo muasmon £¢e3 poms mo3 A80 o.~v x .p.e EU e.~v sEdHoo ouwuomoexoupmn o .poaoom oHo3 msEsHoo xoposmom mdma Houo>om conz omoo onu mo3 mo .ponmoumouoeouno on on onoz cfiououm mo mussoeo Homuoa cos& .ath cowumeuno poo musnuox mo pocuoE onu an poseenouop oHos mcofluouusoocoo swououm one .msoHuooum msoHHo> on» no muosowao HE ~.o mcwms coeuoom oposuoe on» Ge poneuomoo mm commons mo3 ofihuso one .am mmm.o ou mo.ov ouonmmonm Edemmouom mo usoepoum Hoosea HE com m an po3oaaom mo3 Sons one .ucoumcoo no; Canon 5 oemxomm 93 flags Hocmfimoommouosum m 36 mfifimucoo as was manganese Edwmmopom z mo.o suHB cosmos mo3 :EdHoo onu .cfioaoo onu ouco ponuomno moz oaenco on» Houwd .CEdHoo oueuomoexoupmn Hoowmmu o scum omonommconu Iaemonenoammosm pwoo oesHHosHso mo oawmoum coeunamll.v onsmwm 44 (suds) U!w02/ 303,. a a "ISOO aimIdsoud wnrssmoa (mow lb :11 WASH SAMPLE f .1 l I J 20 so 40'50 so 70 so 90 no no N 8 N . “LU/5m) 0191013 IOO IO Fraction Number phosphor the elut Protein I 45 phosphoribosyltransferase and protein. Figure 5 shows the elution pattern obtained with the isoelectric focusing column. The fractions containing enzyme activity were pooled and exhaustively dialyzed against 0.05 M_sodium phosphate (pH 7.0) containing 0.01 M_2-mercaptoethanol to remove the ampholytes. The enzyme solution was then made 50% (w/v) in sucrose and 0.01 M_in dithioerythritol and stored at either -90°C or 4°C. Table 1 summarizes the complete purification procedure. Discussion of the Purification Scheme The pH of the initial extract played an important role in the recovery of enzyme activity during the heat- ing step. When the enzyme preparation was heated at pH 6.5, only 50 to 60% of the activity could be recovered. However, when the extract was adjusted to pH 7.0 before heating 80 to 90% of the original activity could be recovered. Heating the enzyme preparation for periods of time longer than one min at 60°C resulted in a loss of enzyme activity, yet did not appear to cause any further protein precipitation. The DEAE Sephadex column (2.75 cm i.d. x 16.0 cm) gave the best results when 1000 to 3000 mg protein were applied. When columns were run with less than 1000 mg protein, all the quinolinic acid phosphoribosyltransferase activity was invariably lost. On the other hand, when 46 nailliqv ma AIV sot/30m 2:35 3'3 compoum .1553 :msomsuzo can museums no canoes one mesa: ponwfinouop mo3 nowumuucoonoo neououm one .noeuooum nooo no as ea mnems coauoom mponuoE on» cw poneuomop mo poeommo mo3 thuno one .uouoE mm xz Hope: noHoSIunomuom m anew: ousuonomfiou Econ um poneeuouop mos noeuomum HE N nooo mo mm one .uxou on» as poneuomop mo mnOwuoon ooHnu oprHE on» on poops ouo3 nadaoo oueuomoexoupmn onu Eoum oshnno HE m one .moueaonmso Aenm may omnon Sounon on» enema nonmeanoumo moz m.e ou m.v Eoum unoepoum mm d .nEsHoo mnemDUOM oeuuuoHoOme so some omouommnonu mammonwuonmmonm peoo owneaoneso mo oaemoum noeusHMIl.m ousmem 47 Hd 8 F3- 8- 8- T l l l (um/6w) ugarma ' K) s a —. 1R ' '1 25 Fraction Number 20 :- I5 l '0 a .3 (de) uwoe/Zoom IOOO - F‘u‘e A TABLE 1.--Purification of quinolinic acid phosphoribosyl- 48 transferase from castor bean endosperm. " "' autumn, , - Specific . Protein Activity Yield Step Units (m ) milliunits x % 9 mg'1 protein Extraction 3,580 5.6 1.5 100 Heating 2,800 4.4 1.6 79 DEAE 54 3.3 60.0 59 Hydroxyapatite 11 2.2 200.0 39 Isoelectric Focusing 1. 1.1 750.0 20 al unit 1 umole of nicotinic acid mononucleo- tide formed per min. a: A u I a . l .1. .l. AV t.» n » fly vi «to a: Rx. Rbs an 7F Elle ,- o 4: «L 49 large amounts of protein ( > 3000 mg) were used, the specific activity of quinolinic acid phosphoribosyltrans- ferase was not as high. Therefore, when working with larger amounts of enzyme, the enzyme preparation was divided in half and two columns were run simultaneously. Many variations on the elution of quinolinic acid phosphoribosyltransferase from the hydroxyapatite column were tried in an effort to separate it from the protein which started to elute just prior to the enzyme activity (see Figure 4). None of the various gradients tried worked any better than the original linear gra- dient; it was, therefore, used in all the purifications. In order to obtain a large amount of enzyme for the characterization studies, eight individual prepar- ations of the enzyme carried through the DEAE Sephadex column step were pooled and concentrated with an Amicon Ultrafiltration apparatus. This solution was then treated as one preparation during the last two steps. This was necessary because two full days were required for harvesting 150 to 180 gm endosperm, isolating the enzyme, chromatographing the enzyme on DEAE Sephadex, and assaying the column(s). Enzyme Stability Quinolinic acid phosphoribosyltransferase from castor bean endosperm can be considered a very stable enzyme. However, the enzyme was completely inactivated '~ ' .'. Y’.‘ “fl. ! huh; S O . urn on “C a: P'- .U. 0 50 when frozen at -20°C in 0.05 M potassium phOSphate (pH 7.0) containing 0.01 M_2-mercaptoethanol. It lost no activity when stored in the same buffer at room temperature for over 24 hr. When the 0.05 M_potassium phosphate (pH 7.0) was made 50% (w/v) in sucrose and 0.01 M_in dithioerythritol, the enzyme was found to be completely stable for over four months at either 4°C or -90°C. However, when it was stored in 0.05 M_potassium phosphate (pH 7.0) containing 50% (w/v) sucrose but not dithioerythritol, 35% of the original activity was ‘ - lost after only one month at either 4°C or -90°C. One-third of this lost activity was recovered by making the enzyme solution 0.01 M_in dithioerythritol. Enzyme Purity A portion of the enzyme isolated from the iso- electric focusing column was denatured by treatment with sodium dodecyl sulfate (SDS) as described on page 97. SDS-polyacrylamide gel electrophoresis (85) of this denatured enzyme resulted in only one band of protein as seen in Figure 6, A. The electrophoresis was carried out using up to 20 ugm of protein and the gels were stained with Coomassie blue. The molecular weight of this band as determined by comparison with protein standards was one-half that of native quinolinic acid phosphoribosyltransferase, as will be discussed later. 