RABBIT SKELETAI. MUSCLE 5’-A.MP AMINOHYDROLASE: SOME PHYSIOCHEMICAL PROPERTIES AND CHARACTERIZATION AS A ZII‘éC METALLOENZYME Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY CAROL LOUISE ZIELKE , 1970 K $V 1HF am ooasom Ommaoaommocaa4 nfifim .wo mQOHPMngmhm Hahmbmm gnu. Coapmsaammm 9.94 .HOM meMPmQOU OHPmQHM .H mHDME .eeoae aeooeefioee magnoe seam eenfiepno eye: mesame geese .mmwmm pudendum on» pom pom: mag mo godpmnpnoonoo esp pm Soapomam pmonsa esp mo anabdpom canaooam on» > one canon was» Ca oopHOQOH mafia» on» ma am when; zoapmsvm noncozlmaammsoaz esp Scam oopmHSOHmo mos Nma>d Ammo wee edeeem Ammo ma< woo Ammo gee sexedeo Admv Snowman poopmo on +mo .+pm .+m ego eopaeaeeH eemzAn+QZAA+fiq eoeeee on +smz Ammo peemmee seedeaeed +mo .+em .+a wee popeeaeoe +ez n +Hq Ammo me: u m9o.A mBHAntHpo< ooHpOOHosz one Soapmo pSOHmbozoz .N OHQOB 10 Ammv +ez oenaepnoo memmn a: Anny 83 $3 Amflv Aomv +m oendepnoo memme poemme on oen mnoHpeo pneaebonoz oenanoem man no monomeha nH mano oonanooo +ez no +m ho eonewne na poemme oz oendnoem oenanoea pon pep epebapon oendnoea poemwe oapwawaenzm man .men . . a +H1H +M +e: AS Ame... n a: me... poeoee 0: mam +a +nm 8.3 .+ez PHD .86 .93 AAmB< S . +mz ta +VH .+ez +VH .+ez +ez + wee pem nebfiq pappem won one peo onfiom oneanaez eHDSHom neanm meeweoéeuem Dem oHQSHom epeazoaphem 11 ANNV 9H4 nexoano ASSV woe .+o pem Am .mev mos .eee mo AA Lav +sz.<+em.A+Hqu+ez u en paeeem Ammo Hos eH maeu.m.nea< . m 38 +73 M: AA++ezA+3 a eeeeee 0: .+mo +SmZAA+nmAn+a gene noneapoameam :av nEAnEBARSAmoAtL Lees nae memo oHomna .mem mpnenaoo Hopebapon eohnom hoeeeneeoov .N wanes 12 AN: 33 HUM SH +ez A +SA emz m94 oeem eem mHHeo Honda mefiome genera 13 enzyme where lithium is more effective. Although Li+, Na+, Rb+, 03+, and NH4+ can Often substitute for K+, the relative order of effectiveness is not consistent when enzymes from different sources are compared. Monovalent cations are not required for the brain, muscle, and rat liver enzymes since the sameVma was observed at high x concentrations Of AMP in either the presence or absence of cations. However, the enzyme associated with a brain particulate fraction (18), Ehrlich Ascites Tumor cells (45), and the human erythrocyte membrane (19) is reported to absolutely require K+ for activity. The soluble human erythrocyte enzyme (which constitutes 85% of the total AMP aminohydrolase content of this cell) was activated only by K+ and NHL,+ (37); activation by Na+. Li+. and Rb+ required the presence of ATP. ATP alone did not activate the soluble enzyme but did lower the effective concentration for K+ activation. In contrast the cat and dog erythro- cyte enzyme were activated by ATP but not by monovalent cations either in the presence or absence of ATP (37). In the absence of activators, AMP aminohydrolase from brain (46), erythrocytes (41, 47), muscle (43), and liver (27) give sigmoid curves for velocity versus AMP concentration which become hyperbolic after the addition Of monovalent cations, adenine nucleotides, or a combina— tion of monovalent cations and adenine nucleotides. That is, for the rabbit muscle enZyme (43), addition of K+, 14 ADP, or ATP give normal hyperbolic saturation curves for AMP as represented by a change in the Hill lepe, nH, from 2.2 to 1.1; Vmax remained the same. On the other hand the soluble erythrocyte enzyme (47) and the calf brain enzyme (28) required the presence of both monovalent cations and ATP before saturation curves became hyperbolic. In con- trast, the bound human erythrocyte membrane enzyme did not exhibit sigmoid saturation curves and K+ activation was not affected by ATP (19). Inhibition A variety of anions such as inorganic phOSphate (22, 25, 2, 41, 48-50), sulfate (50), nitrate (50), pyro- phOSphate (2, 29), tripolyphOSphate (29), carboxylic acids (32, nu), 2,3-diphOSphoglyceric acid (47), fluoride (12, 25, 2, 36. 48, 49), 3'-AMP (25, 29, 32), GTP (27, 40, 43), GDP (43), and 3-iso AMP (29) have been Shown to inhibit AMP aminohydrolase. GTP inhibits the ATP activa- tion of the enzyme from rat brain, heart and liver, calf brain, and rabbit muscle but had no effect on the elasmo- branch fish muscle enZyme (25). In the case of the calf brain enzyme GTP inhibition appears to be competitive for ATP [Kiapp = 10 HM]. 3-Iso AMP was not a substrate but was an effective competitive inhibitor for substrate in the brain [Kiapp = 60 uM] and muscle enzymes. Inhibition of the erythrocyte enzyme by physiological concentrations of 2,3-diphosphog1ycerate may prevent early depletion of 15 the adenine nucleotide pool, an important physiological factor since mature erythrocytes lack enZymes for synthesis of the adenine ring and AMP (47). Carboxylic acids have been reported either to have no effect (2, 28), to activate (35), or to inhibit (32) AMP aminohydrolase. While the activation of the rabbit muscle enzyme was not thoroughly examined (35), the reported inhi- bition of this enzyme by citrate, succinate, and maleate was most effective in the absence of activators or in the presence of ADP (32). Limited kinetic data were consistent with a competitive interaction between citrate and ADP. The enzymatic activity in intact myofibrills was activated by ATP, ADP, and ITP in succinate buffer but not in citrate buffer (51). With the rat enzyme citrate, succinate, cacodylate, acetate, and lactate decreased Kmapp for AMP and increased the Hill lepe; V decreased only slightly max (1H9. At physiological concentrations of lactate, nucleo- side triphOSphates actually inhibited; ADP and GDP reversed these inhibitions. The inhibition by carboxylic acids, o-phenanthroline, and dithioerythritol led to the discovery of 2.8 gram atoms Zn2+ per 300,000 grams of rabbit muscle AMP aminohydrolase (32). The reported inhibitions by an+ (2, 36), Cu2+ (2, 28, 52), Fe3+ (2), Ag+ (2, 52), Cd2+ and Ni2+ (2, 28), and Hg2+ (35, 36) might be eXplained in terms Of an interaction with a sulfhydral group(S) necessary for catalysis or by a 16 diSplacement of the presumably required Zn2+. Iodoacetate had no effect on the rabbit muscle enzyme (2, 35) but did inhibit the carp muscle and pea seed enzyme (12, 36). Organic mercurials are also reported to inhibit the enzyme from several sources (2, 12, 2h, 25, 36). Except for the preliminary report by Wolfenden.gt a; (30) that mercurials desensitized the rabbit muscle enzyme to allosteric inhibition by GTP, the role of sulfhydral residues in AMP aminohydrolase is not understood. Mechanism Except for the fact that deamination of AMP by the rabbit muscle enzyme was irreversible between pH 6.0 and 9,0 (2) a detailed discussion of the mechanism for 5'-AMP deamination is at present premature. The sigmoidal rela- tionship for substrate saturation and activation by mono- valent cations and adenine nucleotides is consistent with mechanisms involving active site-effector site interaction. The activation brought about by this site-site interaction is a relatively slow ist order process independent of pro- tein concentration (53) and is comparable to observations reported for yeast glyceraldehyde-B-phoSphate dehydrogenase (54) and homoserine dehydrogenase (55). The activation was discussed in terms of a simple scheme similar to those pro- posed by Rabin (56) and Weber (57) which provides a plaus— ible explanation for the sigmoid curve for initial velocities versus substrate concentrationwithout involving 17 additional phenomena such as cooperative interactions between catalytic sites. The hydrolytic deamination catalyzed by rabbit muscle AMP aminohydrolase may be facilitated by Zn2+ in contrast to the mediation of a common purinyl enzyme intermediate for adenosine aminohydrolase catalysis CfiB,59). Considerations of Physiological Function The physiological role of AMP aminohydrolase is not clearly understood. Enzymatic activity in muscle is markedly reduced in the dystrOphic mouse (60, 61), in humans suffering from Duchanne type muscular dystrOphy (62), in hypokaliemic periodic paralysis (63) and upon denerva— tion of normal and dystrophic mouse gastronemii (64). Activity is reported to increase in both tranSplanted and primary hepatomas (48) and in precancerous livers prior to the onset of neOplasia induced by feeding or by intra- abdominal injections of the potent carcinogen 3'-methyl-4- dimethylaminoazobenzene (65). The weak carcinogen 4'— methyl-4—dimethylaminoazobenzene was not effective (65). Increases in enzyme activity concomitant with altered nuclear-nucleolar morphology, nuclear RNA content, and nuclear RNA biosynthesis were also observed after injec- tions of thioacetamide, a hepatocarcinogen (66, 67). AMP aminohydrolase activity was low but distinguish- able in the leg, diaphragm and heart muscle of a 20-24 day old rabbit fetus (68). The activity in the heart remained 18 low in both neonatal and adult life, whereas a rapid increase occurred in the activity of the enzyme in the diaphragm during the 4 or 5 days before parturition reach- ing a maximum activity immediately after birth. In con- trast the enzymic activity of the mixed leg muscles remained relatively constant until 8-9 days after birth when it began to rise steadily reaching an adult value of 7-8 times that of the fetal muscle within 14 days. This increase occurred when the animal began independent move- ment. Similar but qualitatively different effects were observed with guinea pig and rat leg muscle and chicken leg and pectoral muscle. Increases in aldolase, myokinase and creatine phoSphokinase activity were roughly parallel to increases in AMP aminohydrolase activity. Although it has been reported that increased AMP aminohydrolase activity occurred during prolonged stimu- lation of muscle bundles (69-71), the participation of this enzyme in the contractile process seems unlikely in light of the lack of significant changes in the levels of AMP and IMP during a single contraction of frog abdominal muscle (72). This is corroborated by the absence of AMP aminohydrolase activity in muscle of some invertebrates (73-75) and in human uterine muscle (76). It is tempting to consider regulation of the con- centration of AMP, a known effector of several glycolytic enzymes (77), by the antagonistic action of adenine and 19 guanine nucleotides on AMP deamination as a control factor in glycolysis and gluconeogenesis (77). Setlow gt a; (40) suggested the participation of AMP aminohydrolase in a self regulating system for purine nucleotide interconver- sion as presented in Figure 1. AMP % Fumarate Adenylosuccinate IMP GDP + P1 ASpartate + GTP I I I I : XMP : ATP + glutamine I ‘ C : 9 AMP + perphOSphate ' \L + glutamate I I GNP I : I '-—--GTP Figure 1. Purine Nucleotide Interconversions As the GTP concentration decreases, AMP aminohydrolase inhibition is released with a concommitant increase in IMP and GTP which completes the self-regulating system by inhibiting the AMP aminohydrolase. In the case of the rat and calf brain enzymes, the ATP activation and GTP inhibi- tion were observed at the overall inngzg concentrations of these nucleotides and AMP (28, 29). However such a 20 control mechanism based upon kinetically observed changes with an in 31232 system is subject to presently undefined effects by other factors in zlzg. (For example, although AMP could be postulated as a control factor of glyco- genolysis in muscle 333 its activation of phOSphorylase b, Helmreich and Cori could not detect a sufficient increase in AMP concentration in 3112 during electrical stimulation of muscle to eXplain the rapid increase in phOSphorylase activity (78).) At present only preliminary data exist as to (A) the effects of divalent metals such as Mg2+ and Ca2+ upon the activation and inhibition by nucleotides (34, 44) and (B) the effects of anions other than nucleotides (32, 44). An additional factor, the availability of substrate, activators, and inhibitors to the enzyme in the cell has not been investigated. Consequently, the control and func- tion of AMP aminohydrolase remain interesting questions. MATERIALS AND METHODS Enzyme Preparation AMP aminohydrolase from rabbit muscle was prepared by the method of Smiley, §§_§l.