a...“ r. W... noiw . V1. “a. 5,. 3.. .i. L”. h... I. fit ..t. .1... Ca V rm” R mum a 5‘. FM” V! a: r .u “H. “u. a; . J . 1.9.. . v.4 e .u hi. 3» n?- I a .3“ N. w.” .L x. c WE : a w . a r .. ,-. Eu rfi w' a It, fit Mr: wow. i. . flame .. .x a on. N: n. r . a: .u... .l «3 . 1..“ at: an 1.. . 5%.,“ x: Mr. Mm“ mu A a ._ A 43a pm .i C 1.1%: mm D 3 x... a. t: 5M.- ohm ff,- Q .. a "L .L I N. 1:. w.“ r. T: NH mm «W . «i m 5N” 2.1::T-1RY ' Illl“WILDIIIIHIWIlillIIIIHIHNIIIHHIHIIHIIIINI Mich; .... 1 State 1293 10381 3881 University WM Li ABSTRACT Some Biological and Kinetic Properties of S'quenylic Acid Deaminase by Arnold 3. Berry S'quenylic acid deaminase is an enzyme of wide biological distribution which catalyzes the conversion of 5'qAMP to IMP. It is isolated as a complex with myosin from rabbit skeletal muscle and several studies indicate that it may be involved in muscular contraction. This work shows that rabbit skeletal muscle is the best source of enzyme of the animals studied and the enzyme level increases with age in young rabbits. In chicken and pigeon the level in breast muscle is about six fold greater than in leg muscle, The level in heart muscle was very low in all eight species tested, The effect of divalent cations and some organic acids on crude enzyme preparations is reported, An absolute requirement for a monovalent cation is shown and the activation constants for the alkali cations are estimated, 5’2Adeny1ic acid deaminase is shown to be allosteric for AMP and this property is not altered by variation of the K01 concentration or heating the enzyme to 50°C. An apparent Km of 1,8x10'5M was calculated from the Hill equation, Activation by ATP is demonstrated for this enzyme. This was not shown by all preparations and more work must be done in this area before any real conclusions can be drawn; however, a scheme rationalizing this act- ivation is presented. Some Biological and Kinetic Properties of 5'-Adenylic Acid Deaminase by Arnold J, Berry A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1966 :3, ACKNOWLEDGEMENTS The author is indebted to Doctors Eugene Byrnes and Karl Smiley for the supply of purified enzyme, The help- ful guidance of Doctor Clarence Suelter during this project is greatly appreciated, ii II. III. IV. VI, VII. VIII. IX. XI. TABLE OF CONTENTS AcknowledgementS............................ Table of ContentS........................... List of TableS.............................. List of Figures............................. Introduction................................ Historical.................................. Materials and MethodS....................... ExperimentaIOOOOOOOOOCOOOOOOOOOCOOOCOOOOOOOO DiSCUS51onOOOOOIOOOOO00.00.0.000.0.0.0000... Summary...0..........OOOOOOOOOOOOOOOOOIOIOO. BibliograthOOOOCOO0.0.0.000...0.0.0.0000... iii Page ii iii iv 4 m a. h‘ 10 22 26 27 TABLE II. III, IV, VI. VII. LIST OF TABLES The AMP deaminase content of some species and tissues of animaIS..................... The effects of some reagents on low salt treated AMP deaminase at lO-ZM AMP and various ionic strengthS.................... The effects of some reagents on crude deaminase extracts at lO'gM and 10-3M AMP,° The effects of some reagents on low salt treated deaminase at lO'ZM and 10'"3 M AMP... The relative activation and concentration of maximal activation of some monovalent cations on AMP deaminase,,................. The activation constants of some mono~ valent cations and AMP deaminase.,.,,,,,,,. The effect of ATP on AMP deaminase activity iv Page 11 13 14 14 16 18 21 LIST OF FIGURES FIGURE Page 1, Some metabolic reactions involving 5'AMP,,,,., 2 2, Calculation of Ka for NaCl and AMP deaminase,. l7 3, Hill plots of heated and unheated enzyme,,,,,, 20 INTRODUCTION Although 5'-adeny1ic acid deaminase (5'-AMP deaminase) is an enzyme of wide biological distribution, its physiological function has not been elucidated. When extracted from muscle the enzyme is obtained as a complex with myosin (Herman and Josepovits. 1949) and it was speculated that 5'-AMP deaminase is involved in muscular contraction (Parnas, £3 21.. 