51 .peoo oeuoooouoenoeuu woe Ge poneoumop ono unmenuo>o osen oemmoEooo mo noeuseOm o nuez poneoum mo3 neououm one .onou Hod «E m mo unonuso unoumnoo o no ono Uooe no Hn m.e Hom eom mm ne ooEHOMHom mo3 nm.m mmv memouonmonpooem .Amm .mmv me>oa pno neoumnno mo ponuoE on» mnems noeuseOm eouooeem mom o ne oooMHSm eom on» no ponoeoe moB no: men omonowmnouueemoneuonmmonm peoo veneeoneso .m .oeoo oeuooo lou0enoenu woe ne poneoumop poo unmenno>o osen oemmofiooo mo noeuSeom o npe3 noneonm mo3 neououm one .onsu mom 48 m mo unounso unoumnoo o no poo ooem no Mn m.m How UoEHOMHom mo3 memouonmouuooeo ono ooomnsm eom onu no ponoeoe moB COeuSeOm oeenno one .ooooe no nee oe How oouoon mos eouoomem woe ono .Ae.e may ouonmmonm EdepOm.m e.o .eononuoonmoouoelm we .mom we oneneounoo fie: oe mom neououm on one ofihuno on» no n0euseom a .< .nESeoo mnemsoom oeuuooeoOme on» Eoum omonommnouueemoneHonmmonm oeoo veneeoneoo mo meoouonmouuooeo eom oeroemHooMeomll.m ousmem 52 of a a2;r "‘1 ‘L ‘5“ 3.121: ..,;e h M; EU a .5 53 Although the enzyme appeared to be pure by SDS gel criteria, analytical disk gels of the native enzyme resulted in three closely spaced bands as seen in Figure 6, B. Calculations from the densitometer tracing of a typical gel (Figure 7) indicates the relative abundance of the three bands: 70, 25, and 5 per cent for band 1, 2, and 3 respectively. Disk gel electro- phoresis at pH 7.0 gave a profile similar to that obtained at pH 8.3, but electrOphoresis at pH 4.3 resulted in a smear with no discrete bands of protein. Even though the three bands of protein obtained with analytical disk gel electrophoresis were very close, it was possible by slicing the gels with a Canalco Gel Slicer or with a razor blade to obtain two peaks of quinolinic acid phosphoribosyltransferase activity. The two peaks were not always resolved and sometimes the enzyme activity profile had a main peak with a definite shoulder. Both cases are illustrated in Figure 8. Three peaks of activity were never observed and it is not known which protein bands on the gel cor- responded to the peaks of enzyme activity observed. The Ferguson plots in Figure 9 lend further support to the contention that all three protein bands observed on analytical gels were of the same molecular weight. The log of the relative electrophoretic mobility (Rf) of the three protein bands in gels of I ‘l'o"i'. 54 .mneneonmoo Honmo En com no ponnoom mos eom one .onsn Mom «Em mo nnouuso nnonmnoo o no ono Uov no muson m.e Mom eom wm ne ooEuomnom mo3 Am.m may memononmonnooem .noenseom eonooeem won o ne ooom snow eom onn no oouoeoe mos nEdeoo mnemsoom oennooeoOme onn Scum pononme Am: men oaenno one .omonommnounemmoneHonmmonm peoo Deneeoneav mo memon nonmonnooeo eom oeroeeHoomeom mo mneooun HonoEonemnoall.e ousmem 55 E " C 0 L0 to -N 4— ' U Q) U C ' U .Q L O m .0 -—.<[ -O I l I l 1 L0 L0 808L096) (um) ugbuo won aouoisgg 56 .m onsmem ne noom mo meson oonnn onn no one Hoesoennom eno on mne>enoo oEeNno onn onoeonuoo on oenemmom non mo3 ne .eom Hod neonoum mo m: om enema onsn mom <8 m no poEHOMHom mo3 memouonmounooem .nOenoom moonnoE onn ne ponenomop mo Meonoepofise ooeoomo poo REE ¢.e - ~.Hv women scams m nuns one: so no Ass m.ens.ee nooeem emu ooemcmo o enema oooeem ouoB meom one .eom opeaoeenoo m.m mm .wv o Scum >ne>enoo omonommnonneemoneHonmmonm oeoo veneeoneDOIl.m ousmem as? has .8 .5 £83 593 m k m m e.. m N _ _ e . _ J _ -omm -000 1 -2. e e _ _ _ _ . -omm 000 (um) samurwsv/ SONS/203,, 58 .mnnoEoHSmooE mm o3n mo nooE onn me nneom noom .eom nooo on poppo oHoB neononm m: enuene .noenoom mponnoE onn ne ponenomop mo meow onn oon on poms Honoz ono m noenseom mo mnnooao onn mneeno> en poneonno ouoz oeroemuoo mo oomonnoonom nnonom Imep one .onsmew onn ne none: on eoE mo>Hso onn mo o90mm o>enomon one .nEseoo mnem500m oeunooeoome onn Eoum nonoeome Amo pno . O .eov neononm onn mo GOenoHnnoonoo opefioeenooeeom onn osmno> .mm .mneeenoe venouonm Ionnooeo o>enoeon onn mo moe onn mo mnoem nomsmnomll.m onsmem 222284 .23 e. 59 m k m m _ _ _ _ Joe. -8. on no onto MW" .0 // / -oom (oom’a) 501 0o 60 varying porosity plotted against the percentage acrylamide in the gel gave three parallel lines of equal slope. This indicated that the proteins were of the same molecular weight and differed in charge only. This is in agreement with the results of the SDS gels, which indicated that the proteins were com- posed of subunits of the same molecular weight. Assay Characteristics of Quinolinic Acid Phosphoribosyltransferase 2.3 1'27. 5'. ' (“I'- Quinolinic acid phosphoribosyltransferase L activity was linear with both time and enzyme concen- tration under the conditions described in the methods, as seen in Figure 10. This linearity was checked after every step in the purification. The only time when a deviation from the above results was noticed was after the heating and subsequent dialysis of the enzyme prepara- tion. It was assumed that this nonlinearity with enzyme concentration was due to residual unlabeled quinolinic acid which diluted the labeled quinolinic acid used in the enzyme assay. The requirements for the conversion of quinolinic acid and PRPP to nicotinic acid mononucleotide as cata- lyzed by quinolinic acid phosphoribosyltransferase are summarized in Table 2. The reaction was dependent on 2+ Mg and PRPP as well as the enzyme and quinolinic acid. Removal of all potassium phosphate from the enzyme by 61 .pooopoum NOUve onn onenomooe en .n0enoom moonnoE onn ne ponenomoo oncenepnoo puoononm onn moons poEHOMHoQ oHoB mnoem nnon mom whommo one .neE om Mom popoooonm meommo onn .m nuom nH .ea om mo eo>oe o no poms moz ofienno onn .