(l). Since the crystalline enzyme was less stable than enzyme obtained directly from cellulose phOSphate and since it showed evidence of hetero- geneity upon ultracentrifuge analysis, the eluted enzyme in 1 M KCl from the cellulose phOSphate column was used directly for all eXperiments. Although previously reported yields for this preparation were 36%, essentially 100% recovery of enzymatic activity from the column can be achieved if the amount of 0.45 M KCl wash solution is kept to 500 ml or less per pound of muscle. The enzyme was stored as previously described (1). For those eXperiments where it was desirable to remove the activator, K+, (43) the enzyme was chromatographed on Sephadex G-25 equilibrated with tetramethylammonium chloride (TMACl) at either pH 6.3 or 7.1 and 1 mM in mercaptoethanol. Protein Determination Protein concentrations were determined by measurement of absorbance at 260 nm and 280 nm according to the method of Warburg and Christian (79) or from the absorbtion 21 22 coefficient of 9.15 0D for a 1% solution at 280 nm in a 1 cm light path (see page 29 for a description of this determination). Kinetic Assays Kinetic assays were run on a Beckman DU Spectro- photometer equipped with a Gilford Optical density conver- tor with offset and a Sargent SRL recorder. Temperature was maintained at 30° with a circulating water bath. For AMP concentrations less than 0.2 mM the decrease in absorb- ance at 265 nm was measured according to the method of Kalckar (80); at AMP concentrations greater than 0.2 mM and 5 mM the increase in 0D was determined at 285 nm and 290 nm, reSpectively. The change in absorbance per minute was converted to umoles substrate deaminated per minute by the relationship: umoles/minute = llOD per minute/F where F is equal to -8.86 at 265 nm, +0.30 at 285 nm, and +0.12 at 290 nm (53). Specific activity is expressed as umoles substrate deaminated per min/mg protein. Three basic assay systems were used: Assay 1, the KCl activated system, contained 0.15 M KCl, 0.05 M Tris- cacodylate,pH 6.3, and 50 uM Tris AMP unless otherwise indicated: Assay 2, the ADP activated system, contained 0.10 M TMACl, 0.05 M Tris cacodylate,pH 6.3, 100 uM Tris ADP, and 50 uM Tris AMP unless otherwise indicated; 23 Assay 3, the method used to determine activity in the absence of activators, consisted of 0.10 M TMACl, 0.05 M Tris cacodylate,pH 6.3, and Tris AMP at the concentrations indicated. The enZyme was diluted into 0.5 M KCl, or 0.5 M TMACl, 0.05 M Tris cacodylate, and 1 mM mercaptoethanol before assay unless stated otherwise. Reagents All reagents were ACS Reagent grade or the best grade available. Spectropure sulfate salts (Johnson, Matthey and Co.. Ltd.) of zinc, cobalt, ferrous, magnesium, manganese, nickel, and cadmium were obtained from Jarrell- Ash Co. of Waltham, Massachusetts. Stock solutions of 1000 ppm metal were prepared with analytically weighed samples. Harleco atomic absorption standards (1000 ppm) for Cusou and CaClz were supplied by Scientific Products. All solutions were prepared using double distilled water, the second distillation being done in an all glass appara- tus. Trizma Base (Tris), cacodylic acid, mercaptoethanol, dithioerythritol (DTE), o-phenanthroline (0P), ethylene- diaminetetraacetic acid (EDTA), cellulose phoSphate, and Sephadex G-25 coarse were obtained from Sigma. The TMACl, 8-hydroxyquinoline-5-sulfonic acid (8-OHQ5SA), and m-phenanthroline (Alfred Bader Chemicals) were from 24 Aldrich Chemical Co. Tetramethylammonium hydroxide was supplied by Mallinkrodt, methylamine by Eastman Organic Chemicals, Chelex 100 (Na+) 50-100 mesh by Bio Rad, and dimethylsulfate by Matheson, Coleman, and Bell. The TMACl was recrystallized twice from hot abso- lute ethanol and then passed over Chelex 100 (Tris+) to remove divalent metal ions. Carboxylic acids were recrystallized from water or organic solvents, treated with Chelex 100, and adjusted to pH 6.4 with Tris base. A Sargent Model SL pH meter equipped with a 30070-10 com- bination electrode was used for all pH measurements. Zinc, magnesium, and calcium were not detected in the carboxylic acid solutions by atomic absorption analysis. Chelex 100 (NaI) was washed in a sintered glass funnel with three cycles of 1 N HCl and water and then titrated to pH 7.0 or 7.5 with Tris base. Visking Corporation dialysis tubing was boiled twice in NaHC03, three times in sodium EDTA, and rinsed three times in water and stored at 4° in water. Substrate Analog Study Source of Analogs Analogs of the substrate AMP were obtained from the following sources: Sigma (AMP, adenosine, ADP, ATP, adenosine monosulfate, 2'-AMP, 3'-AMP, 3',5'-cyclic AMP, dAMP, GMP, GDP, GTP, and CMP): P-L Biochemicals (adenosine 25 phOSphoramidate and 6-mercaptopurine 5'-ribonucleotide); Miles Laboratories (d,B-methylene Adenosine diphoSphonate); Tubercidine monOphoSphate and tubercidine monophOSphate methyl ester were obtained from Dr. William J. Wechter of UpJohn Co., Kalamazoo, Michigan; 3-iso AMP was kindly donated by Dr. Nelson Leonard of the University of Illinois, Urbana, Illinois; Dr. R. J. Suhadolnik of the Albert Einstein Medical Center, Philadelphia, Pennsylvania, donated toyocamycin monOphOSphate (7-deaza, 7-cyano AMP); Formycin monOphOSphate (8-aza, 9-deaza AMP) was a gift of Dr. S. Nishimura of the National Cancer Center Research Institute, Tokyo, Japan; Dr. F. Rottman of Michigan State University, East Lansing, Michigan kindly SUpplied the 2'-O-methyl ADP (AmDP) and K. W. Rabinowitz of the same institution donated N6-ethy1 AMP and the mixture of 6-amino-9-D-psicofuranosylpurine 1'-ph05phate and 6'- phOSphate. The sodium salt of ADP and the barium salts of 5'- adenosine monosulfate and 6-mercaptopurine 5'-ribonucleo- tide were converted to the Tris salts by passage over Dowex 50 WX8 (Tris+) at room temperature. The acid forms of all other analogs were titrated to the desired pH with Tris base. No attempt was made to remove the less than 5% AMP contaminant in ADP analog studies since the high concentration of enzyme used deaminated the AMP almost as fast as the sample could be mixed. 26 Synthesis of Nl-Methyl AMP Nl-Methyl AMP was synthesized. from AMP and dimethyl- sulfate at pH 4.5 by the method of Griffin and Reese (81). The purified product exhibited only one Spot in Solvent A (see page 28) and the Rf value of 0.75 agrees well with the Rf = 0.76 previously reported (81). In 0.1 N HCl Xmax was 258 mm and Xmin was 232 mu. Synthesis of N6 -Methyl AMP N6-Methyl AMP was synthesized from Nl-methyl AMP by the procedure of Brooks and Lawly (82). The absorption Spectrum at pH 6.7 was identical within eXperimental error to that previously reported for N6-methyl adenosine in water, Amax = 265 mu and Amin = 229 mu (83). Paper chromatography of N6-methyl AMP in solvent system B (see page 28) indicated that the sample contained less than 1.7% AMP and 2.1% N6-methyl AMP methylester. The Rf values for AMP and N6-methyl AMP in system B were 0.23 and 0.41 reSpectively. The change in absorbance for the enzymatic deamina- tion of N6-methy1 AMP was determined using a Cary Model 15 SpectrOphotometer. The Spectrum of a known amount of N6- methyl AMP, 5265 nm = 16.3 x 103 OD/mM (83), was deter- mined from 340 nm to 220 nm before and after the addition of 16 ug of AMP aminohydrolase to sample and reference cells. The absorbance change resulting from deamination of N6-methy1 AMP (at pH 6.3) was calculated in AOD/mM at 27 several wavelengths: 265 nm, -10.65; 270 nm, —11.23; 275 nm, -10.10; 285 nm, -5.45; 290 nm, -3.24; and 295 nm, -1.23. Deamination of Analogs The deamination of analogs of AMP was examined by recording the absorption Spectrum of each before and after the addition of 10-40 mg of enzyme to both sample and reference cells of a Beckman DB SpectrOphotometer equipped with a Sargent SRL recorder. Those analogs deaminated showed changes in their absorption Spectrum within 15 minutes. Assay 1 was used except approximately 60 uM analog was substituted for the AMP. Kinetic parameters were obtained from Lineweaver- Burk plots of initial velocities (in Assay 1) at substrate concentrations from 10'5M to 10‘2M except in the case of adenosine monosulfate and N6-methyl AMP where the maximum concentration of analog was 2.0 x 10'4M. The Hill lepe (nH) was obtained from a plot of Log [(Vmax/v) - 1] versus Log [5]. Purine Product Characterization Substrate analogs and protein (5-80 us) were mixed on a watch glass and incubated 10-70 minutes before the products were chromatographed on Whatman #1 paper or poly- ethylenimine impregnated cellulose MN 300 thin layer plastic Sheets (Brinkman Instruments, Inc.). Impurities 28 were removed from the latter prior to use by ascending irrigation with double distilled water according to method 1 of Randerath and Randerath (84). Chromatograms were develOped with the following solvent systems: (A) saturated ammonium sulfate:0.1 M potassium phOSphate buffer,pH 7.2:is0propanol (79:19:2); (B) 1% ammonium sul- fate:acetic acid:isopropanol (34:20:45) chromatographed on Whatman #1 paper washed with 1% ammonium sulfate; (C) iso- butyric acid:1 N ammonia:0.1 M sodium EDTA (100:60:1.6); (D) 10 ml concentrated ammonium hydroxide added to 32.9 ml water and mixed with 66.1 ml of isobutyric acid; (E) 1 M acetic acid-ammonium acetate buffer,pH 3.8:95% ethanol (30:70); (P) 0.5 M sodium formate buffer pH 3.3. Descend- ing chromatography was used for all systems except for solvents B and F which were used for ascending chromatog- raphy. Solvent F was used to develOp chromatograms on the polyethylenimine cellulose thin layer sheets. Detection of Methylamine Methylamine from N6-methyl AMP deamination was detected with an F & M 402 gas chromatograph equipped with a flame detector. The column, 3 mm x 6 feet, packed with Chromosorb 103, 100/120 mesh (ASpec 00., Ann Arbor, Michigan), was conditioned overnight at 2500 with N2 gas flow. The samples were run at 105° with N2 as the carrier gas. Solutions of methylamine at 1.0 ug/ul and NH3 at 9 ug/ul (ammonium chloride in KOH) were prepared as standards. 