1927). Wajzer. £3 .31. (1956) reported that changes in ultraviolet absorp- tion indicated that deamination of 5'-AMP accompanied a single muscular contraction. Nechiporenko and Ferdman (1953) showed that the amount of S'qAMP deaminase which is complexed to myosin is decreased in myosin extracted from atrophic muscle. Pennington (1961) showed that mice with muscular dystrophy have only one-third the 5'qAMP deaminase level of normal mice. All of these reports seem to indicate a close relationship between 5'-AMP deaminase and muscular contraction. 5'-AMP has been shown to regulate phosphofructo- kinase (Stone and Mansour. 1966) and phosphorylase b (Cori. g: 31., 1943). The importance of S'QAMP deaminase becomes apparent when it is considered that it could be a general control over the metabolic reactions involving S'qAMP. A scheme showing the metabolic involvement of 5*-AMP is illustrated in Figure 1 on pagé 2. 5'-AMP activates phosphorylase b and releases ATP inhibition -1- FIGURE 1 Some metabolic reactions involving 5'-AMP ,--13 ,z"myosin ‘\ \~ ATP 4 ADP + P. \ / / 1 \ / myokinase ‘\ / \ l \ 1 AMP deaminase AMP 4; UMP + NH I \ 3 I \ 1 \ activation \\ I 1 I, \ \ w \ phosphorylase b \ \glycogen :s-G-l-P \ \ \. releasg of inhibition ATP inhibition \ ~~ __ ~ - / ‘~ ‘ V. A . phosphofructokinase -3- of phosphofructokinase. both of which stimulate gly- colysis. Glycolysis increases the ATP level which inhibits phosphofructokinase and, through muscular contraction, forms more ADP. If the myokinase reaction is important in the conversion of ADP to ATP and AMP, activation of AMP deaminase by ATP would close a regula- tory circuit. When muscular contraction causes the ATP level to drop (myosin.ATPase) and this deactivates AMP deaminase, the resulting AMP level increase (myokinase) will stimulate glycolysis increasing the ATP level-again. During muscular inactivity the concomitantly high ATP level could activate AMP deaminase and the resulting lower AMP level would no longer stimulate glycolysis so that ATP production is decreased. This work deals with some aspects of B'eAMP deamin- ase which may someday help to establish its real function. HISTORICAL The first description of the reaction catalyzed by S'aAMP deaminase was given by Schmidt in 1928. He showed that 5'-AMP deaminase from rabbit muscle could be separated from adenosine deaminase by adsorbing the latter on alumina gel. He also studied the substrate specificity of the enzyme and showed that adenine, adenosine, 3'-AMP, guanine, guanosine, and GMP were not attacked. Indeed, this was part of the evidence used by Embden and Schmidt (1929) to show the non-identity of S'qAMP (muscle AMP) and 3'qAMP (yeast AMP). Schmidt showed the reaction products to be ammonia and IMP by isolating them, giving the chemical reaction: AMP + H20 -———- IMP + NH3 No published attempts to purify S'qAMP deaminase were given until Kalckar (1947) showed two methods of purification and a very convenient method of assay. At 265 millimicrons the decrease in extinction with de- amination of AMP to IMP is 60 per cent. Thus, a spectro- photometric assay is possible making the lengthy ammonia determinations unnecessary. In 1947, Herman and Josepovits showed that S'eAMP deaminase was bound to myosin as strongly as ATPase and concluded that both activities were due to the same protein since they could not be separated. Englehardt, Ig£.§1. (1952) were successful in separating the ATPase -4... -5- and deaminase by thermal treatment. When the myosin (ATPase) - deaminase complex is heated the myosin is preferentially denatured. In 1957, Lee reported the crystallization of 5'qAMP deaminase from rabbit muscle with a specific activity of 17.2 and characterized this preparation very extensively. He found that the protein moved in a single peak in electrophoresis (with a variety of ionic strengths, protein concentrations and pH's), in sedimentation and in calcium phosphate gel chromatography. He also de- termined the isoelectric point to be at pH 5.6, a sedi- mentation constant of 12.29x10-13sec, a diffusion 7cmzsec-l, and a calculated coefficient of 3.76x10' molecular weight of 320,000. An Arrhenius plot gave an apparent energy of activation of 10,500 calories. The enzyme exibited a sharp pH optimum at 6.4 in 0.10 M succinate buffer and pH 6.1 in 0.10 M citrate buffer. The Michaelis~Menton constant (Km) was determined to be 1.4x10'3M (0.1M succinate buffer, pH 6.4, 30°C, 2.7x10u5 4M AMP range). Lee reported that orthophos- to 2.0x10’ phate, pyrophosphate