A STUDY OF THE ENZYME XANTHTNE DEHYDROGNASE FROM DROSOPHBLA MELANOGASTER M. S. MICHIGAN STATE UNIVERSITY SHELDON D. PARZEN 1963 3 39 xmmnumu“mm 1%,? 0474 54 IBRARY L Michigan Stan Umvcmty HM \\ *3 )293 1 \\\\\\\\l\\\\\\\\\l\\\ \AEI OVERDUE FINES: 25¢ per day per item gguamue LIBRARY MATERIAQ: Place in book return to remove charge from circulation records ' . ELL-3.:- ‘3 :.w:.‘,.' " '" ?' flaw“ y ‘o“' 1‘1 ln‘ '8‘. C §xmfli¢fiar-"'Wtaq c;';f;z..s-a\il.€£'~:---a an ABSTRACT A STUDY OF THE ENZYME XANTHINE DEHYDROGENASE FROM DROSOPHILA MANGGASTER by Sheldon D. Parzen The purpose of the work reported was to study the biochemical characteristics of xanthine dehydrogenase from Drosomila melanogaster. This involved the development of an assay of enzymatic activity which was linear in relation to enzyme concentration and was sensitive enough to detect activity in single flies. A method of purification was devised, this resulting in a 528 fold purification of the enzyme. The enzyme was found to have a pH Optimum of 8.0. Km's were determined for various substrate and electron acceptors of the enzyme. A study of the stoichiometry of the reaction using purified preparations indicate that 1 mole of NAD is reduced for each mole of hypoxanthine converted to xanthine and another mole of NAD is reduced for each mole of xanthine converted to uric acid. A complementation experiment was performed resulting in the pro- duction of active xanthine dehydrogenase from extracts of two mutants of _I_)_. melanogaster deficient with regard to that enzyme. A STUDY OF THE ENZYME XANTHINEIDEHYDROGENASE‘FRQM DROSOPHILA.MELANOGASTER By Sheldon D. Parzen A THESIS Submitted to Michigan State University in partial fulfilJment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1963 'L\- PREFACE The author wishes to gratefully acknowledge his indebtedness to Dr. A. 3. Fox for his understanding and patience, and under whose direction this work was performed; to Dr. James Kan for his aid in the statistical analysis of some of the data contained in this work; and to his wife for her aid in the preparation of this.manuscript and her patience and understanding during the final.months of the completion of this work. Sheldon D. Parzen August, 1963 ii I. II. III. IV. V. VII. TABLE OF OONTEVTS PEACE . . . . . . . . . . . . . . . . . INTRODUCTION .O........... REVIDVOFTHELITEATURE ....... A. B. GEVETICS 0F ROS! AND MAROON-LIKE BIOCHEMISTRY......... ”ATM AND MEI‘HOD'S . . . . . . . . . A. B. C. D. RESULTS STOCKS............ GROWTH AND COLLETION 0F FLIB PREPARATION OF EXTRACTS . . . BSAIMEI‘HODS ........ DISCUSSIONoooeeoeeooooeo SWI................ Emumm............. iii 0... 53 55 Table l. 2. 3. h. 5. 6. 7. 8. LIST OF TABLE Page Smary of recombination data of an unselected sample of 13 independent mutations of rosy tested against ros in selective recombination tests. . . . . . 6 The differences and similarities between maroon-likeandrosyiz'................... 9 Test of the reliability of single fly assays using several inbred and non-inbred BtOCkB e e e e e e e e e e o 32 Analysis of Variance of enzyme activity in single fly assays among six stocks of Drosophila melanogaster . . . . 3h Purification of xanthine dehydrogenase . . . . . . . . . . 1.3 The stoichiometry of the conversion of hypoxanthine to uric 301d by Within. dehydrogenase e e e e e e e e e o [#6 Results of the complementation experiment with extracts not treated with Norite-A. All reaction mixtures contain 20.2 mg Of protein. 0 e e e e e e e e e 0 1+9 Results of the complementation experiment with Norite-A treat extracts. All reaction mixtures contain18.8mgofprotein................ ‘19 iv Figure l. 2. 3. 1+. 5. 6. 7. 8. 9. LIST OF FIGURES Page Schemes preposed by Glassman and Mitcth (1959a) for the action of the two loci in the production ofxanthinedehydrogenase................ l6 Absorption spectra of AHP, NAD, Thio-NAD, and reduced thio-NADO..0..................... 23 The dependence of enzyme activity on the presence of suitable substrate and electron acceptor. . . . . . . . . 25 The relationship between activity and enzyme concentra- tions, using hypoxanthine as substrate and NAD as electronacceptor..................... 26 The relationship between activity and enzyme concentra- tion using hypoxanthine as substrate and Thio-NAD as electronacceptor...................... 27 The relationship between enzyme activity and the concentration of an extract from a single fly in a reaction mixture containing 0.57 ml. 2 x 10 M hypoxanthine; 0.39 ml 3. 3 x 10-3 M Thio-NAD; 0.1 M TriS,PHBeOeoeeeeeeeeeeeeoeeeeeeee 28 The relationship between enzyme activity and concentra- tion of enzyme obtained from 1, 2, A, and 8 flies homogenized in 1 ml of buffer using a reaction mixture containing 0.57 mls of 2 x 10“F M hypoxanthine and 0.39 mls of 3.1.3 3: 10'3 M Thio-MAD, and 0.16 mls of enzyme, allin0.lMTris,pH8.0................. 29 The relationship between enzyme activity and concentra- tion of extract obtained from 5, 10, 20 and 1.0 flies homogenized in 5 mls of buffer using a reaction mixture containing 0. 7 ml of 2 x 10-h M hypoxanthine; 0.39 ml of 3.1.3 3: 10' M Thio-NAD and 0.39 ml of enzyme prepara- tion;al1i.n0.1MTris,pH8.0.............. 30 Flow diagram of the scheme of purification of xanthine dehydrogenase from Drosophila melanogaster . . . . . . . . 35 Elution pattern of chromatography on a DEAE-cellulose column of a partially purified preparation of xanthinedehydrogenase.................. 37 The determination of the Km of xanthine dehydrogenase for NAD With hypoxanthine 8.3 substrate 0 o e e e o o e e e 39 V 4‘! Figure 13. 1h. 15. Page The determination of the Km for xanthine dehydrogenase Of ThiO-NAD With hypoxanthine as substrate 0 e e e e e e o w The determination of the Km of xanthine dehydrogenase for hypoxanthine with NAD as electron acceptor . . . . . . L1 The determination of the Km of xanthine dehydrogenase for xanthine with NAD as electron acceptor . . . . . . . . 42 Enzyme aetiVity inrelation to pH 0 o e e o e e o o e e e “I. I. INTRODUCTION In recent years several problems have arisen which are of great interest to the biochemist who is genetically oriented. These are the problems of the genetic control of protein structure; the synthesis of specific proteins in cell free systems; and the general question of the nature of the genetically controlled biochemical mechanisms involved in the differentiation of multicellular organisms. The first problem facing one who is interested in these questions is that of finding an organism which is suitable for study with regard to these problems. The organism must, by necessity, be multicellular if the problem of differentiation is to be studied. Secondly the mechanism of genetic control, that is the genetics of the organism, must be well understood if the problem of determination of protein structure is to be studied. Finally, knowledge of the biochemical make-up of the organism should be lmown if cell free systems are to be isolated in which specific proteins can be synthesized. Meeting these criteria, perhaps better than any other organism, is the cannon fruit fly Drosoghila melanogaster, which for years has been under the study of geneticists and is now under the analytical tools of the biochunist. The enzyme xanthine dehydrogenase, isolable from Drosophila, has several advantages which make it ideal as a subject of study for the aforementioned areas