29 Physical.Properties Extinction Coefficient of AMP Aminohydrolase The extinction coefficient of AMP aminohydrolase was determined by the method of Hoch and Vallee (85) using trichloroacetic acid precipitation of protein followed by drying to constant weight. After concentrating the enzyme to approximately 8 mg protein/ml by placing it in a dialysis bag around which dry Sephadex G-200 (fine) was packed to absorb solvent, AMP aminohydrolase was chromato- graphed on Sephadex G-25 equilibrated with 0.10 M KCl at pH 7.0 with potassium phoSphate buffer. The cptical density of the eluted protein was determined at 260 and 280 nm by dilution into 0.8 M KCl, 0.05 M Tris cacodylate, pH 6.3, and 1 mM mercaptoethanol. Three ml samples con- taining a total of 7 to 7.5 OD units (at 280 nm) of pro- tein were placed in conical glass test tubes (previously dried to constant weight), precipitated with an equal volume of 20-30% TCA, allowed to stand at room temperature for 15 minutes, and then centrifuged 15 minutes at t0p Speed in a clinical centrifuge. The supernatant was care- fully removed with a Pasteur pipet. The precipitate was washed once with 2 ml 20% TCA, centrifuged, and the supernatant removed. After a second washing with 2 ml 10% TCA, the precipitate was dried to constant weight at 1050 and weighed to $0.1 mg on a Mettler balance. 30 Molecular Weight of AMP Aminohydrolase The molecular weight of AMP aminohydrolase was determined by the thantis Sedimentation Equilibrium method for dilute protein solutions using the Spinco Model E Ultracentrifuge equipped with Rayleigh interference cptics (86). Enzyme was chromatographed on Sephadex G-25 equilibrated with 0.20 M KCl, 0.05 M Tris Mes, pH 7.2, and 1 mM mercaptoethanol. The protein sample (0.575 mg protein/ ml) and a blank of the 0.20 M KCl buffer were placed in a double sector 12 mm Epon filled cell and centrifuged at 293.0°K and 12,590 RPM. The Rayleigh patterns were recorded on Kodak II-G photographic plates. The fringe displacements of the photograph focused at 2/3 plane were measured at 0.1 mm intervals on a Bausch and Lomb Micro- comparator. The data were treated according to the proce- dure given by thantis. Subunit Molecular Weight Enzyme was chromatographed on Sephadex G-25 equi- librated with 0.1 M TMACl at pH 7.0 with Tris phoSphate buffer and 1 mM mercaptoethanol to remove KCl which inter- feres with denaturation by sodium dodecyl sulfate. The enzyme (96 ug/ml) and standards (bovine serum albumin, RNase, and DNase) were incubated 3 hours at 37° in 1% SDS, 0.1 M sodium phOSphate buffer, pH 7.1, and 1% mercapto- ethanol. SDS polyacrylamide gels and gel buffer were prepared according to a modification of the procedure of 31 Weber and Osborn (87). The gels were pre-electrophoresed to remove ammonium persulfate before application of the protein samples. A total of 20-80 ul of 1:1 mixtures of the enzyme solutions and 50% glycerol was layered on separate gels followed by electrOphoresis at room tempera- ture for 2.5 hours at 7.5 ma/gel. The gels were removed from the glass gel tubes, stained 4 hours with Coomaasie Blue solution (2.5 ml 1% Coomaasie Brilliant Blue, 5 ml absolute methanol, and 10% TCA to 100 ml), and then destained over a 48 hour period with several changes of 10% TCA. Migration distances (mobility) of each standard and sample were measured from the origin. Characterization of AMP Aminohydrolase as a Zinc Metalloenzyme Preparation of Enzyme for Trace Metal Analyses To insure that the enzyme was not contaminated with divalent metal ions during preparation all glassware, pipets, and nalgeneware were carefully washed and rinsed, soaked in 2 N HCl for six hours or more, and then rinsed with double distilled water. A pyrex Waring blender was substituted for the metal meat grinder and stainless steel Waring blender used in the initial stages of the enzyme preparation. All reagents except mercaptoethanol and Tris base were passed over Chelex 100 (Tris+) to remove contam- inating metal ions and were then stored in polyethylene bottles. The cellulose phOSphate was treated as previously 32 described (1) and then soaked in 0.01 M Tris EDTA, pH 7.5. for 1 week, and washed exhaustively with double distilled water before equilibration with extraction buffer. A total of 100-200 mg protein from the crude extract (10-20 ml of the supernatant obtained after centrifugation of the extraction mixture) was dialyzed against three changes (300 ml each) of 0.1 M TMACl, pH 7.0 with 50 uh Tris phoSphate, and 1 mm mercaptoethanol (Buffer A) over a 72 hour period to reduce potassium and phosphate to levels that would not interfere with the metal analysis. The sample was transferred to a 100 ml pyrex Berzalius beaker with pyrex cover glass and evaporated to moist dryness before wet ashing. Two blanks containing an amount of Buffer A equivalent to that in the sample were similarly treated. Purified AMP aminohydrolase (25 ml at 4 mg protein per ml) was dialyzed against 3 changes (250 ml each) of 0.97 M KCl, 0.01 M Tris Mes, pH 7.1, and 1 mM mercapto- ethanol for 72 hours at 4°. In order to remove potassium 10 ml of the dialyzed enzyme was chromatographed on a Sephadex G-25 column (2.5 cm x 22 cm) equilibrated with Buffer A. Before equilibration with buffer all Sephadex columns were washed with 2 to 3 column volumes of 0.01 M Tris EDTA, pH 7.5, to remove metal ions followed by exhaustive rinsing with double distilled water. The enzyme was collected in Nalgene test tubes and the more concentrated 33 fractions were combined (a total of 25 mg protein) and evaporated to moist dryness in Berzalius beakers. Two blanks containing Buffer A collected from the column before protein was applied were similarly treated. After all samples had cooled, 10 ml of concentrated nitric acid (Baker Analyzed ACS Reagent grade) was added to the samples and blanks. The samples were kept at room temperature for 15 minutes and then heated slowly and allowed to reflux until clear and colorless insuring com- plete oxidation of all organic material. Finally all samples were evaporated to moist dryness, cooled, the beaker sides and cover rinsed with 5 ml water, heated and then cooled before quantitative transfer to 25 ml (25 mg samples) or 50 ml (50 mg samples) volumetric flasks. Concentrated HCl (Baker Analyzed ACS Reagent grade) at 0.125 ml/25 ml was used to insure solubilization of all inorganic material. Metal analyses were accomplished with the Perkin Elmer 303 Atomic Absorption SpectrOphotometer at the following wavelengths: Zn, 2138A; Ca, 4227 A; Mg, 2852 A; Fe, 2483 A, and Cu, 3247 A. Samples were aspirated into an air-acetylene flame and the percent absorption of three readings was averaged and converted to OD. Standards for each metal were completed before each analysis using standard solutions made by dissolving analytically weighed SpectrOpure metals or metal oxides (Johnson, Matthey and Co., 34 Ltd.) in concentrated HCl. The standard matrix contained water and 0.5 ml concentrated HCl/100 ml. The ppm of metal were calculated from a standard curve or by pr0portion after subtraction of the blank readings from the sample readings. Ten ml of the samples and blanks for the purified enzyme were sent to Dr. H. Mass of the Union Carbide Corp. (Tuxedo, New York) for Neutron Activation Analysis of Co, Mn, and Cu. Preparation of Apo Enzyme2 ApoAMP aminohydrolase (enzyme from which the bound metal has been removed) was prepared by incubating enzyme at 3-4 mg protein per ml in 15 to 19 mM Tris 8-OHQ58A at pH 7.1 in 1 M KCl for 12 to 15 hours until the activity of the enzyme was less than 10% of the original activity. The chelator (8-OHQSSA) was removed by chromatography on Sephadex G-25zfor metal analysis enzyme was eluted with Buffer A and for reconstitution studies Buffer B (0.5 M KCl, 0.02 M Tris Mes, pH 7.1, and 1 mM mercaptoethanol) was used since the enzyme was generally more stable in this buffer. 2The methods and conditions by which apoenzyme was obtained and by which reconstitution of the apoenzyme was achieved were those that worked but are not necessarily the most optimum. Except for preliminary studies which indicated that reactivation did not occur as readily in TMACl as it did in KCl, the methods and conditions described were the only ones tried. 35 Reconstitution of Apo Engyme Apoenzyme in Buffer B was reconstituted by incubat— ing samples containing 0.10 mg protein/ml in the presence of 0-10 gram atoms of metal (as the Spectropure metal sul- fate) per mole of enzyme. The activity of enzyme at each level of metal was determined with Assay 1 containing 10 mM Tris AMP. The assay mixture was freed of metal ions before use by treatment with Chelex 100 (Tris+). To determine the kinetic constants of reconstituted enzyme, apoenzyme in Buffer B was incubated with 3.7 to 4.0 gram atoms of zinc, cobalt, or manganese sulfate overnight. The enzyme (1.5 to 2.0 mg protein/ml) was chromatographed on Sephadex G-25 equilibrated with Buffer A to remove excess metal ion and KCl and aliquots were diluted in either 0.5 M KCl or 0.5 M TMACl containing 0.02 M Tris Mes, pH 7.1 and 1 mM mercaptoethanol for assay. Apoenzyme (16 mg) reconstituted with 3.7 gram atoms zinc/mole enzyme was chromatographed on Sephadex G-25 as previously described; the recovered enzyme (9 mg) was wet washed and analyzed for zinc. Stability Towards Metal Bindinnggents Inhibition of AMP aminohydrolase by a number of metal binding agents in the presence or absence of KCl was studied by eXposing the enzyme at 0.1 mg protein/ml and 300 to these inhibitors. The loss in activity was followed over a 6 hour period using Assay 1 or 2. The inhibition 36 data are eXpressed as Vi/Vo x 100% where V1 is the activity after exposure to the chelating agent and V0 is the activ- ity before eXposure. The small amount of chelating agent transferred from the incubation mixture to the assay mix- ture did not affect the assay. Correlation of the Removal of Zinc and Loss of Enzymatic Activity Zinc was removed from the enzyme by a modification of the method of Simpson and Vallee (88) using 8-OHQ53A. Enzyme (3.59 x 10'6M) in 0.4 M KCl or TMACl, 0.1 M Tris Mes, pH 7.1, and 1 mM mercaptoethanol was titrated with 8-OHQ5SA and the OD recorded at 370 nm against a blank containing all reagents except protein. The amount of zinc [8-OHQSSA]3 complex formed, calculated from standard curves of OD370 nm versus zinc