and fluoride strongly inhibited the enzyme. No evidence of a metal requirement could be demonstrated by treatment of the enzyme with cyanide, versene or cysteine. The following metal cation effects were reported for the crystalline enzyme (see page 6). The protein was found to be free of the following enzyme activities which exist in the crude actomyosin complex: adenosine diphosphate deaminase, myokinase, -6- nucleotide triphosphatase, and adenosine triphosphate—creatine transphosphorylase. phosphatase, nucleotidases, adenine, Furthermore, no nucleotide pyro- cytosine, or gua- nosine deaminase activities were detectable, Ions +3 +2 Fe , Zn , +2 , Mg+? +2 Cd Ba + Cu ? Ni+2 Ca+2 Co+2 Effects 4. A8 Strongly inhibitory Slightly inhibitory Very slightly inhibitory No significant effects Kaldor (1962) studied 5'-AMP deaminase of rabbit myofibrills (the deaminase of myofibrillar preparations). He found that ATP,ADP,ITP and citrate activated the de- aminase in succinate buffer. The nucleotide activation was not observed in citrate buffer. Lyubimova and Matlina (1954) also reported that ATP and ADP activated their deaminase preparation from rabbit skeletal muscle. Recently, Cunningham and Lowenstein (1965) showed that calf brain 5'-AMP deaminase is regulated by ATP. The purified enzyme did not show an absolute dependence on ATP and a plot of substrate versus rate gave a sigmoidal curve which became hyperbolic in the presence of ATP. Askari and Franklin (1965), working with purified preparations of AMP deaminase from human erythrocytes, showed an absolute requirement for a monovalent cation. , + The enzyme could be activated by K+ or NH4 but not by .+ Na+ + , L1 , Rb or Cs+. In the presence of ATP, which -7- was shown to be chemically unaltered, the enzyme could be activated by K+, NH4+, Na+, Li+, and Rb+ with the activation decreasing in that order. In the case of the enzyme from cat and dog erythrocytes, only ATP showed an activation and the alkali cations had no effect in the presence or absence of ATP. 5*-AMP deaminase has a very wide biological dis- tribution. The existence of this activity has been shown in microorganisms (Angarwala,g£.gl,l954; Saruno, ‘g£.al,l955; Aida,g£'al, 1965), snail, eel, viper and pigeon (Umiastowski,l964), man, dog, and cat erythro- cytes (Askari,§£ 31,1965), rat, rabbit, guinea pig, and ox (Kutscher,g£.al,l948), fish (Nara,£1‘31,1959,1962), and even pea seeds (Turner,g£ 31,1961). This deaminase activity has been shown in a variety of tissues including heart, smooth muscle, brain, per- ipheral nerve, liver, kidney, spleen and lung (Nechip- orenko,§£'gl,1949; Eidelman,l953; Kutscher,§£‘al,l948; Sata,l954) but with much less activity than in skeletal muscle. Kutscher (1948) studied the relative levels of deaminase in white skeletal muscle of various species and obtained the following results: guinea pig 200, man 165, rabbit 123, cat 104, chicken 100, rat 70, and ox 42. The vast distribution of 5'-AMP deaminase in bio- logical systems, including plants, animals, and micro- organisms indicates the possibility of a very signifi- cant physiological role for this enzyme. MATERIALS AND METHODS S'QAdenylic acid (sigma grade) was purchased from the Sigma Biochemical Corporation. All other reagents were of reagent grade. Tetramethylammonium (TMA) ion was used to avoid alkali cations. All animals were sacrificed by suffocation in C02. The desired tissue was immediately excised and chilled in ice. The spectrophotometric assay used was a modifica- tion of that of Kalckar (1947) based on the sixty per cent decrease in absorption which occurs at 265 milli- microns when AMP is converted to IMP. The standard spectrophotometric assay used in this work was: 0.1 M 5M AMP and 30°C. These K succinate at pH 6.5, 5x10" measnements were made on a Beckman DU spectrophotometer. The rates which were measured with the pH stat (Radiometer TTT-l/-SBR2/SBUl/TTA31) were run with no buffer since the liberated ammonia was titrated with hydrochloric acid. This method was especially useful since no AMP concentration limitation existed which was a disadvantage of the spectrophotometric assay due to the high extinction of AMP. Specific activity was de- fined as the micromoles of AMP deaminated per minute per mg of protein. The crude deaminase extract which