of interest. Initially it is known that wild type Drosomg' possess the enzyme and that certain well defined eye color mutants of the organism lack it 3 that is, there is a strong I correlation between the phenotype of the fly and the occurrence of the enzyme. Secondly, the mutants which lack the enzyme have been well studied on a genetic basis and thirdly, these mutants have been studied on a biochemical basis, not only as regards the presence or absence of the enzyme, but also as regards their general biochemical make-up both as adult and larval forms. Hence it was the purpose of this work to initiate a study of the biochemistry of the enzyme nnthine dehydrogenase. This work involved the design of a qualitatively and quantitatively sensitive and reliable assay for the enzyme, purification and characterization of the enzyme, and a study of the nature of the biochemical bases for lack of activity of the enzyme in the mutant forms of the organise. II. REVIEW OF THE LITERATURE A. GENETICS OF RDSY AND MAROON-LIKE R_o_sz (a) is a recessive mutant which was first isolated as a spontaneous mutant in a Canton (Ohio) wild stock by Bridges in 1938 (Bridges and Brehme, 19111.). It was located at 3-511“ and described as having a phenotype of a deep ruby eye color, ocelli slightly diluted, and larval malpighian tubes considerably lighter than wild type (Brehme and Demerec, 19!.2). In 1956 Hadorn and Schwink reported the isolation of an allele of £931, M (£73), from other stocks and also located at 3-51. The mutant lacked isoxanthopterin and was non-autonomous for the red eye pigments (Hadorn and Schwinck, 1956a, 1956b; Hadorn and Graf, 1958). Thus mi eye anlagen implanted into wild type hosts develop a drosopterin phenotype identical to wild type. Moreover, implants of wild type malpighian tubes into ros hosts caused the drosOpterin content in the hosts' head to approximate that of wild type. It was also reported by these workers that wild type eye discs implanted into r_osfi hosts develop non-autonomously, i.e. resemble £o_sfi rather than the wild phenotype. ‘Eye color in this mutant was described as being dark reddish brown due to the partial reduction of the red eye pignents with the color of the ocelli and testes approximating wild type, but the malpighian tubes were shortened and malformed containing in the lumen 3 a. .‘ yellow to orange colored globular inclusions. Aside from the lack of isoxanthopterin, there were increased amounts of other pterins. The viability of the mutant was normal at 18’C, but subvital to semilethal in late pupae and early adults at 25.0. Schwink (1960) also noted that m and mi were both lacking in xanthine de- hydrogenase activity. Further elucidation of the Egg; locus came from the laboratory of Chovnick (Chovnick, Schalet and Kernaghan, 1961a; Chovnick, Schalet, Kernaghan and Talsma, 1962) in their study of recombination at the m locus. Using a series of spontaneous and x—ray induced mutants at the m locus, they applied a recombinational analysis utilizing schemes which are modifications of systems designed for the study of induced crossing over in Drosomila males which selects for crossovers (Hhittinghill, 1950) and which had been applied by Chovnick to other work (Schalet and Chovnick, 1960 3 Chovnick, Schalet and Kernaghan, 1961b). The scheme is based on the utilization of various lethal markers adjacent to the area to be mapped, and used in combinations such that all non-crossovers die and only a fraction of the crossovers survive. The selective efficiency of such a system will be a function of the distance between the lethal markers, this system then making possible investigation of genetic fine structure in a higher organism which previously had been attempted only in lower organisms as exemplified by the work of Benzer (1959, 1961). Using the markers Lug-12d (3-50.0) and karmoisin (3-52.0) which are recessive visibles; Minute-25 (3-1.1..4) and lethal-26 (3-52.5) l“ which are recessive lethals; Deformed (34.7.5), Stubble (3-58.2), and Ultabithorax (3-58.6) which are dominant visibles with recessive lethal effects, Chovnick and co-workers were able to map ll; x-ray induced m1 mutants with map distances ranging from 7.73 x 10““ for the distance between Logfl and r_o_sy_2_é to 5.87 x 10"3 for the distance between M and res 6. Table I shows a summary of the recombina- tion data of an unselected sample of 13 independent mutations of £331 tested against £03123 acquired in these selective recombination tests. Chovnick 91;; 2.1 (1962) attempted a conversion of map distance in terms of percent of recombination to distance in terms of nucleotide pairs in a single double helix molecule of DNA. Assuming that re- combination is uniform throughout the third chromosome of Q. melano tor, Rudkin (1962) has provided a maximum estimate of the number of nucleotide pairs per map unit of 1.3 x 106. Using the minimum estimate of the smallest distance thus far resolved, one emerges with an estimate of 14.0 nucleotide pairs as the distance separating mi? and _r9_sfi. Estimate of the total length of the £031 cistron, using the mximum distances thus far obtained for Log-Macs indicates a value of 11.8 x 103 nucleotide pairs. If one assumes that this structure completely determines the amino acid sequence of that part of xanthine dehydrogenase controlled by the _r_o_sz locus, that the genetic code is a three-letter, non- overlapping, commaless code with no "nonsense" information (Crick g 9_1_., 1961), and that the average molecular weight of an amino acid in this protein is 100, then the molecular weight of the £221 contri- bution to mthine dehydrogenase is estimated to be 390,000. 01’ . Q , \ _ w . I . « . VA . F . \ ° l . l. \ ‘ E ‘ / 5 K . , y a .a u A s - 0-. . .‘ L! a. . . 1.. ._.. ,1 l . s \ ,k - 0....1. ‘, , L r. _. - .. ~. \ , 4 Table 1. Summary of recombination data of an unselected sample of 13 independent mutations of rosz tested against rosfi6 in selective recombination tests. (Adapted from Chovnick gt 9., 1962) .a-. Mutants tested map distance 171 - 1726 7.73 x 10-4 173" - 1126 5.87 x 10’3 :71" - 1726 2.32 x 10-3 175 - 1726 1.42 x 10-3 ry3 .. 1726 2.90 x 10-3 179 - 1726 3A8 x 10’3 1723 - 1726 h.51 x 10'3 - 1726 3.75 x 10"3 n26 - :72 2.60 x 10" ry26 - 176 3.16 x 104‘ 1726 - 177 2.85 x 10" n26 .. r325 3.33 x 10‘“ 1726 - :7“ 3.18 x 10-3 interest is the fact that chicken liver xanthine dehydrogenase has a molecular weight of L80,000 (Remy _e_t_ a_l_., 1955) and that of cow's milk xanthine oxidase, an estimated weight of 290,000 (Avis 93; 51., 1956). Maroon—like (1221-) is a recessive sex-linked eye color mutant originally recovered in a single male from an x-rayed wild type male by Oliver (Bridges and Brehme, 19M). Heliminary mapping placed the mutant locus near vermilion (1-33.0). It was described as having a dullish eye color on emergence which darkened with aging. However, it is brighter than the 9.12219. mutant which completely lacks the drosOpterins. It is not an allele of raspbem (1-32.8). Glassman and Mitchell (195%) reported that maroon-like was closer to 29.9.9925 (1-57) than to vermilion, but close mapping of the locus was hindered by the fact that the wild type allele of m- _l_i_k_g exhibited a maternal effect. Thus, the genetically maroon-like offspring of a female heterozygous for maroon-like showed the wild type phenotype, having normal eye pigmentation, xanthine dehydrogenase activity