< nuom nH .Amv oahuno mo nGSOEo ono Adv oEen mo noenon5m o no omouommnouneemonenonmmonm oeoo veneeoneoo en openooeoononoE peoo venenooen mo n0enoEH0mII.oe onnmem 62 <[ l l o 0 ID 9 L0 Jq/pauuo; NWDN salowu minutes enzyme ul 63 TABLE 2.--Requirements for quinolinic acid phosphoribosyl- transferase activity purified from castor bean endosperm. The enzyme was assayed as described in the methods section, except that the Na+ salt of PRPP was used. Tris- HCl (0. 05 M, pH 7. 0) was used in place of potassium phosphate (0. 05 M, pH 7. 0) in the assays with Ba + an , and Ca2+. Nicotinic Acid Reaction System Mononucleotide nmoles/hr Complete 127.5 -Enzyme 0.0 -I~Ig2+ 0.0 ~PRPP 0.0 -Potassium Phosphate + TrisoHC1 121.2 -Mgz+, + an+ 1 x 10‘2 M 35.7 -Mgz+, + Ca2+ l x 10-2 M_ 0.0 2+ 2+ -2 -Mg , + Ba 1 x 10 IE 0.0 64 dialysis against 0.05 M tris-HCl (pH 7.0) containing 0.01 M 2-mercaptoethanol did not result in any signifi- cant decrease in enzyme activity. This was in contrast to those results with quinolinic acid phosphoribosyl- transferase from a pseudomonad in which all enzymatic activity was lost by replacing the potassium phosphate 5“ with tris-HCl. Subsequent addition of potassium phOSphate to the dialyzed bacterial enzyme did not reactivate it (50). Metal Requirements Quinolinic acid phosphoribosyltransferase dis- played a requirement for magnesium, which is the case 2+ for all known reactions using PRPP. The Mg requirement in this reaction could not be replaced by equal molar quantities of either Ca2+ or Ba2+. On the other hand, Mn2+ was one-third as effective as Mg2+ at equimolar concentrations. The sodium salt of PRPP was used in these experiments to insure that no Mg2+ was present when determining the effect of the divalent cations. Figure 11 shows the effects of Mg2+ concentrations under the standard assay conditions. With a PRPP concen- tration of 0.375 mM, optimal activity was obtained with Mg2+ concentrations between 10 mM_and 50 mM, Gholson EE.E£ (47) reported the quinolinic acid phosphoribosyl- transferase from bovine liver was stimulated by such monovalent cations as K+, Li+, or NH4+ at levels of 65 .neoo +oz onn en poooemou mo3 meme mo neom +mmz onn nonn nmooxo .ooms oHoB omouommnonneemoneHonmmonm oeoo veneeoneso How mnOenepnoo wommo pnoononm .omonommnonneemoneHonmmonm peoo veneeonesv no noenounnoonoo +Nm2 mo noommo oneII.ee ousmem 66 vi T!“ P“: 22v 8:228:00 19>. N.0. .0. v0. _ A _ 0.0. O_ Io I 0 LC :00. .Iq/pauuo; NW 0N se|owu 67 20 mM. The effect of the monovalent cations Li+, K+, Na+, and NH4+ was investigated with the enzyme from castor bean endosperm and the reaction was not signifi- cantly affected. These assays were done under standard conditions except that the 0.05 M potassium phosphate (pH 7.0) containing 0.01 M 2-mercatoethanol was replaced with 0.05 M tris-HC1 (pH 7.0) also containing 0.01 M \ III}. -m—F 2-mercaptoethanol. T Product Identification In order to identify the product of the reaction, nicotinic acid mononucleotide, the following were incu- bated at room temperature for five hr: 1.5 units of enzyme from the hydroxyapatite column, 111 mM potassium phosphate (pH 7.0), 5.4 mM_2-mercatoethanol, 12.5134 MgClz, 3.1 mM_PRPP, 2.5 mM_unlabe1ed quinolinic acid, and 2.1 mM_l6-14C] quinolinic acid (3.82 uCi/umole) in a total volume of 8 ml. The reaction was stopped by adding 1 m1 of 10% perchloric acid and the solution was adjusted to pH 6.7 with KOH. After centrifugation at 10,000xg for 10 min to remove the potassium perchlorate, the super- natant was chilled; the additional potassium perchorate which formed was again removed by centrifugation. The supernatant was then applied to a Dowex 1X2 (50-100 mesh) formate column (1 cm i.d. x 40 cm) along with 10 nmoles of authentic nicotinic acid used as a column marker. The column was eluted with a differential formic acid 68 gradient as described by Ijichi 33 a1 (90) and 7 m1 fractions were collected. The elution profile is shown in Figure 12. The nicotinic acid mononucleotide fraction was concentrated on a rotary evaporator, streaked on citrate-washed paper, and chromatographed in solvent system B. Table 3 summarizes the comparisons made between authentic nicotinic acid mononucleotide and the reaction product obtained from the column. Authentic nicotinic acid mononucleotide (0.5mg) and the reaction product (60,000 dpm) were hydrolyzed in 0.1 M NaOH for 10 min in a 100°C water bath. Alkaline hydrolysis under these conditions completely destroys the pyridinium linkage (12), and as shown in Table 3, the products of alkaline hydrolysis migrated with standard nicotinic acid. Molar ratios for nicotinic acid, ribose, and phosphate were determined for the reaction product and are as follows: 100, 82, and 104 respectively. Nico- tinic acid was determined by its specific activity, ribose by the orcinol reaction (82) and inorganic phos- phate by the micro method of Ames and Dubin (81). pH Optimum and Isoelectric Point The enzyme was active over a very wide pH range as illustrated in Figure 13. The optimum pH was centered between 6.5 and 7.7. This was in contrast to quinolinic acid phosphoribosyltransferase from both bovine liver 69 .1. .3 x 3330882 m LIV E: can no oononHOmn< .mousoooonm n0enooemennooe ne poms ono oonouomo>o ono3 anaenooum z2oz one .0 oesem noenoeeenneom e5 m ne noenooum onn mo eE m.o mnennsoo an noneauonop moz noenooum pnouom euo>o ne >ne>enoo0eoon one .ononxefi noenooou onn nnez poopo ooz AmoeoE: oev neoo oenenooen puoononm .ousnouomson Soon no oonooeeoo ono3 mn0enooum eE e poo anon mom eE ow mo onoH 30em o non nEdeoo one .nnoeponm oeoo oefinom eoennonommeo o nne3 pondeo nEdeoo onoanom AnmoE ooeIomv Nxe xo3oo o mnemo omonowmnouneemonenonmmonm peoo veneeonesv en ooauom mnosoono onn mo noenonomom oenmoumonoEOHnUII.Ne onsmem 8 .0952 c0200.... 