concentration at each concentration of 8-OHQ58A used, was correlated with enzymatic activity remaining. Activity was determined immediately after apprOpriate dilution of aliquots (25 ml) removed from the sample cuvette. The immediate determination of activity after dilution was necessary since a time dependent reac- tivation, presumably due to readsorption of zinc by apo- enzyme, was frequently observed. Aliquots (25 ml) were also withdrawn from the blank cuvette to maintain an equal volume in each cuvette. All calculations were corrected for dilution of the protein during the titration. RESULTS AND DISCUSSION Kinetic PrOperties of AMP Aminohydrolase Substrate Specificity The AMP aminohydrolase purified by Lee (21) was Specific for AMP; dAMP, the only other substrate, was deaminated at 1% the rate of AMP deamination (2). Enzyme prepared by the method of Smiley g} §l_(1) deaminated not only 5'-AMP and dAMP but also adenosine, ADP, N6—methyl AMP, N6-ethyl AMP, adenosine 5'-monosulfate, AMP-NHZ, AmDP, d,B-methylene ADP, and formycin 5'-mon0phoSphate. The kinetic parameters (Km and relative Vmax) for these com- pounds are presented. in Table 3. No data for Né-ethyl AMP, AmDP, a,B-methylene ADP, and formycin 5'-monoph03phate was obtained due to lack of sufficient material. The following compounds were not deaminated under the conditions described in Materials and Methods: ATP, 2i-AMP, 3'-AMP, 3',5'-cyclic AMP, 6-amino-9-D-psicofuranosylpurine 1'-, and 6'-ph03phate mixture, Nl-methyl AMP, 6-mercaptopurine 5'-ribonucleotide, GMP, GDP, GTP, CMP, 3-iso AMP, tubercidine 5'-mon0phOSphate, and tubercidin 5'-monophoSphate methyl ester. pH Optima The pH Optima for deamination of ADP, adenosine, 37 38 Table 3. Kinetic Parameters for Deamination of AMP Analogs Catalyzed by AMP Aminohydrolase Substrate Analog KIn (mM) RelativeVmaxa nH AMP 0.40 100 1.0 AMP-NHZ 49 73.4 1.0 Né-Methyl AMP 1.8 20.4 dAMP 2.3 18.5 1.1 Adenosine monosulfate 3.2 10 to 16 1.1 Adenosineb 20.0 1.1 ADP 0.8 0.20 1.3 aReported as % Vmax for AMP (1240 umoles deaminated/min.per mg) in 0.15 M KCl and 0.05 M Tris cacodylate buffer, pH 6.3. bThe kinetic parameters for adenosine were obtained at the pH Optimum, 6.5, for adenosine deamination. 39 and Né-methyl AMP determined in either Tris cacodylate or sodium acetate buffer are given in Figure 2. The Optimum for ADP deamination occurred between pH 5.0 and 5.5, a marked shift to acid pH from the Optimum (pH 6.3-7.0) observed for AMP deamination.3 Since the pKa for the terminal phOSphate of ADP is 6.44 (89) ADP exists as ADPZ- between pH 5.25 to 5.50 whereas at pH 6.44 it exists as a 1:1 mixture of ADPZ' and ADP3’. The observed shift may indicate that ADP3- is less readily deaminated than ADP2-. The sharp Optimum for adenosine (pH 6.5 to 6.75) and for N6-methyl AMP (pH 6.4 to 6.6) deamination at non- saturating substrate concentrations shows a slight shift to more alkaline pH from the observed optimum (pH 6.3) for AMP deamination under similar conditions. Activation and Inhibition of AMP Aminohydrolase by Purine Nucleotides Activation Both ADP and ATP are allosteric activators for AMP deamination in the absence of the monovalent cation acti- vator K+ (43, 90). Data for activation by other purine nucleotides are presented in Table 4. Of the analogs tested only 6-mercaptOpurine 5'-ribonucleotide and IDP activated the enzyme. Although according to the data in Table 2, 6-mercaptOpurine 5'-ribonucleotide is a better 3Unpublished Observation, K. L. Smiley, Jr. 40 .zodpomoa esp ampmm pad maouop poop mms mmmmm Loco no ma one .,Q .Ha\oamuao my m.o pad .m£< HmSpOaloz :1 om .opmahpoomo mane a mo.o .HQ& 5 mH.o oocfimpnoo chapxda mmmmm opp .sofipdcdamop ma< Hampoaimz hog ..O .Ha\oEmmsm w: om cam ozamocoom as m .ammmSD opmamooomo wage a oa.o .Hox a 0H.o oosampgoo codpmnaammo Osamocopm pop madpxas ammmm one .mpmamooomo mane . I "mumpood E-Soom . D nondpfia momma mo HE H on mammgo .HO w: ma mzappm an oopmapaca mmz :OHpomOh 0:9 .mD« make as N.@ one .ma mandaam> um armada a OH.o .Hom a 0H.o pmcdmpsoo godpmsdamoo mm< pow maauxas known one mas Haroosubz one .osamosoos .mos do sodomsaamoo no we no pooccm one .m unsung 41 INF-METHYL AMP 1&0 '10 _. ._<2 ‘0 l i l I ii 1 .1 <2 I" 0 0 V IO N _ 0 (UIOJOJd 5111/0le UN) All/\llOV OlleBdS T I #f/O' $2 (I) - 2 --2 U I- O " 4 _J2 '- ‘0 . I’ a. q Lain-D“ 2 ~53- 1 ------ ~13“--- . .Ds.~~ D .12 .. a c r I i L ? " N - 0 (0:9de Dw/UIw/wr‘) Ail/\Iiov OlleBdS Figueih Table 4. Activation of AMP 42 Aminohydrolase by Purine Ribonucleotides Purine Nucleotide [A] uM Specific Activity um/min per mg Protein Control 0 0.72 6-MercaptOpurine 5'- ribonucleotide 82 14.1 165 26.0 Inosine diphOSphate 70 2.9 150 7.6 Tubercidin monOphOSphate 36 0.66 72 0.84 Toyocamycin monOphOSphate 47 1.4 94 2.4 CMP 95 0.64 Activity was determined with Assay 3 containing 50 uM Tris-AMP. The protein concentration in the assay varied from 0.2 to 0.5 ug/ml. 43 activator than IDP, activation by this compound was not pursued further due to a lack of material. The Hill plot for IDP activation is presented in Figure 3 along with the data for ADP and ATP activation Obtained by K. L. Smiley. (Due to the high absorbance of IDP at 265 nm, the data presented was obtained at subOptimum concentrations of this activator.) The Kaapp value for IDP is 1.2 mM compared to 10 DM and 50 uM observed with ADP and ATP, reSpectively.At However the observed maximum velocity at 50 uM AMP for IDP activation was 75% of that observed with ADP. Hence the 6-amino group Of ADP, which substantially reduces the con- centration Of activator required for half maximum activa- tion, is not required for the activation per g3. Inhibition In addition to inhibition by GDP and GTP (43, 90), the enzyme is subject to inhibition by 3',5'-cyclic AMP, 3'-AMP, d,B-methylene ADP, and 3-iso AMP (see Table 5). At concentrations of less than 0.3 mM 2'-AMP had no effect on activity. Nl-methyl AMP did not inhibit significantly at 6 times the concentration of AMP in the assay. 3-ISO AMP was a potent inhibitor of AMP, dAMP, N6-methy1 AMP, ADP, AMP-MHZ, and adenosine monosulfate deamination (data not Shown). The Dixon plot (Figure 4) and double reciprocal plot (not shown) for 3-iso AMP ”Unpublished Observation, K. L. Smiley, Jr. .o u 2b}.a>9\>g woq muons hamgu mod mamaop mA>IS>V\>u moqv poaa Hadm esp mo parchopca on» Sony popmsapmo was .aadmx .coHpmbapom wow you poaasvoa aopm>apom mo coHpmapcoosoo one .HOQSSpOOpamOama as H can .m.© ma .opmamooomo made a mo.o .Homae a m.o roar ooomwoaaasoo mmuo woomsoom co ooromnmoomaowro osmuzo mgfims negamuno was acapd>apom mDH Mom mpmo one .m\H no 5> .nopm>apom ho aodpmapgoosoo poaa xasmiao>mmzocaq a Scam oosaaaopmo mm; exp madmam> paw N among wa-ms occampno cams .> .mOHpaooaob HdeaaH mmmHOHUmQOQHS¢ m4< MO Soapm>apo¢ mH¢ U56 .mQ< .mQH how mpoam HHHm .m mhswam 45 .2 Amp—23:03 00.. Qni 06.. 0.0: NV: 9?: Oh: v.0: QB: _ _ _ _ _ _ \o 4 O l .I Nll a .l c O <4 o 9 l A _ I w .. . A A 3...: do. Gun}. .54 I I. o l _ _ _ _ _ b Figure 3. 46 Table 5. Inhibition of AMP Deamination by Purine Nucleotides Inhibitor [1] mM % Inhibitiona 3-Iso AMP 0.01 33 0.10 73 3'-AMP 0.10 14 0.17 41 3',5'-cyc1ic AMP 0.10 14 0.14 23 0.17 46 2'-AMP 0.30 5b d,B-Methy1ene ADP 0.10 21 0.13 41 Nl-Methyl AMP 0.18 15c aActivity was determined in Assay 1 containing 50 uM Tris AMP. In order to Obtain data at total nucleotide concen- trations greater than 0.2 mM where the absorption is = 14.1 x 103), the cuvette greater than 2.5 OD (e was blanked against 50 pH AMP. The reaction was initiated by the addition of 0.2 mg protein/ml to the assay contain- ing the indicated amount of inhibitor. Percent inhibition is reported with reSpect to activity in the absence of inhibitor. bDue to the high Optical density the assay was run in cuvettes with a path length of 5 mm instead Of the usual 10 mm. 0The assay contained 32 uM Tris AMP/ml. 47 .mHobapooamoa .Ha hon campoaa m: 0.5 cam Ha you adopoaa m: :m.o poaampaoo mOHSOxHa :oapomoa mzwo pad mz< one :3 com .< "an era .0 :3 cos .I £3 cm .0 “2% as man .As ”21 cos .AV is: ooa .0 "an on .nc mas "opmemDSw mo macapmapaoosoo wcazoaaow onp pd H momm< maama noncomoa was mpabapo< composasmoo .25 one 82 no 8332.: a: 86A .S 88$ 48 5— 0+0_ x HQEQIOWTMU n. O. 0 O . i 23.8» . :q R 2:00. 1 i0. stanxz 0 low 5:00 nw=¢46u ion .-O| x ,_(ugw/uir1) JV I S. 90. x $24.09an ON 0. AV 2: oo— ' .. a: On 923 2.68 . IILIII Snoom o n: ID 0 4 O l C) .-Ol x |-(U!W/UJ”) All 1 8 Figure 4. 49 inhibition of AMP deamination is characteristic Of competi- tive inhibition. The K1 value for 3-iso AMP Obtained from the Dixon plot was 37 uM. A replot Of the lepes from the Lineweaver Burk plots versus inhibitor concentration was linear (see Figure 5): the K1 Of 32 uM determined from the intercept on the horizontal axis in Figure 5 is in good agreement with the K1 determined from the Dixon plot. The data are consistent with 3-iso AMP exhibiting linear competitive inhibition for AMP deamination according to the terminology Of Cleland (91). Neither AMP nor 3-iso AMP caused substantial changes in the Hill slopes for 3-iso AMP inhibition or for AMP deamination, reSpectively (see Tables 6 and 7). The inhibition of dAMP deamination by 3-iso AMP is not clearly defined. Both the Dixon (Figure 4) and Lineweaver Burk plots were markedly nonlinear. In the Dixon plot the curvature is eSpecially noticeable at low substrate concentration where at increasing concentrations of 3-iso AMP less inhibition is Observed than would be predicted. This could occur by four different mechanisms: (A) iso-AMP is deaminated; (B) iso-AMP is activating the reaction; (C) the ability of iso-AMP to bind to the enzyme decreases upon increasing saturation with the inhibitor; or (D) iso-AMP binds to more than one type of site each with a different binding constant. Possibility(A) seems unlikely in that significant deamination of iso-AMP did not 50 200v- ). Ki=32 11M 1’ o 9 )( Lu 8‘ _I (n 0 ' :00 200 [3-ISO AMP] IJM Figure 5. Replot of SlOpes from Lineweaver Burk Plot of 3-Iso AMP Inhibition Of AMP Deamination Versus the Concentration of 3-Iso AMP 51 Table 6. The Effect of AMP and dAMP on the Hill SlOpe for 3-Iso AMP Inhibition [AMP] mM -nH (3-Iso AMP) [dAMP] mM -nH (3-Iso AMP) 0.05 0.95 0.05 0.70 0.10 0.87 0.10 0.79 0.19 0.87 0.18 1.05 0.52 1.10 0.30 1.04 0.80 1.0 0.60 0.97 The data from Figure 3 were plotted according to the Hill equation (see Figure 1) where V is the velocity of substrate deamination in the absence of inhibitor and v = vi, the activity of the inhibited reaction. 52 Table 7. The Effect Of 3-Iso AMP on the Hill SlOpe for AMP and dAMP 3-Iso AMP 1M1 nH (AMP) nH (dAMP) 0 1.00 1.3 10 1.07 1.2 50 1.04 1.3 150 1.00 1.1 The data from Figure 3 were plotted according to the Hill equation where Vm_is the maximum rate of AMP or dAMP deamination and v is the activity of the reaction as a function of substrate concentration at constant concentra- tions of inhibitor. 