and trace amounts of isoxanthopterin. To circumvent this difficulty, analysis of chemotype obtained by paper chromatography was used (Hubby and Forrest, 1960). The m— _li_1_g_g genotype produces only trace amounts of isomthopterin while the wild type maroon-like allele produces easily discernible amounts of this compound. Thus a division of classes is possible. Classi- fication of this sort may be performed with any marker that contains the wild type amount of isomthopterin. Thus, in a cross involving the eye color mutant raspberry2 (l-32.8), this latter eye color o . \J» \4 I (is ..J u ‘ . ‘J \ I I e s J e - .4 \ , x » "l -‘\ ,1 ‘, a -l a I — - ‘- _ L1 \ , . , \ - .a . .. I. .) ., x ‘7 c - ‘ K - O x we, 5 , ‘ , . \4‘ e 1 . . | n11 . . \_4 V f-\ __ _ I k, L: n 'I . ., -. ,- . . \J ,4 . . . . L mutant was phenotypicaJJy indistinguishable from the double mutant raspberryz, maroon-like; but chemotypically this double mutant lacked the wild type amount of isoxanthOpterin that is characteristic of raspberryz. Consequently, using this technique Hubby and Forrest (1960) made a preliminary cross involving the markers yellow (1-0.0), 9333 (l—36.l), raspberryz (1.32.8), forkeds (l-56.7). and miniature (1-36.l). The results of this cross indicated that maroon-like was situated 12.732 crossover units to the right of forked5. A more exhaustive analysis was then made using Beadex3 (1-59.h) as the most distal, well located marker. From this cross maroon-like was located at 67.2 :1; 0.7 on the X chromosome. B . BIOCHEMISTRY From a biochemical point of view, the deficiencies exhibited by the £931 mutants are quite similar to those shown by the maroon-like mutants in that neither can carry out these reactions catalyzed by xanthine dehydrogenase, with the exception of certain reactions not requiring NAD which can be catalyzed by the £931 mutants but not by maroon-like. In addition, my; exhibits no maternal effect and has no effect on that shown by maroon;l_;ke (Glassman and Mitchell, 1959b). The differences and similarities between maroon-like and my; are shown in Table 2. The eye pigments in Drosophila are a complex of at least three compounds (Viscontini, Hadorn, and Karer, 1957; Viscontini, 1958). Table 2. The differences and similarities between maroon-like and rosy_2_. (Adapted from Forrest gt 9.1., 1961) Reaction wild type ry2 ma-l 2,1e-dihydrolq-u-e 2,L,7-tri— hydroxypteridine + _ - (NAB) Amp-«“4 isoxanthOpterin ' (NAD) + - _ Xanthopterin-n-mt 2-amino-lt,6,7- trihydroxypteridine + - _ (NAB) Hypoxanthine-«u-e Xanthine (NAD) + - - Xanthinen-«e Uric Acid (NAD) + .. _ h—hydroxy-n-ui 2,l.-dihydroxy- pteridine + + _ Pyridoxal-m-«e pyridoxic acid + + .. Maternal effect - .. + Endogenous hypoxanthine .. + + Lack of isoxanthopterin - + + Reduced red pigments - + + 2-mino-A-hydroxypteridine accumulation - + + 10 These pigments have been designated drosOpterin, isodrosopterin, and neodroeopterin by Viscontini 93 _a_._l_. (1957). The three compounds are orange in visible light and fluoresce orange under ultra-violet light. A fourth pigment which is red under both sources is demonstrable upon electrophoretic separation. The chemical constitution of these com- pounds is unknown, though their pteridine nature has been demonstrated by Forrest and Mitchell (1955) and confirmed by Viscontini at 51. (1957). Several attempts to establish the chanical nature of these pigment components have been made (Wald and Allen, 19h6; Haas, 191.8; Heymnn, Chan, and Clancy, 1950; Chen, Roma, and Clancy, 1951). Lederer (191,0) first suggested that the pigments were pteridines. This was denied by Haas on the basis of a low nitrOgen elementary analysis. Later Forrest and Mitchell (1955) determined that these pigments were pteridines and gave rise by photo-oxidation to Lamina- h—hydroxy-6-carbonpteridine. The pteridine nature of the photolysis product of the red pigments has also been confirmed (deLerms and Vincentiis, 1955; Viacontini, Hadorn, and Karrer, 1957). In a series of papers by Forrest and Mitchell (19510., 195kb, and 1955) five other pteridines have been identified and character- ised. This work was stimulated by the discovery of a paper chroma- tographic technique for the separation of fluorescent compounds and other pigments in Drosophila (Hadorn and Mitchell, 1951). This technique was particularly fruitful in the mutant Lapin: in which Hadorn and Mitchell demonstrated the lack of dros0pterin and the occurrence of large amounts of a yellow fluorescent compound. This r. \ xle I a . .V ‘7 A; r) ‘uJ . J . ,, v r a I . . 1- a ~ ‘ s L V , m . v; . . .4 K. (.1 A, _. , ‘ ., ' . ,.1 , _ _ a . I e ‘4 , - ‘ I ~ , '. , , .‘ , v , , _ M .1-.. An- gt... a ( . b ‘ 1 Q s I " -. ‘ . ‘4 > -\ . i, g I 1 u . A ‘a‘ . , _ ~1. 1 ’ \. , ‘ - a . x . s b t , I l l v.1 _ )v \A a, t... 11 compound is present in trace amounts in wild type strains. Forrest and.Mitchell (1954a, 1954b) isolated this pigment in crystalline form from.the mutant ggpi§.and characterized it as 2-amino-6, 7-dihydroeh, 6-dihydroxy-6-1actylpteridine. Shortly after this Forrest, classman, and Mitchell (1956) described the absence of isoxanthoPterin in the mutant maggggi(ggg and.maroon-1ike. Simultaneously, Hadorn and Schwinck (1956a) reported that 5952; lacked isoxanthopterin. In addition the former work described the enzymatic conversion of 2-amino-h—hydroxypteridine to isoxanthopterin'by extracts of wild type and numerous mutant flies, as well as the absence of this enzyme activity in maroon-like. M was also reported to lack enzyme activity (Glass-an, Forrest, and Mitchell, 1957). This was the first report of a lack of enzyme activity associated with a mutant in Drosop_h:L_1a_ melanogaster. Moreover, the mutants in- volved affected a number of well defined and easily identifiable compounds. Forrest, Classman, and Mitchell (1956) demonstrated that extracts of wild type stocks of Drosoggila melanogaster contain enzyme activity for the conversion of 2—amino-h-hydroxypteridine to isoxanthopterin, hypoxanthine to xanthine to uric acid, xanthoPterin to leucopterin, and bensaldehyde to bensoic acid. Later, Glassman and Mitchell (1959a) showed that this activity could be ascribed to a xanthine dehydrogenase rather than to a xanthine oxidase because well dialysed preparations require methylene blue or NAD for activity; Nawa, Taira, and Sakaguchi (1958) derive the same conclusion. In this latter report ‘4‘ 12 it was found that extracts treated with sufficient activated charcoal to remove the last traces of fluorescent materials are essentially devoid of activity without adding an electron acceptor such as NAD. Hubby and Forrest (1960), using an assay based on the reduction of NAD with activity expressed as positive change in Optical density at 340 mu/minute, found a pH optimum for the enzyme at 8.0 with an Optimum molar concentration for MB being unity with respect to hypoxanthine. Glassman and Mitchell (1959a) reported the oxidation of purines, pteridines, and aldehydes by the enzyme. They found the Km for Z-mino-L-hydroxypteridine to be 6.7 x 10"6 M; for xanthine and hyponnthine, 2.5 x 10‘5 M and 2.1 x 10-5 M, respectively; the latter comPound was oxidized 2.5 times faster than 2—amino-h- hydroxypteridine with NAD as the electron acceptor. However, this assay utilizing NAD was found to be undesirable in the measurment of the conversion of 2—amino-h-hydrozypteridine to isonnthoPterin. This is due to the overlapping of the 3m mu peak of deduced MD with an absorbance peak of 2-amino-L-hydroxypteridine, thus making the assay unsuitable for quantitative studies of the reaction (Parson and Fox, unpublished data). A purification scheme for the enzyme was reported by Glassman and Mitchell (195%) which utilized ammonium sulfate fractionations and chromatography on calcium phosphate gel. However, this scheme resulted in a purification of only 10 to 50 fold with a recovery of enzyme activity between 50 to 75 percent. The assay devised by these workers for this work was based on the change in fluorescence when /\ \‘kJ _ k ( I \ O s O a. I IN V. ‘ k.