70 (wuoez) eouoqnsqv co. 0. <1: N g Q ow_ OE ON_ OO_ 8 Om 0v ON . . :4 . . . d o I41 4...... IIIIII I .2. .K . . + -N HG .<.O . . , um..." . 1. m o. w... I@ 2M. . m. <2 a 2202 Im m: r filITll .m 7 1000f «T I OI IOOUI , ( .2 CON EmNO .2 _0.0 I O— e. 71 TABLE 3.--Quinolinic acid phosphoribosyltransferase product identification. The compounds were spotted on Whatman no. 1 paper and developed by descending chroma- tography in the three solvent systems. The enzyme reaction product was identified by scanning its chroma- togram with a Packard Radiochromatogram Scanner. All other compounds were detected visually with a Mineralight UVS-ll lamp. Alkaline hydrolysis was done in 0.1.M NaOH for 10 min in a 100°C water bath. Rf in Solvent System Compound A B D .Authentic Nicotinic Acid Mononucleotide .77 .24 .10 Reaction Product .77 .27 .12 .Alkaline Hydrolysis of Authentic Nicotinic Acid Mononucleotide .37 .73 .65. Alkaline Hydrolysis of Reaction Product .37 .73 .65 .Authentic Nicotinic Acid .37 .75 .66 72 II . Abulov mooZIoneoeem :8 ooe en ono LIV oneoem ono .mmmmm .mmz SE ooe an ooooeoou mo3 nommsn ononmmonm onn nonn nmooxo .noenoom moonnofi onn ne ponenomoo mo ooms oHo3 onceneonoo mommo puoononm .mm «o n0enonsw o no omonomonouneemoneuonmmonm peoo veneeoneso mo mne>eno«II.me onsmem 73 I l.0 I0.0 9.0 8.0 p H 7.0 6.0 5.0 I O In l50~ I00— I O o N . Iq/pauuo; NINDN sa|ou1u 74 and the pSeudomonad which exhibited well-defined optima at pH 6.2 and 7.1 respectively. The isoelectric point of the enzyme was 6.0 and was determined as described earlier in the methods section (Figure 5). This was the average of four determinations in which enzyme isolated from the hydroxyapatite column was used. Temperature Activation Nicotinic acid mononucleotide formation was measured as a function of temperature (Figure 14). The reaction rate increased until 45°C after which point it sharply declined, indicating that some enzyme inacti- vation must have taken place. An Arrhenius plot of this data (Figure 14, A) indicated that inactivation was also occurring at 40°C and 45°C. The Qlo at these two temperatures did not agree with the value of 2.6, which was the Q10 of the reaction between 15°C and 35°C. The energy of activation calculated from the Arrhenius equation was 17.5 Kcal. Substrate Protection Against Heat Inactivation Quinolinic acid was quite effective in protecting quinolinic acid phosphoribosyltransferase from heat inactivation under the same conditions in which the controls lost 48 per cent of their activity when heated at 55°C for 10 min (Table 4). The protection derived from quinolinic acid was probably due to its binding to 75 .onnnouomfion Soon no mo3 noenz onenno en om mo noeneooo onn en oonnonm moz noenooou onn nonn nmooxo .noenoom moonnoE onn ne pononm mo ponnomuom ono3 whommo one .moneo> unom mo omono>o onn we nneom noom .mounnonomnon nnouowmeo no omonomononneemonenonmmonm oeoo veneeoneno mo onou nOenooou onn we mene .m .onnnonomfion onneOmno onn mo eooonmeoou onn noneomo ponnoem me AM mOeV onon noenooou onn mo mOe one .ounmem menn mo m nnom ne no>em onop onn mo neem onenonunn no me mene .n .omonommnonneemonenonmmonm neoo veneeonenw mo onon nOenooon onn no onnnouomEon mo noommo oneII.ee onnmem 76 mm 0.. 0.20.0an ._. I? 1' 0 mm mm e no III/pawns NINDN sanwu é O_x._.\_ mm». m. 0mm 03” 0mm — e — 77 TABLE 4. --Substrate protection against heat inactivation. The enzyme was heated for 10 min at 55°C in 0. 05 M potassium phosphate (pH 7. 0) containing 0. 01 M dithio- erythritol with and without 0. 8 mM quinolinic -acid, 1. 6 mM PRPP, 1. 0 mM MgC12 and with combinations thereof. The samples were then cooled and diluted for assaying. Both the Mg2+ and the Na+ salt of PRPP were used and no difference was observed between the two. Control samples (0% protection) retained 48% of their original activity. . . . . Percentage Additions Prior to Heating Protection Quinolinic Acid 52 Quinolinic Acid + Mg2+ 38 PRPP 4 PRPP + Mg2+ 8 an+ -10 78 the enzyme, although it cannot be said that the binding was at the active site. Neither PRPP (1.6 mM) or its biologically active form, PRPP-Mg2+ (1.6 E! and 1.0 mM, respectively) were significantly effective in protecting the enzyme from heat inactivation. Magnesium (1.0 mM) slightly promoted enzyme inactivation by heat (10%). When fii““ quinolinic acid (0.8 mM) was added in addition to the Mg2+ (1.0 mM), the percentage protection was decreased by 14% from that obtained with quinolinic acid alone. When the l “I ‘ t..— effectiveness of PRPP as a protective agent against heat inactivation was tested, both its sodium and its magnesium salts gave essentially the same results whether or not additional Mg2+ was present. Effect of Divalent Cations The effect of a series of divalent cations on quinolinic acid phosphoribosyltransferase is given in Table 5. The assays were performed in 0.05 M tris-HC1 rather than the equivalent phosphate buffer, as the latter tended to form precipitates with some of the metal ions used. The metals were incubated for 15 min with the enzyme before starting the reaction with PRPP. Ca2+, Ba2+, and Sr2+ had hardly any effect on the enzyme activity even at 1.0 mM. On the other hand Niz+, Coz+, 2+ 2+ 2+ Cd , Fe , and Pb completely inhibited the enzyme at 1.0 mM and caused about 40% inhibition at 0.1 mM, While Cu2+ and Zn2+ at 0.1 mM inhibited the enzyme by 80%. 