53 occur over a 45 minute period at twice the protein concen- tration used in the dAMP study. Mechanisms B, C, and D cannot be distinguished with the data available. Unlike the data for AMP, 3-iso AMP effects the Hill lepe for dAMP and vice versa (see Tables 6 and 7). The Hill lepes of < 1 for iso-AMP suggest that the enzyme is exhibiting negative cOOperativity with reSpect to the inhibitor.5 The Monod, Wymen, and Changeux theory (93) for allosteric enzymes cannot account for nH < 1.0 (94) although Levitzki and Koshland have pointed out that enzymes with Hill lepes of less than 1 for some effectors and greater than i for others are common phenomena (95). For example aSpartate transcarbamylase, which diSplays positive cOOperativity (nH > 1) for aSpartate (96), exhibits negative COOperativ- ity for the inhibitors GTP and CTP (95): from the theory of negative cOOperativity the incomplete inhibition by nucleotide triphOSphates at concentrations which should have completely inhibited the reaction was rationalized. AMP aminohydrolase may be yet another example of both posi- tive and negative cOOperativity, the former exhibited by substrate and nucleotide activators and the latter by the 5Negative COOperativity is characterized by decreas- ing affinity of the enzyme for ligand upon saturation and by nH <:1. It may arise from ligand-induced conformational changes in a single protein Species or from a mixture of two or more proteins or subunits (isoenzymes) with differ- ent intrinsic binding constants. For instance, glyceralde- hyde-3-pho3phate dehydrogenase exhibits negative COOperativ- ity for MA binding as a result of conformational changes upon binding of successive molecules of NAD+ (92). 54 inhibitor 3-iso AMP, at least with reSpect to inhibition of dAMP deamination. One Enzyme ReSponsible for Deamination of all Substrates The less Specific behavior Of AMP aminohydrolase towards purine nucleotide analogs could be due to contam- ination by other aminohydrolases. In addition, deamina- tion of ADP might be effected by tranSphOSphorylation Of ADP to ATP and AMP with myokinase followed by deamination of AMP by the Specific AMP aminohydrolase. However, deamination of various substrates by a single enzyme, AMP aminohydrolase, is consistent with the results obtained from studies of enzyme homogeneity, heat inactivation, elution from cellulose phOSphate, and product characteriza- tion. Homogeneity AMP aminohydrolase was homogeneous by ultracentri- fugal and electrOphoretic criteria (1). Heat Inactivation The rates of heat inactivation Of ADP. AMP and adenosine deaminating activity were identical within experimental error when examined under 2 conditions, 62° in KCl and 40° in TMACl (Table 8). In the latter system the slightly greater rate of heat inactivation for AMP than for ADP and adenosine activity may be the consequence 55 Table 8. Heat Denaturation Of Adenylic Acid Aminohydrolase Rate of Heat Inactivation k (min’l) b Substrate (CH3)¢NC1 Systema KCl System AMP 2.2 x 10-3 7.3 x 10-2 ADP 1.5 x 10'3 7.0 x 10"2 Adenosine 1.5 x 10‘3 ND0 3Conditions of incubation in the (CH ) NCl (TMACl) system: Protein which was passed over a Sepga.ex G-25 column equilibrated with 0.10 M TMACl, 0.05 M (CH )hNicacodylate, RH 7.2, and 2 mM mercaptoethanol was incubgted at 1.3 ms/ml, 0°C in a closed test tube from which aliquots were removed for assay. Protein concentration per ml of assay was 0.26, 13, and 32.5 us in the AMP, ADP and adenosine systems, reSpectively. Assay solutions contained 1 mM Tris AMP and 0.05 M Tris cacodylate, pH 6.4; 1 mM Tris ADP and 0.05 M Tris cacodylate, pH 6.4; and 0.1 mM adenosine, 0.10 M KCl. 0.05 M Tris cacodylate, pH 6.4. bConditions Of incubation in the K01 system: Deaminase (0.48 mg/ml) was incubated at 62°C in 1 M KCl, 1 mM mercap- toethanol, and 0.10 mM potassium phOSphate, pH 7.0. The ADP assay contained 0.1 mM Tris ADP, 0.10 M TMACl, and 0.05 M Tris cacodylate, pH 6.3. The AMP assay contained 50 uM Tris AMP and 0.10 M potassium succinate, pH 6.5. The protein concentration in the ADP and AMP assays was 4.8 ug/ml and 0.24 ug/ml, reSpectively. 0Not determined. 56 of an additional dilution required before AMP aminohydro- lase could be assayed which could introduce an additional inactivation. Elution of AMP and ADP Aminohydrolase Activity from Cellulose PhOSphate The ratio of Specific activities for AMP and ADP deamination remained constant within the protein peak eluted from a cellulose phOSphate column with a linear gradient from 0.h5 M KCl to 1.0 M KCl (Figure 6). The recovery of ADP aminohydrolase activity with reSpect to the crude extract was not determined due to the presence of interfering enzymes, i.e. myokinase, in the extract. Product Characterization The purine ribonucleotide products of adenosine, AMP, and ADP deamination were characterized by paper chromatography (Table 9). The product of ADP deamination chromatographed with the same Rf value as commercial IDP in solvent systems A and D. The product for adenosine deamination exhibited the same Rf value as inosine in systems A, D, and E. No IMP was detected in either of the ADP or adenosine reaction mixtures. Since the relative roles of deamination of ADP and adenosine are only 0.2 to 1% that observed for AMP deamination, any AMP formed by reactions involving contaminating enzymes such as myokinase would be immediately deaminated to IMP at the concentra— tions of protein used. Since this was not observed, the 7 5 .pozmmmm no: max xmma same on» wcaooooaa Mama £393 325 are .20 mg .32383 3.5 2 3.0 sea 338. z 3.0 E on: 3.3. :5 oa.o pm oocaamxo mm: cofipmcaamop mm< .mz4 :1 AK“ 62m 1.0 mg no mpmzaoozm azammmpoa z oa.o ca mpdbapom mmmaohvmsosdao was now domMmmm paw copooaaoo ohms as 0.: mampmaaxonaam mo mmaaamm .o.m ma pm Hocmspoopamohma 2a H mndnfimpzoo aux z o.« op m:.o aoam pzmapmnw Hammad as co: a Spa: confide was mamucm opmnmmonm omoHsHHoo aonm mmapabfipo< mmmaoapmsozfia< was cum mz< mo madmohm soapsam .0 masmHm 58 o—o 9.01:: umuonmvoo mwmzaz m4¢2oa pump pom oom.Hm 35 .ommzm mu om Amv “Amwv oom.mH 32 .ommzm w: om va "mzoaaom mm oopnowoaa was one» son as m.m pm mdmoaosaoapooao masoz m.m scams mamw opaamamaommaoa mam no ommaonumsocaam mz< use mUHSUQMpm ooanp pom cobaompo mcaoppma capoaonaoapooao one name ooHEMHmaoomHom mam so zdasnaw adhom ocabom dam .ommzm .mmmzm .ommaoaemnocdaa has UoHSpMSoQ mam Mom msaoppmm capoaosaoapooam .m madwam 77 Q -hm-fi *“fl Figure 9. 78 AMP aminohydrolase is estimated as 69,000. Enzyme incu- bated in both urea and SDS buffer to insure complete denaturation (F) still moved with the same mobility as enzyme incubated in SDS buffer (D, E). Even at high pro- tein concentrations (D) only one band could be detected for the aminohydrolase. The data are consistent with AMP aminohydrolase being a tetramer of like chains, at least with reSpect to molecular weight. The molecular weight calculated for the tetramer is 276,000 which is within 2% of the 278,000 obtained by equilibrium centri- fugation. The Divalent Metal Component of AMP Aminohydrolase The Effect of Carboxylic Acids Upon Activity Since the ADP activation of AMP aminohydrolase was not observed in the presence of Tris succinate buffer, the effect of several di- and tri-carboxylic acids on activity was investigated under three different conditions (Table 13). Enzyme assayed at 0.52 mM AMP in the absence of activators was eSpecially sensitive to inhibition by succinate, maleate, and citrate while the ADP-activated enzyme was inhibited by citrate, maleate, fumarate, and malonate. The K+ activated enzyme was less sensitive to carboxylic acid inhibitions: only citrate inhibited more than 20% at 0.01 molar. Acetate, a monocarboxylic acid, did not inhibit. 79 mm mm as 0H m: 0H m a oahpao mm oH ms ma m.s ma : :N H ofioamz mm oH ab NH m.m ma m H oneness om am on OH OH 6 a oaamxo Auden nasal ooopsbapoermme poopssasoer+m maze swam Azsv .osoo oaoe oaasxonsmo nodpaDaSSH unmohmm .Ha\w1 mm.o mos momma on» :« scandap Isoosoo Sampoaa one .HopdanpaaooHSpHc as N was .m.m ma opmamuoomo make 2 no.0 .maopwmm penchapow adfiwmwpoa on» now Hum use aopmam comebapom mn< on» one seems as<_smas oso sea Hezehmmovg same a m.o oped ooosaao mos assess any moses adamaonsso an omoaosessosess was so soapansssH .ma manna 80 .8: new no bozoaaom mums mmdmm< .m.w ma .mpdameoomo maps a mo.o one .Homze a H.o .mm< mane a: ooa .m2¢ wane an on cosampSoo aopmmw movabfipomummd 0:90 .Ec new no mozoaaom ohms msoapomom .m.o ma .opmasooomo mane a mo.o one .Hoa z ma.o .msq mane :1 on ones sowed son msoapaosoop .Es mmm pm posoaaom ohms msoapowmm .oESHob as a :H .m.o ma .opmasooomo mass 2 mo.o one Homes 2 oa.o .ms< mass as ~m.o eossmosoo ammmm seems ma om as 0 SN OH OH H odsoaoz mm ma mm OH NH 0 a oHHms 6 ma 0 m 0H . canoes as ma ma 0 mm OH 0 0 ma m enhances em am we oH NH m 6 ma H osssoosm 81 The inhibition of this enzyme by 01- and tri- carboxylate anions contradicts previously reported results obtained in Na+ or K+ succinate buffers. According to Nikiforuk and Colowick (35) citrate, acetate, and lactate activated AMP aminohydrolase assayed at pH 5.9: Lee could not reproduce their data (2). Their failure to observe inhibition may be attributed to the presence of Na+ or K+ in the assay Since in this case the K+ activated enzyme was also relatively insensitive to carboxylate anions. Furthermore, the failure to observe activation by ATP or ADP in the earlier studies may have been due to the presence of succinate (2), which at 0.1 M completely inhibits the ADP activation. The insensitivity of the calf brain AMP aminohydrolase to such anions (28) may reflect Species or tissue differences. The effect of citrate on ADP activation is pre- sented as a Woolf plot of [ADPJ/vi versus [ADP] at con- stant levels of citrate and a subOptimal substrate con- centration of 50 uM (Figure 10). While interpretation of such data for an allosteric enzyme is difficult, citrate does not change the observed maximum velocity of the ADP activated AMP deamination as indicated by the identical SlOpes obtained in the Woolf plot. Furthermore the lines are linear except for slight curvature in the absence of citrate. Thus the data are consistent with a competitive interaction between citrate and ADP. From the intercept 82 Figure 10. The Effect of Citrate on ADP Activation of AMP Aminohydrolase The data are presented in a Woolf plot, [ADP]/v1 versus [ADP] at constant levels of citrate where v1 represents the initial velocity of the inhibited reac- tion. The reaction was followed at 265 nm in 0.15 M TMACl, 0.05 M Tris cacodylate, pH 6.3, 50 uM Tris AMP and variable amounts of ADP. Tris citrate concentra- tions were as follows: 0 - no citrate; .- 0.1 mM; EJ- 0.175 mM: l- 0.25 mM; and A- 0.50 mM. Each point represents the average of 2 or more assays at 0.15 Mg enzyme per ml. The insert represents a plot of the [ADPJ/vl intercepts versus citrate concentra- tion. The K1 for citrate was determined from the intercept on the citrate axis. 83 q _ 4 LM _1m .1m 13 4M 16 13m ' m. A!