- 13 2-amino-h-hydroxypteridine is converted to isoxanth0pterin. Parzen and Fox (unpublished data), however, found this assay undesirable because of secondary interactions between the fluorescent emissions of isoxanthOpterin and 2-amino-L-hydroxypteridine when together in reaction mixtures. Consequently, this assay was also nonpusable for quantitative work with the enzyme. The maternal effect exhibited by the maroon-like genotype has the result of causing.maroon-like progeny from females with a wild allele of maroon-like to exhibit a wild type phenotype. Hence, the male progeny from a cross of an attachedqx female (homozygous for the wild allele of maroonplike) with maroonplike.ma1es unexpectedly showed a wild type eye color although genetically they were maroonplike. Similarly all progeny of a cross of heterozygous maroon-like females 'with maroon-like males were phenotypically wild type, even though a 1:1 ratio of wild type to maroonplike was expected. These effects 'were noted and studied by Glassman and Mitchell (1959b), Hubby and Forrest (1960), and Glassman and McLean (1962). The maternal effect involved not only eye color, but also morphology and function of the malpighian tubes. In.maroon~like flies not exhibiting the maternal effect there is aberrant morphology and function of the malpighian tubes (shorter, irregularly shaped, puffed up, containing yellow to orange globules). However, in ma- ternally affected flies these abnonmalities are not exhibited (Schwinck, 1960). This maternal effect exhibited itself only in maroon-like flies which emerged in the first six to eight days after the first appeared; r1 . ._ >4 s ’ ‘ » 9. u . . ‘ , ‘ I ‘ G <1 .- . _ . i. ‘ ' . \ I I ‘ . a _ Av . I \'~ I. - s . . i. A ‘L~ —- \ ‘ I \ . - v N a ' l r . ' r - . .. V as k. r 1 h I . \ ‘ - I. u kl _ ,JA‘ - - ‘l ) . .. ~x ‘ ' N ll. after this a short period occurred during which flies emerging had eye colors intermediate between maroon-like and wild, but later none were maternally affected. However, when the egg-laying female was transferred to new food, the maternal effect again appeared (Glassman and Mitchell, 195%). This would indicate that the previously men- tioned change was not due to depletion of the maternal substance in the aging female, but to some environmental cause. Glassman and Mitchell also pointed out that the maternal substance was probably not xanthine dehydrogenase, since females homozygous for _r_o_sl, and hence lacking the enzyme, can still have maternally affected maroon- __lik2 progeny. Glassman and McLean (1962) also found mthine dehydrogenase activity in maternally affected larvae, but none in eggs, the amount of enzyme slowly declining during development. This observation indicates either activation, complementation, or synthesis 91; m of xanthine dehydrogenase during early developnent of maternally affected maroon-like flies. These workers also found xanthine dehydrogenase activity in maroon-like larvae dericed from doubly attached-X, scarlet, 321$ females which indicated to them a type of complementa- tion _i_n 123.12 in which the product of the wild type allele of m- _li_k_e_ in the maternal parent reacts with the product of the wild type 1991 gene in the progeny to produce active xanthine dehydrogenase. An manple of this type of maternal influence is that of some egg color mutants in m (Kikkawa, 1957) in which the pigmentation of the egg is passively passed from the female parent into the egg, but is diluted out during developnent. In contrast to this observation 44 s -a I V l i \J . V 4 _ n -a . . V 't . s ‘ \‘ . e. . ‘1 a I) I .. b . s v \ “ . . 1 '7 t h .. .4 — \ ' A, r .J I a n - —' . . -‘ ‘ ‘ . > ‘ . . '4 . _- 7 . - . . K \ . . ‘1‘ ‘ ' '- ‘ —- IJ—‘h. \ . . r h a I ‘ _. r 7‘ . . . - l v ' «x y-.. ‘ I - ' l I - v l H d ‘l 4 t ._ ‘-b - .. \ 7 ‘ t 2’ x ' ‘ a , ’ A ’ . 4 . ,2. . “ “' ~ ’ x . - ' i “ ‘ I ~ .I Q . \‘l p I s --. — 15 . is the finding of Fox (1958, 1959) and Fox, Zoom, and Head (1962) wherein they report that a segment of the Y chromosome in Q. melano ter, when present in the oocyte of a female, has a struc— tural effect on a protein in her offspring even though the latter lack the chromosome segment in question. Since the effect persists through ontogeny, it implies the existence of a self-perpetuating information transfer mechanism in protein synthesis. This is quite dissimilar to the maternal effect of maroon-like in which there is a progressive dilution and eventual disappearance of the characteristic passed from parent to offspring. The presence of mthine dehydrogenase activity in maroon-like larvae derived from attached-X females having the MI]; gene indi- cates a type of complementation in _v_i_v_o_, in which the product of the wild type allele of maroon-like in the maternal parent reacts with the product of the wild type £19. gene in the progexv to produce active xanthine dehydrogenase (Glassman and McLean, 1962). Thus, there are at least two loci which control xanthine dehy- drogenase in Drosomila melanogaJter. The fact that either locus can cause the deficiency of the enzyme while the other is normal indicates that each locus has a different function in the genetic control of this enzyme. This is further indicated by the fact that the maroon-like and m mutants can complement each other in L112. Glassman and Mitchell (1959a) have proposed two possible schemes for the action of the two genes in the production of xanthine dehydrogenase (figure 1). Xanthine dehydrogenase could have two sites of enzyme activity each controlled by a different gene. (Dual (a \J ‘4 '5 L. ’\ ‘4' (4 16 Figure 1. Schemes proposed by Glassman and.Mitchell (1959a) for the action of the two loci in the production of xanthine dehydrogenase. .ma-l?‘ 11T+ a) l ; X : Ehzyme activity " ma-l+ b) 4 x 17 functions for the classical xanthine oxidase and chicken liver dehy- drogenase, where purines, pteridines, and aldehydes are oxidized at one site and reduced NAD is oxidized at another site, have been suggested by other data (De Renae, 1956)). Thus the mutant Egg-ya would affect the dehydrogenase site (perhaps the site where the co- factor is bound), leaving the site of oxidation unaffected but probably with a markedly reduced efficiency. On the other hand, the mutant maroon-like would necessarily affect the site of substrate binding, consequently interrupting both the oxidase and dehydrogenase activities. Alteration of the substrate site would be assumed to have little or no effect on the protein's ability to react with specific antibodies. Hence, cross reacting ability was found by Forrest, Hanley, and Lagowski (1961) with maroon-like and wild type extracts to antibodies formed to the enzyme itself. This explanation would be more in accord with the second of Glassman and Mitchell's schemes for the action of the two genes on the activity of the enzyme (figure lb). Thus, maroon-like flies would contain the product of the wild type £291 allele, 1, having all the cross-reacting ability but no enzyme activity. In contrast, £991 flies would contain the product of the wild type maroon-like allele, 1;, having no cross—reacting ability, but which would be ensymatically active with an efficiency, however, less than that of wild type flies. In fact, by an extension of this scheme, it becomes somewhat analogous to the system in Mherichn coli controlling tryptOphan synthetase (Crawford and Ianofsky, 1958), where two proteins, A and B, are necessary to make a fully functional enzyme, although each, by itself, , ‘4 ‘# l 7‘ — > \. . . ‘ - .LI _‘, x ‘l g, . v_ V - J , rV . .