2+ The percentage inhibition caused by Mn might have 79 TABLE 5.--The effect of various cations on quinolinic acid phosphoribosyltransferase activity. The assay con- ditions were the same as described in the methods section, except that the enzyme was dialyzed against 0.05 M tris-HCl (pH 7.0) with no mercaptan present. TheFe was no appreciable loss in activity. The following salts of the cations were used: Ca2+, Ba2+ Sr2+, Fe3+, and Zn + chloride; Mn2+, Niz+, Cu2+, and Fe + sulfate; and Pb2+ acetate. Relative Activity Percentage Metal 1 x 10"5 1 x 10‘4 1 x 10'3 1 x 10’2 Ca2+ 104 90 Ba2+ 105 110 an+ 59 33 Sr2+ 100 83 N12+ 100 88 2 o C02+ 81 79 20 1 Cu2+ 68 18 o o Cd2+ 69 61 4 o Fez+ 96 64 4 o Fe3+ 64 0 pb2+ 99 n.d. 1 o Zn2+ 82 21 o o 80 been competitive as previous results indicated that Mn2+ could partially replace the requirement of the enzyme for Mgz+. Quinolinic acid phosphoribosyltransferase was stabilized over long periods of time with the mercaptan, dithioerythritol. Studies with the classical sulfhydryl inhibitors, NEM, and p-CMB at 0.1 mM, however, did not lead to any significant inhibition. Effect ofguinolinic Acid Analogues jut: .vv The effect of quinolinic acid analogues on the rate of quinolinic acid phosphoribosyltransferase activity when assayed together with quinolinic acid is shown in Table 6. The assays were carried out in triplicate as stated in the methods, except that lower levels of quino- linic acid (37.5 EM) and PRPP (62.5 EM) were used. The inhibitors were incubated with the enzyme for 15 min at 30°C before adding the quinolinic acid to start the assay. The assays were stopped after 5 min. From the data given, it was apparent that a com- pound having a carboxyl group, a carboxylamide group, or even a cyano group at the 3 position of the pyridine ring was an ineffective inhibitor of the enzyme. However, if the latter two compounds had a carboxyl group added at the 2 position, they were very effective inhibitors of the reaction. When the added carboxyl group at the 2 position was converted to its methyl ester, it no 81 TABLE 6.--The effect of substrate analogues on the rate of nicotinic acid mononucleotide formation. The standard assay for quinolinic acid phosphoribosyltransferase as described in the methods was used, except that lower levels of quinolinic acid and PRPP were present. The assay contained the following reagents at the final concentrations given in a total volume of 0.8 ml: 2 milli- units of enzyme; 115 mM_potassium phosphate (pH 7.0) with 2-mercaptoethanol; 12.5 mM_MgC12; 37.5 0M [2,3,7,8-14C] quinolinic acid; 62.5 uM PRPP and 1.0 and 10.0 mM_additions of the various compounds listed in the table. The assays were equilibrated for 15 min at 30°C, started by the addition of quinolinic acid, and then stopped after 5 min. The average value for three reactions are reported as percentages of the control. Percentage of .‘d emanate Am. Compound Structure Control 10'311 10'2M Nicotinamide CijycouHa 105 102 N 3-Amid0pyridine-2- ONH2 carboxylate .N OOH 39 5 Methyl 3-amido- pyridine-2- Erjjcouna carboxylate N COOCHS 86 43 0 O I 0 CN Nicotinonitrile l n 101 91 3-Cyanopyridine-2- cu carboxylate I COOH 52 22 Methyl 3-cyano- CN pyridine—Z- E::B ' carboxylate N C00033 95 85 Methyl 3-carboxy- OCOOH pyridine-2- COOCH carboxylate N a 69 30 TABLE 6.--Continued. 82 Compound Structure Percentage of Control 10'3M 10’2M H — — 4-Hydroxyquinolinic E::]EOOH Acid 00H 48 12 . . . . 00H Nicotinic Ac1d E:i]c 101 91 N Picolinic Acid 20 8 OOH 2-Hydroxynicotinic OOH Acid H 78 50 Trigonelline OOH 90 88 Ha 2,5-Pyridinedi- H00 carboxylic Acid coon 100 90 OOH 2,4-Pyridinedi- carboxylic Acid CODE 88 73 H 3,4-Pyridinedi- /’ COOH carboxylic AC1d \\ 61 20 2,6-Pyridinedi- OCE:;] carboxylic Acid HO COOH 43 6 83 longer served as an effective inhibitor. In fact, methyl 3-cyanopyridine-2- carboxylate was about as ineffective as its parent compound, 3-cyanopyridine (101% and 95% relative activity at lmM_and 19% and 85% relative activity at 10 mM respectively). The 2-methyl ester of quinolinic acid inhibited more effectively than either methyl 3-amidopyridine—Z-carboxylate or methyl 3-cyano- pyridine-Z-carboxylate, which indicated that the enzyme showed some preference for the group at the 3 position (COOH > CONH > C E N). 2 Picolinic acid (2-pyridine carboxylate) proved to be one of the best inhibitors tried. The 2-hydroxy- nicotinate inhibited to some extent (50% at lOmM), but it was not as effective as the 2-carboxy analogues. The 4-hydroxyquinolinic acid was also one of the more effec- tive inhibitors, which might imply that a certain degree of steric interference at the 4 position did not exclude the molecule from the active site. The four positional isomers of quinolinic acid tested can be divided into two groups based on their effectiveness as inhibitors of this reaction. The 2,5- and 2,4-pyridinedicarboxy1ate were relatively ineffective, whereas the 3,4- and, to a greater extent, the 2,6-pyridine dicarboxylate were more effective. 