was used for some of the experiments was prepared as follows. Each gram -8- -9- of tissue was ground with 7 volumes of cold Guba~Straub (Szent-Gybugyi, 1951) solution ( 0.3 M KCl, 0.09 M KH2P04, 0.06 M K HPO4, pH 6.5 ) and stirred for an hour 2 in the cold. The mixture was centrifuged and the super- natant solution was called the crude AMP deaminase ex- tract. The crude actomyosin extract was prepared by grind- ing each gram of tissue in 3 ml of cold Weber-Edsall (Szent-GyBrgyi,1951) solution (0.6 M KCl, 0.01 M Na2C03, 0.04 M NaHCO pH 9.0). The homogenate was allowed to 3. stand overnight in the cold and centrifuged. The super- natant solution was called the crude actomyosin extract. A low salt treatment was defined as the process of diluting a protein solution to 0.05 M KCl at which point the myosin and deaminase precipitate. The precipitate was then dissolved in 0.5 M KCl again. This separates the deaminase from the low salt soluble proteins. The enzyme of specific activity 25 was prepared by Dr. Eugene Byrnes (unpublished) and that of specific activity 50 was prepared by Dr. Karl Smiley (unpublished). The paper chromatography solvent system used to separate nucleotides was; saturated (NH4)ZSO4 : 0.2 M Na acetate (pH 5.9) : isopropanol, 79:19:2 (v/v). EXPERIMENTAL A study of the 5'qAMP deaminase levels of several tissues of a variety of animals is of interest for -several reasons. This type of study will reveal the best source of enzyme of the animals studied and, the results of species and tissue studies may give some insight as to the physiological function of the enzyme. The data of such a study are given in TABLE I. The level of AMP deaminase is seen to be very low in heart. The standard 5M AMP) failed to show any activity; however , assay (5x10' higher substrate concentrations demonstrated activity in pigeon and rabbit hearts. As further evidence for'the; existence of the enzyme in heart tissue, a paper chromato- graphic study showed that spots which have the same R as f IMP and AMP appeared from a sample of the reaction mix- ture: lO'ZM AMP, 0.10 M K succinate (pH 6.5) and heart deaminase extract. No IMP appeared in the controls lack- ing the heart extract or the AMP. The data in TABLE I also show that the enzyme level is much greater (about six fold) in breast than in leg muscle in both chicken and pigeon. The significance of this is not known. Rabbit muscle is seen to be the best source of enzyme of the tissues studied. The enzyme level increases with age in young rabbits and the data show that mature rabbits have about eight times the level of very young rabbits. -10- TABLE I The AMP deaminase content of some species and tissues of animals Activity, units: per gram crude deaminase crude actomyosin tissue extract extract * frozen dog heart 0.0 0.0 * frozen mouse heart 0.0 0.0 * frozen rat heart 0.0 0.0 * frozen guinea pig heart 0.0 0.0 * refrigerated beef heart 0.0 0.0 * fresh bullhead' fillet 8.6 6.5 * fresh chicken breast 11.2 10.6 * fresh chicken leg 1.6 2.4 + fresh chicken heart 2.1 1.8 * fresh pigeon breast 9.7 9.6 * fresh pigeon leg 1.7 2.2 + fresh pigeon heart 1.9 3.3 + fresh honey bee muscle 0.4 1.0 fresh rabbit heart #7.3 +4.0 fresh rabbit skeletal muscle: * 9 day old rabbit 2.5 - *16 day old rabbit 9.8 - *72 week old rabbit 24.3 - *94 week old rabbit 18.8 - ----------- -IIQ-fln-I-HQ"- I'll-- nfl-C---. -Q-u-c-c-q-n .n-n--------. * standard assay + 10'2M AMP, pH stat assay # 2x10-2M AMP, pH stat assay ~11- -12.. The importance of Ca+2 in muscular contraction and the occasional observafion.of divalent cation activation of AMP deaminase preparations led to a study of the effects of some of these cations on rabbit muscle AMP deaminase at different stages of purity, different AMP concentra- tions, and various ionic strengths. The effect of some organic acids were also studied since conflicting reports on these effects appear in the literature. TABLE II shows these effects on low salt treated deaminase extract at lO'ZM AMP and various ionic strengths (adjust- ed with KCl). Divalent cations and ionic strength have no effect. Citrate, succinate and lactate activate slightly with lactate being the best of the three (134% of non-activated rate). TABLE III and TABLE IV compare the effects of some of the same reagents at two AMP levels