‘ .‘ . , l , ~ L1 2.1 a, s _ V x . '_ _, _ + ‘1 \. . ~ g c 1.4. , \ . . . r w, . . .1 \- U K- a: \ _. . a '4 ~ . _ U . . .. n . - a I ‘ s - u \. up; —- ‘ ‘1 ~ u.< a 4 LJ - V - ‘ - v I .. .. ‘ -- . x l s. . ‘1 ,, a ‘ . x - i c . -7 , -, , H, ‘xo ' - ‘ b \ I _.a —A x A ' s\ . - .I V '_J .. ‘ . r . . .i , f ) '.l u ‘ w . ‘ . - ' .. tv ' w 'y '4 __ _ c . _ . , ,. (,4 Q g _ . . . 4 “ , ‘ .. . . y- a ~ ..... *7 I, .‘ ~ 7 . ( a r. - -- . ‘l , ~4 - a - .... - .4 ‘4 t , . at i in. u . . _. .._,,-‘. _ r . a- l. i , . I, . . . w - . . ‘ I 7 a a I ‘7 L - 3., W 1 A . . m . ‘- —_ A v. I ' ‘1 I ._. - A. . t . , , 1 '— , \, v ‘ g ‘ R; U _ '_ _ . l; . ' a . ‘ , ‘ ‘J »_, _ _ hi 2 _'. _ l‘ - 1 3 . , a - -,._. t - \. - ' a ‘ . — . D n ( . .4-s~iz. 1 \-A v \ a \J 18 has some enzyme activity in the half reactions involved in the pro- duction of tryptophan. Cross-reacting activity, in contrast to this, is confined to one of the proteins (Lerner and Yanofsky, 1957). On this basis, extracts of the two mutants when incubated to- gether in specific fashions should result in xanthine dehydrogenase activity if this incubation allows for assumption of proper tertiary and quaternary structure necessary for enzymatic activity. Glassman (1962) reported Just such an experiment, utilising a fluorometric assay supposedly sensitive enough to detect activity of the enzyme in single flies (Glassman, 1962). Results of this experiment indicated that although the mutant extracts incubated alone exhibited no xanthine dehydrogenase activity, incubation of mixtures prepared by combining extracts from the mutants of each locus results in the production of ensyme activity. III. MATERIALS AND METHODS A. STOCKS l. Oregon-R (99:3). A wild type stock maintained by Dr. a. S. Fox. The stock contains a slight ebony allele, a slight branching of the posterior crossvein and an occasional scooped wing. It is homozygous for Df (2) Qr__e_-_-_I_i_ at the tip of 2R. 2. Oregon-Edam (Ore-R-I). A wild type stock originally iso- genized by J. Schultz and subsequently maintained in this laboratory by single pair, brother-sister matings for 168 generations at the beginning of this work. 3. Maroon-like (99:1). A recessive sex-linked eye color mutant. It has a dullish eye color which darkens with aging but is brighter than m which lacks all the drosopterins. This stock was obtained from Dr. Arthur Chovnick. h. M (a), 13.213 (i). Recessivo eye color mutants having deep ruby eye color. These stocks were also obtained from Dr. Chovnick. 5. White-apricot no. 2 Lwfi), white-anricot-Smgy (fi-Syg). Two eye color mutants in the white region typified by orange pink eye color which is darker in the males. 6. Swedish and Samarkand and Oregon-116M. Three additional wild type stocks. Oregon-R-Syggey, Oregon—R-I, and white-apricot-Sflggz are all inbred isogenic stocks. Samarkand, Swedish, and white—agicot no. 2 are non-inbred isogenic stocks. 19 (s 20 B. GROWTH AND COLLECTION OF FLIEB A11 stocks were grown in half-pint milk bottles at 25°C. on a standard corn meal-molasses-agar medium enriched with brewer's yeast and seeded with living yeast. Flies were collected by light etheri- nation after a period of three to four weeks, and either used im- mediately or stored in a freezer at -20‘C. Cs HiEPARLTION 0F EXTRACTS Extracts were prepared by homogenising the flies in a 2.5 w/v ratio of 0.1 M Tris (hydroxymethyl) amino-methane ("Tris") buffer, pH 8.0 which was 5 mg/ml with respect to crystalline serum bovine albumin in an all-glass, conical homogeniser at 5'0. The homogenate was then centrifuged at 30,000 x g for 30 minutes at 0°C. To the resulting supernatant was added N orite-A to give a concentration of 100 mg/ml. This was allowed to stand in the cold for one hour with occasional stirring, immediately after which the mixture was centri- fuged at 30,000 x g for 30 minutes at O'C., and the resulting super- natant was poured through a coarse sintered glass filter to remove any remaining charcoal. The resulting filtrate, designated extract, was then assayed for enzymatic activity. The preparation of extracts of single flies for enzymatic assay duplicated the foregoing assay with the exceptions that the fly was homogenised in 1 ml. of the buffer, 10 to 20 mg. of Norite-A then being added to this homogenate. This was then allowed to stand for one hour at 5'0., and was centrifuged. e 21 D. ASSAY METHODS Assay of the enzyme is based on the conversion of nicotinamide- adenine-dinucleotide (NAD) to reduced NAD, or thionicotinamide- adenine-dinucleotide (thio—NAD) to reduced thio-NAD, with the con- commitant increase in absorbance at 31.0 mu in the former case and at 395 mu in the latter case. Thio-NAD was purchased from Pabst laboratories. 2-amino-lt- hydroxypteridine (AI-LP) was purchased from General Biochemicals. Other samples of AHP were gifts from Lederle laboratories and from Dr. Arthur Chovnick. 2-amino-lt, 7-dihydroquteridine (isoxanthop- terin) was also a gift of Dr. Chovnick. Tris (hydroaqvlmethyl) aminomethane, "Tris," was purchased from Sigma Chemical Co. All solutions were made up in 0.1 M Tris buffer, pH 8.0. Hypoxanthine was prepared in concentrations of 5.1 x 10'”3 M and 2 x 10-1» M; This-NAB, 3.1.3 3: 10-3 H; NAD, 10-2 M; xanthine, 3.3 x 10-3 M; NADH, 10-1 M; uric acid, 7 x 10-5 M; AHP, 2 x 10-“ M. Reaction mixtures contained either hypoxanthine or xanthine and a suitable electron acceptor in the form of NAD or thio-NAD to follow the reaction in the forward direction. Increase in absorbance at 3A0 mu or 395 mu was then followed on a Beckman DU spectrOphoto- meter with a Gilford Recording attachment at 25°C. This-NAB was used at these times when increased sensitivity was desired. The increased sensitivity is due to the fact that reduced thio-NAD has an am of 11.3 x 103 in comparison to the am of reduced NAD of 6.22 x 103. C ., .c,.~ '0 n n " ' ' § 'm 22 Conversion of AHP to isoxanthOpterin was followed by using thio— NAD as the electron acceptor and recording the increase in absorbance at 395 mu as the thio-NAD was reduced. In this case the use of thie- NAD was.mandatory due to the overlapping of absorbance curves of NADH2 and AHP (figure 2). One unit of enzyme activity is defined as equal to an increase in Optical density at BAG or 395 mm.(depending on electron acceptor) of 0.001 per minute per m1. of enzyme preparation. Study of the reverse reaction, that is, the conversion of uric acid to xanthine to hypoxanthine, with the conccmmitant oxidation of reduced.NAD, was attempted by using an assay mixture containing uric acid and reduced NAD and observing the decrease in absorbance at 340 mu. VI . '23 +- -h 3:: Lacgmga OWN? . . Com 00m _ . 