84 Effect of Potential Inhibitors on Quino- linic AcidP505phoribosyltransferase The effects of a series of possible inhibitors including the pyridine nucleotides (NAD, NADP, NMN, and NaAD), the pyridine nucleotide precursors (nicotinic acid and nicotinamide), the pyridine alkaloids (ricinine and trigonelline), and two other reported inhibitors of the E enzyme (ATP and azaleucine) are listed in Table 7. The I! _1_1U'_'.' LP". possible effectors were assayed using lower levels of E PRPP and quinolinic acid, as were used with the quino- linic acid analogues. None of the compounds tried inhibited the enzyme to a great degree; NAD, however, caused a slight inhibition of about 19% and 15% at ngg and 10 mM levels respectively. ATP was reported to significantly inhibit the bovine liver enzyme (47) and to partially inhibit the enzyme in crude preparations of castor bean cotyledons (41), but it had no effect on the purified enzyme from castor bean endosperm. Waller et a1 (69) recently suggested that azaleucine might act as an inhibitor of quinolinic acid phosphoribosyltrans- ferase in castor beans based on the results of in vivo studies. The purified enzyme used in these experiments was not inhibited even at 10 mM levels of azaleucine. Kinetics Double reciprocal plots of the initial velocity Versus the concentration of one substrate at a series of 85 TABLE 7.--The effect of possible inhibitors of quinolinic acid phosphoribosyltransferase. The standard assay for Imiinolinic acid phosphoribosyltransferase as described 111 the methods section was used except that lower levels of’HuoQO0H H ham .2 .0 .m mm>uao How 2 mIOH x m. N can .2 mIoH x mum.H .2 IOH x mN. H .2 mI IOH x mNm. 0 mac? mQOHumuucmocool UHom OHGHHocHav l was .WHm>HuommmmH m cam Am .0 .m .d mw>uao map How .2 mIOH x o. m can .2 mI IoH x mm. m .2 mI IoH x m. N .2 mI IOH x mum. H .2 mIOH x mN. H mums mGOHumuu Icmocoo mmmm was .muscHE mom mmmummmcmnuHmmonHHozmmonm cHom UHGHHOOHc mo mmHOEc mm pwmmwumxm mH .> .muHoon> was .mNMmmm manna mo mmmum>m may mH ucHom comm .mmmm can pHom OHcHHocst mo mGOHumcHnEoo paw mGOHumuu Icmocoo HHm um 6mm: poHnmm wEHu msu Mom HmmcHH muw3 mummmm may umsu wusmcH ou pmfiuomumm mnmz mucmEHummxm mumummmm .hmmmm ucHompcm cm :H ucmHmncH mcoHumuHEHH may mo mwnmomm .cHE m cho mm3 08H» coHuommH on» can soHoa cm>Hm mm pmHHm> mum3 mcoHuHccoo momma pumpcmum .GOHumuucmocoo mumnumnzm umchmm muHoon> HMHuHcH mo muon HmooumHomu manooII.mH mucmHm 88 mt. 2.0.x TEES Om mm Om. Sung x Izod. 22:05:81 Om (DI—I _ H l — moow OO_ _ J S3 quI/salouI60| x A/| 8 89 changing fixed substrate concentrations, -l/Ka and -l/Kb can be read from the abscissa and l/V is the intercept on the ordinate. The slope of the curves in Figure 15, A and B, were determined and are plotted in Figure 16, B, as a function of the reciprocal of the changing fixed substrate concentrations. The two curves obtained with - this replot had identical slopes equal to KéKb/V. The #3, values of these kinetic constants are given in Table 8. The Km values were very similar to those observed with 5 5 the bovine liver enzyme, 6 x 10- g_and 5 x 10- §_for . j I“[_A quinolinic acid and PRPP respectively (47). The same was true for the bacterial enzyme with respect to PRPP, 5 7.4 x 10- E, but the Km of this enzyme for quinolinic 4 acid was significantly higher, 1.2 x 10- g (50). Physical Characteristics Molecular weight Estimation Using a Sephadex G-200 Column The molecular weight of quinolinic acid phos- phoribosyltransferase in 0.05 §_potassium phosphate con- taining 0.01 g 2-mercaptoethanol and 0.1 g KCl was estimated by the method of Andrews (83) with a Sephadex G-200 column. The enzyme had been purified through the hydroxyapatite column step as described previously. The molecular weight of castor bean endOSperm quinolinic acid phosphoribosyltransferase was estimated by this method to be 68,000. The standard curve is shown in Figure 17. 90 . AOIIOV UHom UHGHHOCHsv can. AIV ammo mo .mH mnsmHm mo mGOHumuucmocoo mumuumasm pmme 939350 on» wo meOHm on» no uon humpcoomm .m . AIV pHom UHGHHocqu cam AOIIOV ammo mo . Hm_\H .mGOHumuucmocoo HmHoE 93 mo mHmooumHomu on» momHm> .m can 4 .mH musmHm mo mumchuo may Scum Umchqu muHUOHw> GOHuommH HmEmeE on» no HmooumHomu may no HOHQ mumccoomm .2 .mump muHoon> HMHuHcH on» no muon mumccoowmII.mH musmHh 91 ISO 8'57O O -50/ - 5g ‘ 8 a, «a ‘ ' _.' 0' 8 9 uglu/saIOLusolx‘wA/l I/UIUU 90' x adoIs 92 TABLE 8.--Kinetic data for quinolinic acid phosphoribosyl- transferase as determined from initial velocity studies in the absence of product. Constant Values V 7.1 nmoles/min -5 KQA 1.2 x 10 M -5 KPRPP 4.5 x 10 M E x 1.1 x 10’9 M2 a b : 3. ant-II-.. ! i 7 i YTQ. ?' 93 .muH>Huom mahncm no a: OMN um chuoum now pmmmmmw cam pmuomHHoo wum3 mcoHuomum HmuHHHHHHEIoza .ccu mm3 cEdHoo on» mEHu 20mm mEdHo> pHo> on» mcHEHmump on com: mm3 GOHusHOm cmuuxmv man A>\3V wN.o fl .Aooo.OMHV umEHc cHeanm Eduwm mcH>on Ame can NonnummmcmuuHhm IonHuoammoam Baum oHcHHocH2a mo much ~.o .Asc “loco.mmo cHsanm asumm mcH>on as m .lme “loco.m¢a :Hesnam>o as ea .lmc “loco.m~o ammocammuuoemaouu m8 o.n .AHV "mGOHumcHEHmumc owns» mo Hmuou o How mGOHumcHQEoo mSOHHm> cH mEHu m um 03» cEdHoo on» on UmHHmmm mum3 HE mco mo mEdHo> Hopou m cH mmOHOSm A>\3V wON cH pmummwum mpumpcmum mcHonHom one .Hox_m H.o cam .HocmsumoummonEIN.m Ho.o mchHmucoo Ao.n may mumnmmonm Edemmuom meo.o nuH3 pwumunHHqum noon to: :EsHou one .250 m.mm x .U.H EU m.NV cESHoo OONIU xmpmcmmm m Eoum pwusHm mchuoum cumpcmum meHHm>_mo muanmz HMH IsomHoE map mo moH on» umchmm .mEdHo> cHo> on» mH 0> can 0E5Ho> coHusHm on“ mH m> muons .o>\m> mEdHo> coHusHm m>HuMHmH mo uonII.>H musmHm 94 O.