“ i‘ n .unw ... ”Jr a o .2 xi§§?::5ich=-&$u .. smears. . b - P p b m m .m m 0 #25051 as 73522 [ADP]: IO°M Figure 10. 84 on the [citrate] axis of the line drawn through a plot of the [ADPJ/vl intercepts versus citrate concentration, a K1 for citrate inhibition of 0.11 mM was determined. The Hill SlOpe for ADP activation in the presence of increasing citrate concentrations decreased from 1.7 to 1.1 (Figure 11) while the Kaapp for ADP calculated from the Hill plot increased: Kaapp for ADP was 22 0M, 30 0M, 36 uM, 42 MM, and 63 uM at 0, 0.10 mM, 0.175 mM, 0.25 mM. and 0.50 mM citrate, reSpectively. Perhaps citrate is binding at the ADP site eliminating the cooperativity observed for ADP activation. Citrate and ADP, although dissimilar in structure, do possess common metal binding pr0perties; consequently citrate may inhibit ADP activation by interaction with an enzyme bound cation necessary for activator binding. Although other mechanisms of inhibition could be proposed such as dissociation of subunits or conformational changes, the presence of a metal ion component seemed, intuitively at least, a good possibility. Inhibition9 of AMP Aminohydrolase by Metal Binding Agents AMP aminohydrolase, in either KCl or TMACl at pH 7.1 was inhibited by several metal binding agents including 91h this thesis inhibition is defined as any loss of activity caused by metal'binding agents. Theoretically there are two mechanisms by which an inhibition can be realized: (A) binding of the inhibitor in situ or (B) removal of the metal from the protein. In either case 85 Figure 11. Hill Plot for Citrate Inhibition of ADP Activation The data from Figure 8 for ADP activation at various levels of the inhibitor, citrate, are plotted according to the Hill equation: Log [(Vmax/vi) - 1] versus Log [ADP] M, where Vmax is the observed maximum velocity of the reaction at 50 0M Tris AMP and v1 is the velocity of the inhibited reaction. 86 5.5 5.0 4.5 -LOG [ADP] M 4.0 NT .5 63 .62; Figure 11 . 8? o-phenanthroline, ethylenediaminetetraacetate, citrate, 8-hydroxyquinoline-5-sulfonate, dithioerythritol, and mercaptoethanol (see Figures 12-15). In all cases enzyme in the absence of K+ was more sensitive to inhibi- tion by the metal binding agents. For example, 0.18 mM 0P inhibited the enzyme in TMACl to the same extent as 1.0 mM GP in KCl: in 0.67 mM 8-OHQSSA the inhibition in TMACl was 78% versus 37% in KCl during the same time Span. Although 50 mM citrate inhibited the enzyme in TMACl, this concen- tration of citrate did not inhibit the enzyme in KCl (Figure 13). This greater sensitivity to metal binding agents in the absence of K+ may reflect a weaker binding constant of the enzyme for its metal component or greater accessibility of the chelating agent to the metal as a result of a conformational change. A second possibility is that a significant amount of the inhibitor(s) may be complexed with K+ thereby reducing the available concen- tration of inhibitor(s). No stability constants for potassium complexes with the metal binding agents used in this study were available; however, if one assumes a Kf 2— for KCitrate similar to KADPZ' (n5 M'lL) (107) the free inhibition may be reversible or irreversible. The loss of enzyme activity may be restored upon exposure to environ- mental conditions similar to those under which the enzyme was exposed to the inhibitor. In mechanism (B) the rever- sibility is dependent upon the ability of the apoprotein to reassociate with the metal once dissociation occurs. Irreversible inhibition (sometimes referred to as inactiva- tion) results from an irreversible loss of activity y denaturation or by covalent binding at the active Site. For further discussion see Reference 119. 88 Figure 12. Semilogrithmic Plot of oéPhenanthroline Inhibition of AMP Aminohydrolase as a Function of Time AMP aminohydrolase (0.10 mg protein/ml) was incu- bated in 0.5 M salt (KCl or TMACl), 0.10 M Tris Mes, pH 7.1, and 1 mM mercaptoethanol at 30°. Aliquots were removed at the intervals indicated for assay in Assay 1 containing 50 0M Tris AMP (A) or Assay 2 containing 50 0M Tris AMP and 100 uM Tris ADP (B). The concentra- tions of metal binding agents in the KCl incubation mix- tures (A) were c)- control, X - 0.6 mM m-phenanthroline, A- 1 mM GP, and EJ- 2 mM 0P. For the TMACl incubation mixtures (B) the concentrations were 0- control, + - 0.18 mM m-phenanthroline, ‘- 0.05 mM GP, and I - 0.18 mM UP. The degree of reactivation during the assay is represented by the broken lines: --- 1 mM 0F and --- 2 mM GP in A. I I I t {r— L U #1 fl )- t _>_ § .— 2 _____________________________ U Control 0 0.6 mil m-Phononnwolino x I .O M” o-Phononthrollno A 2.0 III“ o-Phononthrollno a 3 4 5 6 TIME (hours) I I I I w v‘= + >- A ‘ t 2 E E TMACI Lu .— 8 Control 0 31' 0.13 m m-Pbumtmnm + - 0.05 mm o-Phononthrolino A O.IB mu o-Phononthrollno I l l l 3 4 6 6 TIME (hours) Figure 12. 90 .Sosmsm Hesse one mHonamm pomoao on» use aopmmm How on» paomohaoh maonamm noao one .maobap uooaoos .39 mass as or one as 03 .. 4. 4 ”banana 3.5 as on .. I . o ”Honpnoo I 0.0 ”mzoaaou no one: oHSpNHB :oapwoboca on» a." “Emma @5933 Hence mo meodpmapcoosoo .ma oaswam ca eonaaomoe one mSOapaeaoo made no cosponsm a ma ommaondhnosaad mam mo noapandan «saw one opmnudo Mo poam caESpdnmwoaaaom .ma ohswdm 91 3.35 ms; m q .1 4 «Bu :5 8 4 «Eu :5 8. I a 22.8 :2 on o o .888 .043 an 4 .1 O. O¢ All/\llOV .LNBOHBd Figure 13. 92 .ampmmm H0¢28 0:» pammmhamh maopamm ommoao map find ampmmm HUM m2» unommhaon maopamm ammo mne .qmmamoé as $5 .. 3.0 93 Jmmamoum 2a m6 ud 395:3 .. o. o «szHHOh mm mpoz mnsumaa nodpmpdoza 0:» :« Houanagna mo mnodpmppnmocoo was .NH mmswam ca omnahommv mm vmmmmmm cam Umpdmhp was mahuzm mafia mo soaponsm a mm mmdaondms tended mz< mo neapanannH «mmdmoam mo poam oaanpanmwoaaamm .aa mnswam 93 “9:35 mg... O. u n c d u u T I 9 38.5.0 2.5.3 4 «8.6.» :53 I o .6528 u .65: "am. I. D 9.1 V All/“1.0V .LNBOHBd Figure 14. 94 .98 2a 3 .. 4. 4 Eu £8 2a H u I. u Sommfimopamonwa 2a 3 ab “29583 Hogpmopmmohma as H .. O . 0 ”down 0.33 mugoaaoo H329 mo mzoapmnpnmocoo wzaxoaaom mns .ma mnswdm ca umnanommv mum zoapansozd Mom macapavnoo made we codpogm a ma mug—09600 Hedge E wmflongsofiaa m5. go 8332”: on» go no: Sasfinmwoflamm .3 93m; 95 52:05 NEE. o n. c n N . o . I . ‘ u q q 0. D 1m! 35L ‘ 4 who 250.0. I a US 2.5.. 4 u I o is 2.8.. .. om _. . i». g... . D _l l 0' 4 O r! I! 511]! r j % All/\liOV .LNBOHBd Figure 15. 96 citrate concentration at 50 mM total citrate and 0.5 M KCl is reduced to 15 mM. Such a reduction in free citrate concentration might account for the decreased sensitivity of the enZyme to citrate inhibition in KCl. In the study of GP inhibition in KCl the enzyme was reactivated upon dilution in the assay mixture; the extent of reactivation depended upon the concentration of OP in the incubation mixture. Although this reactivation phenomenon was not explored further, two eXplanations can be proposed: either the inhibition by OP is truly rever- sible upon dilution or the presence of a trace metal(s) in the assay mixture may have removed some of the OP inhibi- tion through competition with the metal-enZyme for OP or displacement of a metal[O‘P]x complex from the enzyme (119). Meta-phenanthroline did not inactivate AMP-amino- hydrolase. thus binding of the phenanthroline ring system to the enzyme is not reSponsible for the observed inhibi- tion by OF. The inhibition by GP via sulfhydral oxidation. as was shown to be the case with rabbit muscle aldolase (108), seems unlikely since mercaptoethanol which protects against such inhibition was always present in the protein incubation mixtures. Although.AMP aminohydrolase absolutely requires thiols such as mercaptoethanol for stability (1), an excess of either DTE or mercaptoethanol caused a time dependent inhibition (Figure 15). The contradictory effects of 97 thiols can be explained by (A) a sensitive sulfhydral protected from oxidation by low concentrations of thiols and (B) the relative ability of thiols and dithiols to bind metals (109, 110). An additional possibility that high concentrations of thiols reduce disulfide bonds can- not be eliminated with the present data. Mercaptoethanol, a monothiol, was a less effective inhibitor than DTE, a dithiol. For example, after one hour in the TMACl incuba- tion mixture, 10 mM UTE inhibited the enzyme 78% whereas 40 mM mercaptoethanol inhibited only 50%. The data are consistent with the ability of dithiols to form stable bidentate chelates with divalent metals (109). The inhibition of AMP aminohydrolase by metal bind- ing agents possessing widely different ligands is consis- tent with the enzyme requiring a metal for activity. Since many known zinc metalloenzymes are subject to inhi- bition by thiols such as DTE, mercaptoethanol, cysteine, and 2,3-dimercaptoethanol (111-115), zinc is a good possi- bility for the metal component of AMP aminohydrolase. Direct evidence that the enzyme contained zinc or another metal was obtained by quantitative metal analysis. Quantitative Metal Analyses of Meta;_Content of AMP Aminohydrolase Purified AMP aminohydrolase and an aliquot of the crude extract were analyzed for the presence of seven transition metals by atomic absorption Spectroscopy and 98 neutron activation analysis. The average of the analyses for two preparations are presented in Table in. In order to eliminate metal ion contamination, the enzyme was purified using the precautions outlined in Materials and Methods. Zinc increased from 0.? gram atom per mole enzyme in the crude extract to 2.58 gram atoms per mole after purification based on a molecular weight of 278,000 for the native enzyme. Extrinsic metals such as calcium, magnesium, and iron, although present in significant quantities in the crude extract, were not found in stoi- chiometrically significant amounts in the purified enzyme. Neither were significant amounts of metals found in the TMACl solutions used for dialysis and Sephadex chromatog- raphy. The neutron activation analyses for cobalt, manganese, and copper were reported as minimum detection limits for an instrumental analysis without radiochemical separations. In each case the detection limit for the blank was higher than that for the sample: cobalt, <35 ppb blank and.