0mm . v i1::..!-31l- f 3.1.. 41% :3 ,._.. I--s§T.i.i§ 4- :13..4Il£../i.+. q . O , d— u fir- q asuvqmsqv M. “DE: )2 1 AV 4.Z<2..£r_.r . 22.2: a ~19: a o\.\o tut/e... \ -. 0\ 2mm \\\ o . :Ov .o.w In. .2? 23 ME: md o. _E 0.0 E2... eE:_o> E frag SENS v.5 0.34-05 2 yo. x E... b .E no 323%} 2 b. x m .5 _E am gees 22.me : Seam lounge college me 92125 t e 2.22% mm . SEE/.8»; , Em: cofizcfice eExNE was 32% 502.33 958223 EH .0 .235 28 353% eExNCe .5 =2 9.0 , - $0....Jmoo -. v0.0 .I’It.’u.1||l‘. 0.5." I ,3. av o (shun) AgAy \ 5S! 31:; Ed 92.0.5 379 x3“ he :53 ecfzex 18x: EVIQ x N .6 .550 Ecficoe c938. s cm x: 20ch e E9... sate Ce .5 cpEEScS .2: E» 3.2%» ethE c352 nfimcozge ac: .0 2:5 29 8x33 Em Eastwoceoz _E\$_c e6 EEJZ m, 1 v ._ N _. 1 11fi1 11,. q 1 \ \ e\ .oe I. .5 3.0 e c._ .ExNE .5 19.22-05 zn..o_.xme.m no .Emmd . Em ecEEexOQ z EVIQXN .6 E30 .ocfficoU BJEE 8.88. a Dawn. 5:33 .5 _E_ c. ENEeooEocmeE m are .e .N._ 88 8:58 eExNE e0 65:85 .Ee bite. eExNCe c8353 95828 E» N .59: 0 N (I; V V W a 8 MW: (sum) 0 00 .r ON— 30 Exewwm we UeramoEon _E0\me_c 00 set: ow ON. 0. m. . Fl. 4 q 000 . .. . . .3 In eetgzé 2.0 . E E. “£23385 egg... .6 .830 Em. o4znofih Emsgx med Lo E500 Hecéceéef 2 v.19 Xmao _E No.0 EEEES .2:an c253. e aces. 5:2 .6 EB c. ENEevoEoL we: 0e. use .0m.0. .0 80¢ , 8:58 case we smears..ch Ea 55% 890:: 59,23 . 258233. 2.: .w 232... 0 V LWDV (sum) A 0 co ..0m_ :09 31 Test of the reliability of single fly assays were carried out using several inbred and several non-inbred stocks. Flies used for these assays were all one day old, each being collected and immediate- ly frozen until homogenization. The results are given in Table 3. An analysis of variance of these data has been performed by Dr. James Kan and is given in Table h. To be significant at the 1% level, an F value must equal or exceed 3.31.. This is true only in the case of variation between stocks 3 i.e., there are significant differences in the enzyme activities exhibited between stocks, but not between sexes in general or between sexes within each stock. Enzyme Purification: Due to the extrce likility of the enzyme, a method of purification had to be designed which would treat the enzyme in the most gentle way. Accordingly the following scheme was evolved which resulted in a 528 fold purification of the enzyme (figure 9)- Flies were homogenized in a 2.5 (w/v) ratio of 0.1 M Tris buffer, pH 8.0, which was 5 mg/ml with respect to crystalline .m. bovine albumin. All procedures were carried out at below 5’ C unless other- wise specified. The resulting homogenate was centrifuged for 30 minutes at 30,000 x g in an International Model HR-l centrifuge. The precipitate was discarded and Norite-A was added to the supernatant at a concentration of 100 mg/ml. This was allowed to stand with occasional stirring for 60 minutes at the end of which time the solution was recentrifuged at 30,000 x g for 20 minutes. The result- ing supernatant was poured through a coarse sintered glass filter to Ll- ,\ t 'W ‘1 \, \e C , J. L\' "\ _ ‘74 l ' l ., r. . “ r7 P» k. .. 1. ,.. ‘ L. ; Ii _ \ rm ._,’A‘ . \_‘~ A ‘. . (_; \ ' I .- a '3 x,“ J ’1 TJ 32 Table 3. Test of the reliability of single fly assays using several inbred and non-inbred stocks. Stocks and Sex Activity of single flies Hean.and StandardjError Samarkand male 28.h 27-3 27.5 28.h 26.0 25.0 i = 27.100 3: 3‘ Oe56 Samarkand female 2A.0 28.0 23.9 27.8 28.0 28.0 ‘i' = 26.617 Si '3 0.85 Swedishuiale 27.6 27.6 25.h 28.2 26.6 28.6 'x‘ = 27.333 31 a 00h? Swedish female 28.6 28.6 29.3 26.3 28.7 28.7 i = 28.033 32 = 0075 "a2 male 15.h 15.8 17.8 16.3 17.5 18.0 i. = 16.800 31 g 0.115 w‘2 female 19.0 17.0 19.3 16.0 15.0 18.8 i = 17.517 Si ‘7‘ 0e73 \J \l L '- ‘n-vr - Table 3.-- continued 33 Stocks and Sex Activity of single flies Mean.and StandardiError Oregon-R-Sydney male 27.6 27.4 27.4 27.7 27.8 28.0 i = 27.65 S: = 0e096 Oregon-RqSydney female 28.0 28.1 27.6 27.6 27.6 28.0 i = 27.783 3x = 0.12 Oregon-Rplnmale 2h.5 23.7 25.8 28.8 26.3 26.5 if = 25.267 51 = 0075 OregonAR-I female 24.3 26.7 26.9 28.3 20.0 22.0 i- z: 2‘6e367 Si = 1.18 113- Sydney male 18.0 23.0 28.6 27.6 28.0 29.0 if = 25.700 3x e- 1.78 w3- Sydney female 17.0 18.3 25.6 27.6 28.0 23.0 i = 23.217 Si 2 le92 -0-- 36 Table A. Analysis of variance of enzyme activity in single fly assays among six stocks of Drosoghila melanogaster. Source of variation Degrees of Mean F Freedom. Square Between sexes l 2.68 - Between stocks 5 190.96 33.2 Sex and Stock 5 6.41. - Error 60 5.76 - 35 Figure 9. Flow diagram of the scheme of purification of xanthine dehydrogenase from Drama melanogaster. Hecipitate (discard) Procipitate - (discard) Precipitate (discard) Precipitate (discard) Supernatant (discard) Precipitate ' (discard) Supernatant (discard) Homogenate (l) Centrifuged, 30,000 x g, 30 min. Supernatant (2) Horite-A’ 100 mb/mo' 60 Kline Centrifuge 30,000 x g, 20 min. Supernatant (3) Filter through sintered glass filter. Filtrate (1.) 50's, 10 min. Centrifuge 30,000 x g, 20 min. Supernatant (5) Adjust to pH 5.0 Centrifuge 30,000 x g, 20 min. Supernatant (6) Adjust to pH 8.0. Brought to 80% (NH )280 saturation. Centrifuge 30,000 x g, 20 min. Precipitate (7) DiSCOIVO in 0e]. 1‘! Tri', pH 8eOe Brought to 25% (NHQgSO saturation. Centrifuge 30,000 x g, 0 min. Supernatant (8) Brought to 50% (NHhhSOh saturation. Centriguged 30,000 x g, 20 min. Precipitate (9) Dissolved in 0.1 M Tris, pH 8.0. Passed over DEAR-cellulose column. Purified Emma (10) ' re 36 remove any remaining charcoal. An increase in total enzyme activity was characteristically observed after passage over Norite. ‘ The filtrate was then heated to 50' C for 10 minutes, cooled immediately, centrifuged as above, and the precipitate discarded. The supernatant was then adjusted to pH 5.0 with l M acetic acid. This was inmediately centrifuged, the precipitate being discarded and the supernatant being adjusted to pH 8.0 with 0.1 M NaOH. A saturated solution of ammonium sulfate was then added to give a final concentration which was 80% saturated. This was allowed to stand for 60 minutes at the end of which time the solution was centrifuged, the supernatant discarded, and the precipitate redissolved in 0.1 M Tris buffer, pH 8.0. To this solution was added a saturated solution of ammonium sulfate to give a final concentration of 25% saturation. After 1 hour the solution was centrifuged, the precipitate discarded, and the supernatant brought to 50% saturation with ammonium sulfate. This was allowed to stand for 1 hour at the end of which time the solution was centrifuged, the supernatant discarded, and the pre- cipitate redissolved in 0.1 M Tris buffer, pH 8.0. Samples of this solution were then added to a DEAE-cellulose column (1.5 cm x 26 cm) previously equilibrated with 0.1 M Tris buffer, pH 8.0. The column was then eluted with 50 ml of 0.1 M NaCl in Tris, and then with 50 ml of 0.15 M NaCl' in the same Tris buffer. 5 ml samples were collected, protein being determined by the method of Warburg and Christian (1942). These samples with protein were assayed for enzymatic activity. Figure 10 shows the elution pattern of the column chromatography. Fractions 11, 12, and 13 were pooled '4 37‘ a/( p P53; [Lul'Q/SHUH 19101 (a 9 8 Q (a) 00. NJ 9— Q A 35% ecEEex ao confine awnnwglfx .@.t: a PC my .oC cmutumi Quest. éfiatflhfldf‘ll [3.2.5. i .\i. / i. .3..