¢ 0.. J ‘2 °/\/ 9A 95 Sucrose Density Gradient CentrifugatiOn Another estimate of the molecular weight of quinolinic acid phosphoribosyltransferase was obtained by sucrose density gradient centrifugation according to the method of Martin and Ames (84). Five per cent and 20% sucrose solutions containing 0.05 M potassium phos- phate (pH 7.0) were used to make 5 m1 linear gradients. Quinolinic acid phosphoribosyltransferase in 0.1 ml containing, either together or separately, bovine liver 5 catalase and yeast alcohol dehydrogenase was layered on top of each gradient. The gradients were centrifuged for 15 hr at 4°C in a SW-39L rotor at 38,000 rpm. Twenty-six 6 drop fractions were then collected and assayed for enzymatic activity as seen in Figure 18. The molecular weight of castor bean endosperm quinolinic acid phosphoribosyltransferase as determined by this method was 72,000 with a range of i2000. The enzyme used for this determination had also been purified through the hydroxyapatite column chromatography step. Electrophoresis in SDS- Polyacrylamide Gels It was shown by Shapiro et_al (85) that during electrophoresis in SDS-polyacrylamide gels proteins are separated into individual subunits which migrate as a function of their molecular weight. Enzyme from the isoelectric focusing column containing 1% SDS, 1% 96 .AHmV mmmcmmonphcmp Hosoon How ooo.omH can mmMHmumo Mow ooo.omN mumz muanm3 HMHsomHOE pmfidmmm one .uxmu mnu cH cm>Hm mum mcoHuHccoo HmucwEHummxm one .wmmcmmoupmnmp Honoon ammo» can mmmumwmcmuuHamonHuonmmonm pHom UHGHHocHsv can mmmHoumo 0cm mmmnmm ImcmnuHmmonHuonmmozm UHom UHQHHOGHDq Umchucoo mucchmum Hmnuo 039 .30Hmn pmHSuOHm H .oc ucmemum co nonm>MH mums Ame h.ov mmmcomoupmnwp Hosoon can AmE m.ov mmMHmumo suH3 musuxHE m cH ”HE H.o Hum muHccHHHHE ONV GESHOU wUHu Immmmxoupwc wcu Eoum omuMHOmH monummmGMHUHhmOQHHocmmocm UHum oHcHHOCHso .mmmcmmoncmcmo Hosoon ammo» can .mmMHmumo Hm>HH mcH>on .mmmummmcmuuHamonHH Iocmmocm UHom OHGHHocHsv mo cumuumm GOHuoucmEHUmmII.mH musmHm 97 _Eo_o.\8mom\se<. $228 .5 0.3 882 550.882.522.18 .282. _eoo_.\ 52828... 28.»qu F E fiI-oll 1| ..... L HIII II.II.II .l L i .§£§_§§89@1 ....... L 1. 28 25.8.8 .I ........... J .1. .l ............. J — r lllll I... «8.83550 I .282 _,..II1 in J .. _ . . .IIIL qu """"" .Hl I4 03 o 1IIII _ .8 Finn-.. a .I lllllll 4 PIIIII|'J J .J H _ H 47 _ _ _ _ p . 5 5 O 5 4 n4U 3 m 2 m I I 24680I2I4|6|82022242628 Fraction Num be'r- 98 2-mercaptoethanol, 0.1 M_sodium phosphate (pH 7.1), and 10% glycerol was incubated at 100°C for 10 min. The enzyme (12 ug protein), along with the similarly treated standards a-chymotrypsinogen, ovalbumin, bovine serum albumin, and bovine serum albumin dimer, was electrophoresed in 5% acrylamide gels containing 0.1% SDS for 3.5 hr at 8 mA per gel tube and stained in 0.4% Coomassie blue as described in the methods. A plot of the log of the molecular weight of the standards against the distance of migration gave a straight line as illustrated in Figure 19. The molecular weight of quinolinic acid phosphoribosyltransferase as determined from this and from the data of two other similar experi- ments gave an average value of 35,000 with a range of 1'2000. This indicated that the enzyme was probably composed of two subunits, since a molecular weight of 70,000 would be in very close agreement with the molecular weight obtained by the two previously described methods. Physiological Studies Ricinine Biosynthesis In the interval of six to seven days in a warm moist environment, the germinating castor bean mobilizes the energy stored in the fatty endosperm tissue for use by the developing seedling attached to it. Accompanying 99 .mH musmHm mo pcmmmH map cH Umuuommu mmozu mm meow may mumz bump chu mcHquHm cH com: muzme3 AMHsomHoE meSmmm was .cmoochmmuu08220Ia Ame cam “mmmuwm ImcmnuHMmonHuonmmonm vHom UHGHHOGHDU .avv “QHEdnHm>o .Amv “GHEdnHm Edumm mcH>on .ANV «umEHU GHESQHM Edumm mcH>OQ .AHV "m30HHom mm mum pmuonmp mckuonm one .uxmu may cH cm>Hm mum wcoHqucoo HmucmEHummxw one .mHmm mpHEmHmuommHom mom :0 GOHumumHE mo wUGMUmHU “Hosp umchmm pmquHm mCHmuoum pumocmum mo mucme3 umHsomHoE may no mOAII.mH musmHm Id’nrn. Eli-HIII 1.5-E. 100 2.3 3:322 n v m _ _ — Ow I 0.? 0.0 'lM'W 501 101 this period of active deve10pment are many diverse bio- chemical phenomena, not the least of which is the bio- synthesis of the alkaloid ricinine. Its rate of pro- duction during early seedling development is shown in Figure 20. This data agreed well with the results of Waller (91), who found that the ricinine content of ?‘i- castor seedlings increased 30-50 fold during a 48—72-hour period between the third and sixth day following planting in moist sand at 30°C. After this period of rapid for- mation, the absolute amount of the alkaloid increased; j however, its concentration (mg ricinine per gm fresh weight) remained constant (92). To date, much of the experimental investigation of ricinine biosynthesis has involved feeding eXperiments with intact plants. Little work has been done on the biochemical processes related to ricinine biosynthesis. lg_zigg studies showed that the pyridine nucleus of quinolinic acid, nicotinic acid, and nicotinamide was directly incorporated into ricinine; however, the actual precursor remains unknown (14, 57). These three com- pounds are related in that the quinolinic acid formed by the dg £932 pathway is eventually converted to nico- tinamide and nicotinic acid via the Preiss-Handler pathway. Waller gt_al’(l4) demonstrated that quinolinic acid was two times more efficient than either nicotinamide or nicotinic acid as an in vivo precursor of ricinine. 102 .HH mHnma cH @mnHHommp ucmEQOHm>mp HMUHmOHOHmmnm mo mmmwum msoHHm> on» um mmCHHUmmm uomucH moan» mo ucmucoo cHonme on» so comma mmz 05Hm> comm .coHuomm moonume map cH cmumum mm UmuMpHucmsq can Doom um muHHsoHEum> umHOE cH c3oum mmcHHcmmm Hoummo scum pmuomuuxw was mchHOHH 0:9 .mmcHHpmmm comb Houwmo mcHumcHEHmm mo acmucoo wchHonII.ON musmHm 103 l l I l O. O. Q 0. ~cm 00_ me n Om mm _ _ ON 0.. $301 0.. m .0 _ 0M pauuog NINDN 39'0qu 109 .CHE 03n now nnon Honos mCHHHon o CH noon CoHnoooH onn mCHoon an Commonm oo3 COHnoooH onn Hn oCo nonm< .oonooHoCH mnCCoEo onn CH CoHnoHomonm ofimnCo onn ooCHonCoo whomoo one .m .oEHn onoHHmonmmo onn no CHE o3n How nnon Honoz mCHHHon o CH nooHn CoHnoooH onn mCHoon en commono mo? COHnoooH one .COHnoHomon oeeuCo H: ov ooCHonCoo eommo noom .C .oOHCOm oE>Nco onn mo onoComoEon Coon nonmoo oosno o mCHoC CoHnoom moonnoE onn CH ooanomoo mo oomommo mo3 oaeuCo one .onoComOEon Coon Honmoo ooCHo oCo oEHn no CoHnoCCm o no omooHEoCHnOOHC en ooHEoCHnOOHC mo COHnooHEoooII.NN oHCmHm 1i. 110 m <[ l l J l 0. t0. 