<20 ppb sample; copper,‘<250 ppb blank and <<100 ppb sample; and manganese, <350 ppb.blank and <300 ppb sample. The detection limits for copper and manganese were too high to rule out the presence of 0.4 gram atom copper or 1.5 gram atoms of manganese in the samples. However, for cobalt the detection limit was low enough to eliminate the 99 m oz :z m “92 co m no.0 n so smmaw $3 .85 N u m... mm 8.3. w v 9.0 m H o: w: mo.ov oHv no.0 :H H mm so mm.m OH H moo mos.o ma H boa as mamnzm macs sampoam_amaw mahusm maoz sampohm Esau Hmpmz H09 H09 awn Hog wsops swag macaw oaoaz maops swam macho oaoaz oommHoavmsosdaw mzq omdmaasm noosapxm ocsao soapomam mommaoacmsoaaa< mz< soamdasm was pomapwm coupe on» no pampsoo Hmpcz .ea canoe 100 .Umsaahmpod #0: .929 .soammSomau noanSm you vamp mom .moHQEMm on» pom comamppo omonp ads» ampmmaw who: msoapsaom xssap on» how comadpno mosamb one .noapmbapod soapzoz mp commaond macs mmamssmm .asaasaa was» op fiasco no can» mmma mm oopaoaoa ma pnopsoo mm on» .Ha\2amponm we a pm copompoo no: was om mondm .Bam mo.o was ob ace paaaa coapompop Hansoasapmsa aged .meSpoz was masdaopsz ma confluence no main Noodzmom no pmsamamopmaoano one aommsn HUM unnawwo douaadav one: sasaoo cumsamosa omoafiaaoo on» Sony msodposam uoaooa case .mamhamzm onomop moonpoz dam masdaopmz ma conaaomop mm condos» was pooapxm oaomsa moans on» mo soapmwsudapzoo nouns pampmshmgzm ms» mo pondaam can .mfihuno shapes on» How ooo.mmm mo psmaos asasomaos o no woman mnoapmnsnoaa «amuse or» you muoapssdanopoo esp no owwaobs on» ma oopaoammo 101 presence of greater than 0.1 gram atom cobalt per mole enzyme. The enzyme containing 2.58 gram atoms zinc per mole enzyme was fully active with reSpect to previous preparations of the enzyme: the KIn for AMP and‘Vmax were 0.4 mM and 1345 um/min/mg protein, reSpectively (Assay 1). ADP activation was not as great since the Specific activ- ity in Assay 2 was only 70 um/min/mg protein versus the specific activity of 110-130 um/min/mg protein usually observed. Considering the non integral amount of zinc found upon metal analysis, it is conceivable that some zinc was lost during the purification procedure with a resultant decrease in ADP activation. Loss of ADP acti- vation without a concurrant loss in maximum activity for the K+-activated enzyme is consistent with two different types of zinc. Divalent Metal Ion Requirement for Enzymatic Activity fiemoval of Zinc from.AMP Aminohydrolase and the Concurrent Loss of Enzymatic Activity The trace metal analyses and inhibition by metal binding agents suggest that AMP aminohydrolase contains strongly bound zinc. However the data does not establish that zinc is required for activity. Therefore it is necessary to establish that the enzyme loses activity as a function of the loss of zinc. 102 In the presence of excess 8-OHQ5SA, zinc and 8-0HQ5SA form a 1:3 complex which exhibits a distinct absorption maximum at 370 nm (see Figure 16) with an extinction coefficient of 11.1 x 103 OD/mm and a stabil- ity constant, Log 83, of 20.10 The absorption obeys Beer's law; i.e., at excess 8-0HQSSA the absorption of the complex is a linear function of zinc concentration (data not shown). Since AMP aminohydrolase was rapidly inhibited by 8-0HQ5SA, the method described by Simpson and Vallee (88) was adapted to correlate the loss of enzymatic activity with formation of a ZnEB-OHQ58A13 complex. AMP aminohydrolase (3.59 x 10'6M) was titrated with Tris 8-OHQSSA as described in Materials and Methods. Addition of this chelator resulted in a rapid inhibition the extent of which was dependent on the amount added (see Figure 14). The extent of inhibition is presented as a plot of Percent Activity Remaining versus Moles of Zn[8-0HQSSA]3 formed (Figure 17). Ninety percent of the activity was lost after formation of 3 moles of Zn[8-OHQ5SA]3 per mole enzyme. After the loss of 3.5 moles of zinc turbidity prevented further measurements. There is some disagreement between the 3 to 3.5 gram atoms of zinc titrated by 8-0HQSSA and the 2.58 gram atoms of zinc per mole of native enzyme as previously determined. However, the theoretical absorbance for the Zn [8-0HQSSA13 _# 1oPrivate communication, B. L. Vallee. 103 Figure 16. The Visible and UV Spectrum of 8-0HQ5SA Before and After the Addition of Zinc Sulfate The spectra were recorded from 500 nm to 220 nm with a Beckman DB Spectrophotometer equipped with a Sargent Recorder. The sample and reference cells con- tained 0.# M TMACl and 0.1 M Tris Mes, pH 7.2. The represents the spectrum of 0.18 mM 8-0HQSSA and the ----- represents the Spectrum of 0.18 mM 8-0HQSSA plus 0.02 mM zinc sulfate. 101+ 260300340380420460500 l .0 0.2 - 0.0 WAVELENGTH (nm) Figure 16. 105 Figure 17. CorreSpondence Between Activity of AMP Aminohydrolase and the Formation of the Zn[8-0HQ58A]3 Complex The percent enzymatic activity remaining as a function of the Zn[8-0HQ58A]3 complex formed is presented. Enzyme was chromatographed on Sephadex G—25 equilibrated with 0.5 M salt (KCl or TMACl), 0.10 M Tris Mes, pH 7.1, and 1 mM mercaptoethanol. Increments of 8-OHQSSA were added to 3.6 x io-6M enzyme under conditions described in Materials and Methods and the absorption at 370 nm was recorded; aliquots were withdrawn and diluted for assay after each addition. The activity of the enZyme incubated in KCl (--()--) before the addition of inhibitor was 108 um.AMP deaminated/min per mg protein at 50 0M AMP: the Specific activity of enzyme incubated in TMACl (--.--) was 65 um/min per mg protein in the same assay. The assay mixture was passed over Chelex 100 (Tris+) prior to use to remove contaminating metal ions. 106 I l I l00 - 0 Incubation g 0 KC! 2 O _. so .- 0 TMACI .. <1 2 Lu 0: >_ 60*- t: Z 23 q 40- *— E o 20.. 0: Lu a. O MOLES Zn (SOHQSSAL, MOLE ENZYME Figure 17. 107 complex, assuming 4 gram atoms zinc per mole enzyme (at 3.59 x 10"6 mm enzyme per ml) is only 0.17 0D units. With such a low total absorbance some error can be expected. The data are consistent with zinc being required for enzymatic activity and that loss of activity is due to complexation by 8-0HQ5SA. To establish that zinc is indeed removed from the enzyme in contrast to formation of an inactive ternary complex, purified enzyme incubated in 8-OHQ5SA was chro- matographed on Sephadex G-25 to separate the enzyme from excess 8-0HQSSA and the Zn.[8-0HQ5SA]3 complex. The recovered enzyme generally exhibited V45% of the activity of the native enzyme at 10 mM AMP and contained 0.45 gram atoms of zinc per mole enzyme as determined by atomic absorption analysis. The 15% residual activity closely corresponded to the 17% residual zinc based upon the 2.58 gram atoms zinc per mole native enzyme; thus the lower activity is consistent with the loss of zinc from the enzyme rather than formation of an inactive ternary complex. Reactivation of Ago-AMP Aminohydrolase by Transition Metal Ions Since AMP aminohydrolase could be obtained relatively free of zinc, reconstitution of the enzyme by the addition of zinc and other transition metals was attempted. The apoenzyme was titrated with SpectrOpure sulfate salts of zinc, cobalt, manganese, iron, nickel, magnesium, 108 capper, and cadmium; activity at each concentration of metal was determined at 10 mM AMP. The gram atoms of metal added per mole of enzyme varied from 0 to 4 or 10. Zinc reactivation (Figure 18) approached 70-80% that observed for native enzyme. Cobalt, iron, and manganese, in order of decreasing effectiveness, also reactivated the apoenzyme (Figure 19). Nickel, magnesium, copper, and cadmium were ineffective in restoring activity to the apoenzyme. Kinetic Properties of the Zinc, Cobalt, and Manganese AMP Aminohydrolase Since the replacement of zinc by other transition metals alters the kinetics and Specificity of other zinc metallo enzymes (116, 117), the kinetic parameters for AMP deamination in KCl and for ADP activation in TMACl were investigated for the zinc, cobalt, and manganese reconstituted enzyme and compared with those of the native and apoenzyme (see Table 15). The ferrous enzyme was too unstable to obtain reliable data. An aliquot of apoenzyme reconstituted by addition of 3.7 gram atoms metal sulfate per 278,000 grams of enzyme was chromatographed on Sephadex G-25 to remove excess divalent cations and potassium chloride. The zinc and cobalt enzymes were 20 to 30% more active after chromatog- raphy, however only 60-70% of the protein applied to the column was recovered. These observations are consistentv 109 .mcoa Hopes wsHmeHEmpsoo oboaoa op A+mda9v ooa Noaoso spa: Uopmoap noon on: chapxfia momma onp wSHUSHona mucowmoa HH4 .mz< mane as 0H wadsdmpsoo H momma Spa: poasmmoa mm: mpabapom mammsm .osau yo undead assoamma was» how oopooaaoo ma spade Soapmapap map oaowmaoSp .oamuno oHoa pom scam maopm swam m:.o posdopcoo oBhuSooao on» pomp popooH8:« mamhamnm scapaaownm oaaopq .mUOSpmz one mamaaopmz ad oonaaomop mm H.m ma pm popdbapomoa mos cahNamoam one mammasm oaaw masaoapooam Spa: Soapmapae an omoaoapmsocaa< mzwloa< mo coapmbapomom .mH shaman 11.0 A}: C) 3-0l X (ugaimd bui/ugw/um) Ail/\LLOV OlleBdS Figure 18. GRAM MOMS Zn2+ ADDED/MOLE APO ENZYME 111 .mpdbapom oamu stoHSp mmma no: 08 mpoaa onp men» use osHN HmSpHmoa you ompomaaoo no: cams dump on» .oSHN mo oosommaa on» pom commamsm pom mm: mpSpm maze ca poms camusooam can moaam .mponpmz use mHmHaopmz use ma maswdm :H Uopaaommo was nodumpapomoa mo macapapsoo woudmadm Hope: :oHpHmsmae mHSQOApomam Spa: coapmapda mp ommaoaomsoaaas madloas ho noapmbapommm .ma oaswfim 112 usimzw 84 3023804 822 929.4 54mm m on c n m _ o o no me n N . o fill: a a , JL 4 4 d £36.80 £532.32 £380.32: In! X a 3209.6: 0 f :2. I .o_. :88 o . oEN a 25 a L b p P h L P h 0 All/\llOV 0|:Jl03dS C) 3-0l X (ugeimd bw/ugw/um) Figure 19. 113 with selective binding of the denatured enzyme (which perhaps was incapable of being reactivated by zinc) to the Sephadex resulting in recovery of enzyme with a higher Specific activity. The activity of the manganese enzyme reverted to that of the apoenzyme after chroma- tography indicating a weaker binding of manganese with AMP aminohydrolase. The observed Vmax for the zinc reconstituted enzyme (4 gram atoms zinc per mole enzyme) was 1550 um/min per mg protein or 13% greater than that observed with the native enzyme (Table 15). Both enzymes exhibited the same Km for AMP. The data suggest that the 4th atom of zinc is not necessary for maximum enzymatic activity in the K+-activated system. Enzyme which contained 4 gram atoms zinc per mole enzyme had a Specific activity of 150 um/min per mg protein when assayed at saturating ADP and 50 MM AMP; however enzymes with an estimated 1.85 gram atoms of zinc, although still activated by ADP, exhibited a consid- erable reduction in observed maximum velocity at 50 uM AMP. It was previously pointed out (page 101) that the native enzyme which contained 2.58 gram atoms of zinc (by atomic absorption analysis) also exhibited a lower relative activity in the ADP-activated system. Although the kinetic results are only preliminary, they suggest that zinc functions in more than one capacity; i.e., zinc may be necessary for both enzymatic activity and for ADP activation. 114 com mm.o any a.m SE :.N on on coma no.0 Asa s.m oo m.a oma em omma pm.o va 0.: a.m mm ma Acv mw.a com mm.o Adv me.o memH oe.o aov mm.