- _ a o\ oi! _ i _ 1 _ i . _ i o d _ 1 / m. _ i . o .u.. _ ii m u \ md. _ \ w ID _ i / m x W _ a O. 1 Q1 cm A . 83 die ”531:6 61:13 ea EESQ 32:8 3:31 ,- who m :0 56:8 :83E .9 2%: 38 to give the final purified enzyme preparation. Table 5 indicates the steps in the purification and the assay of each stage of purification of the enzyme. Specific activity is in terms of units of actifity per mg of protein. Kinetic characteristics of the purified enzyme: Using samples of this purified preparation, Michaelis-Menten constants were determined for hypoxanthine, xanthine, NAD, and thio—NAD. The Km for NAD was found to be 2.5 x 10-6 M (figure 11) 3 for thio-NAD, 2.3 x 10-5 M (figure 12); for hypoxanthine, 2.0 x 10’5 M (figure 13); for xanthine, 2.36 x 10-5 M (figure 11.). By way of comparison, the Km's exhibited by crude extracts were as follows: NAD, 3.25 x 10“ M; hypoxanthine, 2.032 x 10"5 M. The maximum velocity of reaction attained using xanthine as sub- strate was found to be 1.0% of that with hypoxanthine as substrate. The pH Optimum for the enzyme was found at 8.0, decreasing in activity above and below that pH (figure 15). Studies involving the stochiometry of the conversion of hypoxan- thine to uric acid were also attmpted. In this instance a known concentration of hypoxanthine was added to a reaction mixture contain- ing a known excess of thio-NAD and the reaction, after addition of enzyme, was allowed to run to chletion. From the change in absor- bance at 395 mu, the amount of reduced thio—NAD formed could be cal- culated. In the specific case, 1.0 x 10'"7 moles of hypoxanthine and 3.43 x 10'6 moles of thio-NAD were allowed to incubate together in a reaction mixture of 3 ml with excess enzyme. The progress of the f‘ 39 0006 mw\g ooom coco OOON Equnz X ©.NnuCLX .3233 8 msg#90283 £3. .o o _. E EOIQ X @VM H X :00. , . 59:8: 8333 use x2. 8:: £233 imam. 65:53 me ecfEexaxf £3, 2262,: .6 6058638 fiLEex 5*. Ex. a: 10 cozggemu Eh .m. 2391 41 Q. x W\: N . e me- .Ib.lues . lla1.’.lesr“1‘ \ . 20.0. x canes 2:3 0m uer> . .5388 85% me. 942 £3, 0:51:6an? .8 3880638 ecEEeX .6 Ex 3.10 EQEEEEE er: .9 239.... o. x. m: mi... 6. e 1 .‘ .03 g at. g «Q. 2 To. x new nEx 35 mm. ux§> 588.8 :02er me 042 2:3 Eric? .8 3882338 ecEEex a0 Ex 8: 10 8:28.28 2: 3332.1 6N .VOt 00A mo. 0.. .N_. 43 Table 5. Purification of xanthine dehydrogenase W 22° .2222. 221 2222 22222; W2 .222. l 20 10 200 48.87 0.202 - - 3 17 618 10,506 39.68 15.6 100 1 5 lb 571 7,990 20.63 23.0 76 1.L7 6 13 577 7,501 15.29 27.7 71 2.42 7 5 IAOO 7,000 8.88 157.5 66.5 10.1 8 6.5 1000 6,500 5.43 186.5 61.8 11.8 9 5 1000 5,000 1.46. 695 A7.5 4h.5 10 5 990 4.950 0.12 8,250 47.0 528 *Refer to Figure 9. 44 .1“. 2 bum .r. - 0% i2 .2 on To. / ‘on IA /. . . w ./ o‘l‘lo\ a D. . .\ 20mm. / \ , a. 0(0\ rOWW 6 9 8:22 2 53:3 mEchm .9 2:9 .1 #5 reaction was followed at 395 mu. When Optical density reached a maximum the change in absorbance was noted, and from the known molar absorbancy of reduced thio-NAD the amount of this compound formed was calculated. The molar absorbancy of reduced thio-NAD is 11.3 x 103. The change in absorbance at 395 m was found to be 0.710. This is equivalent to 1.89 x 10"7 moles of reduced thio—NAD formed. 0n the basis that 1.0 x 10-7 moles of hypoxanthine were initially added, this would imply that 2 moles of NAD are required for the conversion of 1 mole of hypoxanthine to uric acid (Table 6). Similar experiments were carried out with xanthine as substrate and NAD as electron acceptor (Table 6). The results of this experi- ment indicated that the conversion of 1 mole of nnthine to uric acid involved a concomitant conversion of 1 mole of NAD to reduced HAD. The reverse reaction, that is the conversion of uric acid to hypoxanthine with the concomitant oxidation of reduced NAD was attempted using reaction mixtures containing various concentration of uric acid and reduced .NAD. Activity in the reverse reaction would be indicated by a decrease in optical density at 340 mu. In no case was this observed. It is interesting, however, to note that this phenomenon did take place in certain extracts prior to treatment with Norite-A. This finding could be indicative of a systan capable of oxidizing reduced NAD in the extracts. Flielsin and Schon (1957) have previously described such a system in 2. melanogaster. Reaction mixtures containing uric acid in concentrations as high as 7 x 10-5 M were also used to test for the possible inhibition of the forward reaction by that compound. Such inhibition would be 1+6 Table 6. The stoichiometry of the conversion of hypoxanthine to uric acid by xanthine dehydrogenase. Experiment Substrate Electron Reduced electron Mole of Elec- Added acceptor acceptor formed tron Acceptor added reduce per mole of substrate 1 Hypo- thio- 1.89 x 10"7 1.89 xanthine NAD moles 1 x 10-7 3.43 x 1.0-'7 moles moles 2 Xanthine, NAD, 0.33 x 10-7 1.1 0.3 x 10-7 0.33 x 10-5 moles moles moles . 47 suggested by a lessened increase in absorbance at 31.0 mu in compari- son to assay mixtures having the identical concentration of substrate, electron acceptor, and enzyme, but lacking uric acid. No such inhibition was indicated in any .2... _I_n_ 22.9. complementation: An in £5.53 complementation experiment involving extracts of the two non-allelic mutants deficient in ‘ xanthine dehydrogenase was also attempted. Extracts were prepared from maroon-like and M mutants on the presummion that maroon- 1ike should contain the normal rgsfi substance and 225.3112. extracts would contain the normal maroon-like substance. By incubating the two extracts together under specified conditions, it was hoped that the two pieces would come together in the structure necessary for xanthine dehydrOgenase activity. Flies of both mutant types were homogenized separately at 5' C in 0.1 M Tris buffer, pH 8.0 in the usual fashion. The homogenates were then centrifuged at 30,000 x g for 30 minutes and the resulting supernatant was adjusted to pH 5.0 with 1 M acetic acid. They were centrifuged immediately, and the resulting supernatant was readjusted to pH 8.0 with 0.1 M NaOH. This preparation was modified in some cases by treatment with Norite-A prior to the pH 5.0 fractionation. This involved adding 100 mg of activated charcoal per ml of homogenate. The miicture were allowed to stand with occasional stirring for 1 hour and then centri- fuged to remove all charcoal. The charcoal-free supernatants were then adjusted to pH 5.0, centrifuged, and readjusted to pH 8.0. ~v 1+8 In each case, the extracts were adjusted to approximately equal protein concentration. 