0. l0. 9 I\ In N 9904105 pw OIUIIOOIN salowu 20 3O 4O Enzyme Concentration (ul) IO “up. ' '3. r. g" I I. A 111 nicotinamide (3.52 uCi/umole), 4.8 E! ATP, 8 mM_MgC12, 76 mM_potassium phosphate (pH 7.0), and 8 mM_2-mercapto- ethanol in a total volume of 3 ml. (It was later shown that Mgz+ and ATP were not necessary for nicotinamidase activity.) After 4 hr, the reaction was stopped by heating the reaction flask for 3 min in a boiling water V” bath. The deproteinized solution was applied to a Dowex 1X2 (200-400 mesh) formate column (1 cm i.d. x 40 cm) and eluted with the same gradient as described in the r o-hunm.‘ legend of Figure 12. The nicotinic acid was well separated from nicotinamide, which did not adhere to the column. The fractions containing nicotinic acid were concentrated on a rotary evaporator and an aliquot was co-chromatographed with authentic nicotinic acid in solvent C. The radioactive material isolated from the column migrated to a spot having an Rf identical to that of the authentic nicotinic acid. After adding non-radioactive nicotinic acid to the isolated product, it was recrystallized to constant specific activity * as seen in Table 10. * . The product was identified as nicotinic acid by Anne Bosch, a 1971 summer NSF student. 112 TABLE 10.--Recrystallization of nicotinic acid to constant specific activity. The isolated [7-14C] nicotinic acid from the Dowex 1X2 formate column was added to 200 mg nonradioactive authentic nicotinic acid, dissolved in water, and recrystallized four times, as reported below. A few crystals were dissolved in water and diluted to 5 ml with water each time. Two 0.5 ml aliquots of this material were counted in scintillation fluid C; 0.5 ml aliquots were diluted to 10 ml with water and the absorbance was determined at 260 nm with a Hitachi spec- trophotometer. Absorbance . . cpm per 260 nm Ratio crYStalllzat1°n 0.5 ml of a 1:20 Dilution Abs/cpm #1 105 .58 5.5x10‘3 #2 106 .61; .61a 5.7x10'3 #3 162 .91; .92a 5.6x10'3 #4 85 .48 5.6x10'3 aA reading from a duplicate dilution. 113 The Level of Quinolinic Acid and Nicotinic Acid Phosphoribosyltransferases in Developing and Mature Castor Bean Plants In Figure 23 is shown the level of quinolinic acid and nicotinic acid phosphoribosyltransferases in both the endosperm and the cotyledons of etiolated castor bean seedlings at different stages of develop- ment. The corresponding visible physiological changes which occurred during seedling development are depicted in Table 11. The specific activity of quinolinic acid phosphoribosyltransferase in the endosperm increased dramatically during days three and four with a gradual leveling off at day five. The rapid drop in activity on days six and seven probably reflected the general wasting away of the endosperm tissue, as shown in Figure 24. The peak level of quinolinic acid phosphori- bosyltransferase in the endosperm was a little less than three-fold higher than the peak level of the enzyme in the cotyledons. The specific activity of nicotinic acid phosphoribosyltransferase in the endo- sperm remained approximately constant during days two, three, and four, but started to decrease on day five. The level of this enzyme in the endosperm was approxi- mately equal to its level in the cotyledons. Quinolinic acid phosphoribosyltransferase activity was lOO-fold higher and lZ-fold higher than nicotinic acid 114 Figure 23.-~The specific activity of nicotinic acid phosphoribosyltransferase (A) and quinolinic acid phosphoribosyltransferase (B) in the endosperm and cotyledons of germinating castor bean seedlings. The seedlings were selected for their physiological age rather than their chronological age. Any one flat contained a random population of seedlings, which, although they were planted at the same time, did not germinate at the same rate. Table 11 gives a descriptive daily characterization of the castor bean germination. The 27,000xg supernatant was obtained as described in the section on enzyme isolation and was used to assay these two enzymes. Three to four plants were used for each series of determinations. The cotyledons were not separated from the endosperm for the enzyme preparations made on days two and three. (0—0) Endosperm (O——-—0) Cotyledon 115 2 3 4 5 6 7 2 3 4 5 6 7 Age of Seedlings (days) I w 5205 95.52202 $.08: IOOI- 50- 116 TABLE 11.--Physiological development of the etiolated castor bean plant grown in moist vermiculite at 30°C. Moisture and temperature were very important factors in castor bean development. Castor beans germinated at 28°C instead of 30°C required two to three days longer to develop. Day Observations 2 Seed coat had just started to crack open. The radicle was from 0.3 to 5 1.0 cm long. The radicle was from 1.5 to 4.0 cm long with secondary roots. The radicle was from 5.0 to 9.0 cm long and the hypocotyl was from 1.0 to 2.0 cm long. The secondary roots were well developed. 7" The hypocotyl was from 3.8 to 5.2 cm long with an average of 4.5 cm. At this point, the endo- sperm was starting to liquify. The average length of the hypocotyl was 6.7 cm. The endosperm was transparent and slimy and had been nearly consumed. 117 Figure 24.--Total soluble protein of the coty- ledons and endosperm of castor bean seedlings. The pro- tein was determined by the method of Lowry et a1 (76) and was based on the amount of protein present*1n the enzyme extract of either four endosperm (O—-—O) or four cotyledons (o—-—-0) all at the same stage of development. 118 l 1 l l J cg 9 co 0