~ an ac amo azmv mos ama> azav ea oaamam oaoz Hmpoz II. o D hog coaemoaooc mo< msoapdaaadoo axe Hosea maopq amen myopoamamm oapmSam camusm Umpfipapmaooom ommcmwsmz cam .pawnoo .oSaN o£p was .ommaoaomsoaaaw mzsnoa< .mmdaoaoms nosaas mzd whapmz you whopoamamm capoaam was pampSOU Hope: no mamaadm .ma canoe 115 .msamamopmaoaso mmuo Nopmsamm ohomop mamncooas on» on mouse Hopes mo mEOpm Echo: .Amomhamss N no owonbmv 393.38 22993de oaaopm an 5393 m\+NnN mm :mm pm:«dpdoo o6»u:o one .mmnu Noomnamm so anamawopmSoaso machon Amy oaaucoo m op ends mo maopm swam m.m mo Soapapum cap an Umsamppollmamuso Umpfipapmsoommw .« Howmsm ad mmlw Hopmsaom no msamawopmaoano an doonHow Amy mamuaooam op ends mo msopm swam :.H mo soapapow on» an vocampDOIloamNSo downpapmSooomm .mamzawsm noapaaomns odaopo an sonaaaopmo :Hopoaa w SN m: .moa pampaoo cadmiumsmucooa< +N m 0 .AHH canoe ommv mamaamsm soapaaompm OHSopm an donasaopoo psopsoo osaunnmaamco obapmzo .coaec>apom one cow oooam Haam on» ma a when; a was \a mamamb >\H mo poaa xasmnambmozoaaq poamaooa a Scam popmaapmm mos Andopoam ma non :aa\popoaaamop mzw 21 Gav occampno mafia» one .mm< mp scumbapom moss mz< make 21 on us mpaooaob adaaxma ombaomno on» ma >0 .m maswam SH confluence mm Amz< make 2: om Spasamomommwv aoaocaaocm age not scan Haam one acne cocaaaopco not and ace ma oxen .sHopoaa we hog maa\sopm2HESop opmapm anew 21 mo commoaaxo ma xda> . mz< \H msmaob >\H mo mpoaa Mafimlhobmozoaaq Scam comaahopmo cams AH zommwv soapmaaammo mad you mampoamama caveman ones 116 The first is consistent with the low activity found in the apoenzyme versus the high activity of the native and reactivated enzyme: the second is consistent with citrate inhibition of ADP activation. Since the enzyme has 4 sub- units of identical molecular weight, the binding of 4 atoms of zinc to the apoenzyme is consistent with one zinc per subunit. However very careful binding studies are necessary before the actual relationship between the total number of zinc atoms bound and enzymatic activity can be established. The reconstitution of the apoenzyme with divalent cations other than zinc affects both the maximum rate of reaction and to some extent the Km for AMP in KCl. 'The cobalt reactivated enzyme showed an increase in Km for AMP from 0.36 mM to 0.63 mM and a decrease in maximum velocity relative to enzyme containing 4 gram atoms of zinc. Furthermore the cobalt enzyme was not as effectively acti- vated by ADP: the observed maximum velocity at 50 0M AMP was 0.53 V(Zn)' A greater increase in Km from 0.36 to 0.93 occurred with the manganese enzyme; the relative maximum velocity in KCl was 0.36 V(Zn)’ ADP activation of the manganese enzyme was not studied since the enzyme lost activity when chromatographed on Sephadex G—25 equilibrated with TMACl. Since atomic absorption analyses of the cobalt and manganese reactivated enzymes were not done, the 117 stoichiometry of metal to enzyme used for determination of Km and‘Vmax is not known, eSpecially with reSpect to the replacement of the residual zinc (<0.5 gram atom per mole enzyme) in the apoenzyme. Therefore the kinetic parameters obtained for the cobalt and manganese enzymes are only approximate values. Until the present no AMP aminohydrolase has been shown to be a metalloenzyme. Lee (2) and Nikiforuk and Colowick (35) observed no dependence of activity upon divalent cations. However their judgement was based upon (A) the observed inhibition rather than activation of the enzyme by Cu2+, Zn2+, Fe2+, and Ag+ and (B) the failure of known metal binding agents such as cyanide, EDTA, and cysteine to inhibit the enzyme. Trace metal analysis was not attempted by these investigators. Indeed the rabbit muscle enzyme prepared according to the method of Smiley gt a; (1) is also inhibited by divalent metals added to the assay. At 0.1 mM, Cuz+ and Zn2+ inhibited 98% and 8%fl reSpectivelmrwhile 0.5 mM N12+ or Cd2+ inhibited 23%. 2+ 2+ 2+ 2+ 2+ Inhibition by Co , Mn , Fe , and Ca was less . Ms than 10% at 1.0 mM. Similarly yeast alcohol dehydrogenase and beef liver glutamate dehydrogenase are actually inhibited by Zn2+ the very metal essential for catalytic + activity (118, 111). The ions Cu2+, Fe2+, Ag+, Cd2 , and Hg2+ also inhibited (118). Hence inhibition of zinc metalloenzymes by transition metals is no criteria for 118 eliminating a metal cofactor requirement. Furthermore failure to observe inhibition by EDTA, a strong metal chelator, is not justifiable evidence that a metal ion is absent. The high association constants for metal EDTA complexes apply only for the tetradentate complex and their association constants are altered if some of the coordination sites of the metal are already occupied by protein ligands. Therefore inhibition may not be observed (119). The failure of EDTA to inhibit yeast alcohol dehydrogenase is attributed to such steric hinderance (118). The preparation of AMP aminohydrolase used here required higher concentrations for inhibition than would be eXpected indicating steric factors may be interfering with binding of EDTA to the metal ion. Setlow and Lowenstein observed slight activation of the purified calf 2+ and Mn2+; however Zn2+ brain AMP aminohydrolase by Mg did not activate (28). The nonSpecific adenine nucleotide aminohydrolase from Red Marine Algae is reported to be activated by divalent metals: Ca2+ was the most effective activator of this enzyme (120). No metal analysis for this enzyme was reported. The data presented in this thesis partially fulfill the postulates first enumerated by Vallee (121) for iden- tification of a metalloenzyme: (A) The enzyme is homo- geneous by physiochemical techniques (1); (B) Metal analysis performed on the purified enzyme show an increase 119 of zinc, "intrinsic metal," to protein ratio with reSpect to the crude extract and a decrease in "extrinsic metal" to protein ratio; (C) Iron, calcium, magnesium, and cobalt were present in stoichiometrically insignificant amounts (the analyses for copper and manganese were ambiguous due to insensitivity of the analytical method); (D) The zinc content of the enzyme correlated with the Specific activ- ity; and (E) Enzyme activity was inhibited by a variety of metal binding agents. Three additional postulates have not yet been fully satisfied. The stoichiometry of zinc to enzyme has not been absolutely established. The ratio of gram atoms of zinc per mole of enzyme, although a small number;2.58, was not an integral number. It seems possible that the pure enzyme is not isolated with its full zinc complement eSpecially in light of the fact that the enzyme consists of four subunits of the same molecular weight. Although the prevention or reversal of the inhibition by metal binding agents was not investigated, enzyme from which zinc had been removed by incubation in 8-0HQSSA followed by chromatography on Sephadex G-25 was found to be readily reconstituted not only by Zn2+ but also by C02+, Mn2+, and FeZ+. Reconstitution by metals other than zinc resulting in enzymatically functional proteins is a common phenomenon among zinc metalloenzymes (88, 116, 117). The replacement of zinc, a diamagnetic ion, by paramagnetic Coz+ or Mn2+ makes further investigation of the role of the 120 divalent metal in the enzyme possible through Spectral and nuclear magnetic resonance studies. Investigations along these lines are of Special interest in light of the kinetic data which indicate (but do not prove) that the zinc atom(s) perform two roles: (A) The zinc atom(s) is absolutely necessary for enzymatic activity although its function remains unknown (for example zinc may participate by bind- ing AMP, by orientation of either substrate, by polariza- tion of the bond to be broken, or by maintaining the enzyme in an active conformation); (B) Zinc may be required at the activator site. Precedence for two classes of tightly bound metal atoms has been established in alkaline phospha- tase, a zinc metalloenzyme where 2 of the 4 zinc atoms are essential for activity (88). Horse liver alcohol dehydro- genase also possesses two classes of zinc (122): two of the four zinc atoms are essential for activity while the remaining zinc atoms stabilize the quaternary structure of the enzyme. Although the data are not sufficient to prOpose a molecular model for the enzyme and the mechanism of action of zinc, it is tempting to suggest that zinc is directly involved in the hydrolysis of the 6-amino group from.AMP in a manner analogous to the hydrolysis of peptide bonds by carboxypeptidase A, a known zinc metalloenzyme. In the latter enzyme recent x-ray crystallographic studies indicate that zinc may participate in polarization of the carbonyl of the peptide linkage to be hydrolyzed (123-125). 3 UN NA RY Rabbit skeletal muscle AMP aminohydrolase deaminated the following analogs in order of decreasing Vmax‘ AMP, AMP-NHZ, Né-methyl AMP, dAIVTP, adenosine mono- sulfate, adenosine and ADP. The enzyme also deamin- ated Né—ethyl AMP, formycin 5'-mon0ph08phate, AmDP, and d,B—methylene ADP. 6-Mercaptopurine 5'-ribonucleotide and IDP (Kaapp = 1.3 mM) activated the enzyme. 3'-AMP, 3',5'-cyclic AMP, and 3-iso AMP inhibited AMP deamination. 3-Iso AMP was a linear competitive inhibitor for AMP (K1 = 32 mM). With dAMP as the sub- strate the enzyme exhibited negative 000perativity with reSpect to this inhibitor. Deamination of adenosine, AMP, and ADP by a single enzyme was consistent with the results obtained from studies of enzyme homogeneity, heat inactivation, elution from cellulose phoSphate, and product charac- terization. Analysis of the products of N6-methyl AMP by paper and gas chromatography indicated that only IMP and methylamine were formed. The data favor the direct hydrolysis of substrate rather than a Dimroth-like rearrangement. 121 7. 10. 11. 12. 122 The Aégo nm (1 cm light path) for AMP aminohydrolase was 9.15 0D on a dry weight basis. A molecular weight of 278,000 for the native enzyme was obtained from sedimentation equilibrium centrifu- gation in 0.2 M KCl and 1 mM mercaptoethanol at pH 7.2. AMP aminohydrolase consists of 4 subunits of identical molecular weight (69,000) by SDS polyacrylamide elec- trOphoresis. Citrate, succinate, maleate, fumarate, malate, and malonate strongly inhibited the activity of enzyme assayed in the absence of K+. Citrate was a competi- tive inhibitor (K1 = 0.11 mM) for ADP activation and eliminated the coOperative kinetics observed for ADP by decreasing the nH for ADP from 1.7 to 1.1. The enzyme was strongly inhibited by metal binding agents such as o-phenanthroline, 8-hydroxyquinoline- 5-sulfonate, ethylenediaminetetraacetate, citrate, mercaptoethanol, and dithioerythritol. Atomic absorption analysis demonstrated that the native enzyme contained 2.58 gram atoms Zn2+/278,000 grams protein. Only zinc showed an increase in metal to protein ratios during purification. 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