0.5 m1 of each extract alone was then assayed to test for enzymatic activity. Gunplementation experiments were performed by mixing equal volumes of £3 and m_a_.;]_. extracts, incubating for 1 hour at 30’ C, and assaying 0.5 m1 of the mixture. In addition to 0.5 m1 of extract (single or mixed), the complete assay mixture contained 0.1. m1 of 5.1 x 10-3 M hypoxanthine and 0.1 ml of 1.37 x 10'“3 M thio-NAD. Enzyme activity was measured by rate of increase in optical density at 395 mu. The results obtained with the extracts that were not treated with Norite-A are given in Table 7. As may be noted, a slow increase in optical density was observed in the presence of enzyme and thio- NAD even without added substrate. Such an increase was remarked above in discussion of the dependence of enzyme activity on substrate and electron acceptor, and probably reflects the presence of endo- genous substrate in extracts prior to Norite treatment. There occurs, however, a marked increase in enzyme activity when the mixture of i and m_a_,:_]_._ extracts is preincubated prior to assay. This increase is even more marked when Norite treated extracts are used (Table 8). In this case no endogenous reduction of thio- NAD occurs, and activity is observed only with preincubated i and ma-l mixtures in the presence of both hypoxanthine and thio-NAD. \ r. A9 Table 7. Results of the complementation experiment with extracts not treated with Norite-A. All reaction mixtures contain 20.2 mg of protein. Enzyme activity (units) i m__a_-;1_ Mixture Complete 3.0 12.0 16.25 - Hypoxanthine 5.3 4.0 1.25 - Thio-NAD 0 O O - Hypoxanthine and Thio-NAD 0 0 O Table 8. Results of the complementation experiment with Norite-A treat extracts. All reaction mixtures contain 18.8 mg of protein. Enzyme activity (units) i ma__;___-1 Mixture Complete 0 0 22.0 - Hypoxanthine 0 0 0 - Thio-NAD 0 O 0 - Hypoxanthine and Thio-NAD 0 0 0 V. DISCUSSION The results indicate the successful development of an assay method for the enzyme xanthine dehydrogenase from Drosgphila melanogaster. This assay is sensitive enough to detect enzymatic activity in a single fly; the measured activity is linear with respect to enzyme concentration. Furthermore, the assay has none of the shortcomings of the fluorometric assay previously described. The assay was utilized in the testing of enzymatic activity of single flies from several different stocks. Statistical analysis of the results of these assays indicate no significant differences in enzyme activity between miles and females of any stock, but a sig- nificant difference between stocks. The causes for this variability are probably genetic, but do not involve the n or ma___-_-_l_ loci. A 500 fold purification of the enzyme was accomplished and deter- minations, using this purified preparation, were Iade of the Michaelis- Menten constants of various substrates and electron acceptors. Using NAD as the electron acceptor a Km of 2.0 x 10'"5 M was found for hypoxanthine; 2.36 x 10'“5 M for xanthine. This is in close agreement with Glassman (1959) who reported a Km of 2.1 x 10"5 M for hypoxanthine and 2.5 x 10-5 M for xanthine. Using hypoxanthine as substrate, Km" were also detemined for NAD and thio-NAD. These were found to be 2.5 x 10‘” M and 3.1.6 1: 10"5 M respectively. The difference in Km's suggests that the enzyme has a higher affinity for thio-NAD than for NAD. The explicit causes 50 . l e .. . ' u . . ‘ a .I ‘ . . . K . . - ‘ I \J a. \ 2‘ —A. .- l ‘. - A .. ~ . , i ‘ I ' ,» L J . . v . 0‘ A - -‘ . k g , “or. _ ‘ . . . _ . O I I O . , . O. . . k4 51 for the differences observed are unknown. The stiochiometry of the reaction catalyzed by xanthine dehydro- genase was also studied using the purified enzyme. The results indicate that conversion of a mole of hypoxanthine to a mole of uric acid requires the reduction of 2 moles of NAD. Similarly, the con- version of a mole of xanthine to a mole of uric acid requires the reduction of only 1 mole of NAD. The inability to run the reaction in the reverse direction suggests that the equilibrium for the reac- tion lies far to the side of uric acid. This is perhaps also indi- cated by the fact that relatively high concentrations of uric acid in reaction mixtures do not inhibit the forward reaction. Results of the complementation experiment involving extract of my: and maroon-like, each of which is deficient in regard to xanthine dehydrogenase, suggests that each locus in wild type form synthesizes a necessary part of the enzyme. This is also in agree- ment with the findings of Glassman (1962), though it in no way indicates the exact commsition of each part. The data could be interpreted in terms of a "combining-subunits“ hypothesis in which each locus produces a protein, both proteins being necessary for enzymatic activity; or in terms of a "catalytic-activator” hypothesis in which the product of one locus is activated by some product or action of the other locus. Unfortunately, no method has been devised to test either of these hypotheses. Forrest, Henley, and Lagowski (1961) have reported enzymatic activities in extracts of 50% which are not present in extracts of maroon-like, e.g., the conversion of pyridoml to pyridoxic acid and 52 h-hydroxypteridine to 2, h-dihydroxy pteridine; neither of these reactions require NAD. These reactions may be able to serve as an assay for the product of the wild type maroon-like locus. However, no reaction has been found which is restricted to maroon-like extracts. It is known that at least 2 different loci control the formation and activity of xanthine dehydrogenase in DrosopL . However, the situation may be much more complex. Glassman (unpublished data) has reported the existence of strains of wild type 2. melanogagter which exhibit different levels of enzyme activity. This perhaps suggests some form of quantitative inheritance; that is, quantitative genes controlling xanthine dehydrogenase levels in the flies. The results of the test of reliability of the single fly assay reported in this work may also indicate the same situation. Schepers (1962) has also reported an interaction in pteridine metabolism involving alleles of the mutants gang}; and 111219. in Droflhila. He reports that Z-mino-A-hydroiqpteridine is present in both single mutants, but in lower concentration than in wild type forms. The activity of xanthine dehydrogenase in those mutants is the same as in wild type. However, flies which are mutant with re- spect to both of these alleles completely lack 2-amino-lt-hydroxypteri- dine and exhibit a reduction in xanthine dehydrogenase activity as compared to wild type. This observation suggests some type of mechanism controlling xanthine dehydrogenase activity. If such is the case, it will be the first found in Q. melanogaster and 1011 be invaluable in the study of genetic control mechanisms. ~r‘ VI. SUMMARY 1. The purpose of the work reported was to study the biochemical characteristics of xanthine dehydrogenase from DrosOphila melanggaster. This involved the develOpment of an assay of enzymatic activity which was linear in relation to enzyme concentration and was sensitive enough to detect activity in single flies. The assay is based on the change in optical density at 31.0 mu as NAD is reduced or at 395 mu as thio-NAD is reduced, with either mthine or hypoxanthine as sub- strate. Tests of reliability of the assay were performed on single flies from various inbred and non-inbred stocks. 2. A method of purification was devised resulting in a 528 fold purification of the enzyme, the purified enzyme having a pH Optimum Of 8000 3. Using this purified preparation of the enzyme, Kn's were deter- mined for hypoxanthine, xanthine, NAD, and thio-NAD and found to be 2.0 x 10-5 M, 2.36 x 10-5 M, 2.5 x 10-4 M, and 2.0 x 10-5 M respec- tively. it. Stoichiometry of the reaction was studied using purified prepara- tions and the results indicate the reduction of 1 mole of NAD for each mole of xanthine converted to uric acid and the reduction of 2 moles of NAD for each mole of hypoxanthine converted to uric acid. 53 54 5. A complementation experiment was carried out using extracts of maroon-like and ros . Prior to incubation tagether, neither of the extracts exhibited any xanthine dehydrogenase activity. 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