on crude deaminase extract and low salt treated deaminase extract, respectively. These data show that only citrate and lactate have any significant effect and both activate slightly at 10‘2M AMP with both the crude and the low salt treated deaminase extract. At 10-3M AMP, lactate still activates slightly; however, citrate activation is greatly increased (236% of the non-activated rate with crude extract and 145% with low salt treated extract). Since the activation is greater on the crude extract than on the low salt treated extract the effect of citrate on AMP deaminase is probably not a direct effect. TABLE II Effects of some reagents on low salt treated AMPckmminase at 10"2 M AMP and various ionic strengths * specific activity reagent(M) ionic strength KC1(.10) 0.10 CaC12(.01),KCl(.o7) 0.13 MgC12(.Ol),KC1(.07) 0.13 MnC12(.01),KC1(.07) 0.13 MgC12(10'2),KC1(.10) 0.16 MgC12(10-3),KC1(.10) 0.11 MgC12(10-4),KC1(.10) 0.10 Kcitrate(.01),KCl(.04) 0.10 Kcitrate(.01),KC1(.l4) 0.20 Kcitrate(.01),KCl(.24) 0,30 Klactate(.01),KC1(.09) 0.10 Klactate(.01),KC1(.l9) 0.20 Klactate(.01),KC1(.29) 0.30 Ksucc.(.01),KC1(.07) 0.10 Ksucc.(.01),KC1(.l7) 0.20 Ksucc.(.01),KCl(.27) 0.30 6.18 6.94 6.37 ppt. formed 6.54 7.12 6.43 7.50 8.13 7.55 8.29 8.29 7.36 6.89 7.07 7.37 2 * determined with the Radiometer pH stat at 10- M AMP abbreviations: succ.(succinate) -13.. c. a I r . v t I I a I I O O .- nth—n. ,1 r‘ The effects of some reagents on crude deaminase extract reagent (M) none - Cac12 10--2 CaC12 10-3 Cac12 10-4 K citratelO”2 K 1actate10-2 none - CaCl 10-2 K ci rate1o-2 TABLE at lO‘ZM and AMP conc.,M III 10-3M AMP . . . . * spec1f1c act1v1ty 10"3 10'-3 10-3 10-3 10-3 10- 10"2 10“3 10'“2 3.23 3.52 3.48 3.18 7.62 3.85 8.20 7.45 9.08 ----------~--“ ---~~ --- ~“--------- -‘--‘---------~------ *determined with Radiometer pH stat and each reaction 'contained 0.10 M KCl TABLE IV Effects of some reagents on low salt treated deaminase pat 10‘3M and 10~3M AMP . . . . * reagent (M) AMP conc.,M spec1f1c act1v1ty none — 10-3 8.85 CaCl 10'2 10"3 8.02 K ci rate 10"2 10"3 11.58 K lactate 10"2 10"3 9.04 none - 10-2 12.19 C801 10"2 10-2 13.18 K ci rate 10"2 10-3 12.77 K lactate 10"2 10-2 13.10 ----------.- ----“-----. ------‘---------- ---~-~‘---- ---~ * determined with Radiometer pH stat and each reaction "contained 0.10 M KCl -14- fl.- . | 1 The activity of purified rabbit muscle enzyme (specific activity 25 or 50) always seemed to increase with increasing KCl concentration when this was used to adjust the ionic strength in several experiments. Suspecting that KCl was activating the enzyme, a group of experiments were done to determine the effects of some monovalent cations on AMP deaminase. TABLE V shows the relative activation and concentration of maximal activation for some monovalent cations. K‘I is the best activator and it is seen that AMP deaminase has a definite monovalent cation requirement. The activation constants (Ka) for the monovalent cations were determined spectrophotometrically. Lineweaver-Burk plots were made and the activation constants were calculated from these plots. A sample is shown in FIGURE 2 for NaCl and the complete data are given in TABLE VI. Lineweaver-Burk plots for AMP and purified rabbit muscle AMP deaminase (specific activity 50) repeatedly exibited upward curvature. This suggested that the enzyme may be allosteric; thus, Hill plots (Monod, 23.21-: 1963) were determined for AMP and AMP deaminase. The slopes varied between -1.2 and ~1.4 with most at -l.3. This slope did not change when the Kcl concentration was varied between 0.05 M and 0.15 M. .An attempt was made to desensitize the enzyme (destroy the allosteric nature) by heating to 50°C for 5 minutes which denatured 1:; TABLE V The relative activation and concentration of maximal activation of some monovalent cations on AMP deaminase reagent rel.activation* conc. of max,activationfM KCl 100 0.11 t .02 NaCl 54 0.12 3 .02 LiCl 26 0.10 NH4C1 23 0.10 RbCl l4 - CsCl 0 - TMACl O - none 0 - * .Standard assay at 0.15 M cation and enzyme specific activity of 25 # Standard assay with enzyme specific activity of 25. Based on one determination for LiCl and NH4C1. n16- -- FIGURE 2 * The calculation of Ka for NaCl and AMP deaminase 600m 1 slope u Ka xi; = 2.30 9 m 510 e 2 30 K = = -%3- = 0.037 M 400w a 1 Vm