[65] AMP deaminase from rat skeletal muscle

490

[65]

PURINE METABOLIZING ENZYMES

analog nucleotide, 8-azaGMP, displayed significant activity. The enzyme also appears highly specific for ADP or ATP. Since the reaction mechanism follows a "random" bi-bi reaction sequence that does not involve the formation of a phosphorylated enzyme intermediate, it may be concluded that the enzyme contains two specific binding sites: one for GMP or GDP and the second for ADP or ATP. The formation of an abortive ternary complex between GMP, ADP, and hog brain guanylate kinase has been demonstrated. 1~The high specificity of guanylate kinase for GMP, dGMP, and ATP has made guanylate kinase a useful biochemical tool for the development of highly specific and sensitive assays, often involving enzyme cycling for biologically important molecules such as 3',5'-cGMP, GMP, and ATP. 2°'~1 A point of interest is that in most tissues examined, the activity of guanylate kinase is relatively low and, to date, no evidence has been found that indicates that it is subject to allosteric regulation or metabolic induction. It should be noted that recent advances in the use of affinity chromatography with a substrate, an inhibitor, or a reactive dye coupled to cross-linked agarose beads for enzyme purification have not been applied to date to the purification of guanylate kinase. The use of these techniques could lead to major modifications in the purification scheme described aboye. laR. P. Miech, Ph.D. Thesis, University of Wisconsin, Madison (1963). 2°N. D. Goldberg, S. B. Dietz, and A. G. O'Toole, J. Biol. Chem. 244, 4458 (1969). ~IR. P. Miech and M.-C. Tung., Biochem. Med. 4, 435 (1970).

[65] A M P

Deaminase

from Rat Skeletal Muscle

By CAROLE J. COFFEE AMP + 1-120 ~ IMP + NH3

AMP deaminase (EC 3.5.4.6, AMP aminohydrolase) catalyzes the hydrolytic deamination of Y-adenosine monophosphate as illustrated in the above reaction. The enzyme is widespread in animal tissues, although a considerably higher concentration is found in skeletal muscle than in other tissues including cardiac and smooth muscle. 1 Moreover, the distribution of the enzyme in skeletal muscle varies greatly, with white muscle having significantly higher concentrations than red muscle. ~ Evidence has been presented recently which suggests that different 1 E. J. Conway and R. Cooke, Biochem. J. 33, 479 (1939). 2 A. Raggi, S. Ronca-Testoni, and G. Ronca, Biochim. Biophys. Acta 178, 169 (1969). Copyright© 1978by AcademicPress, Inc.

METHODS IN ENZYMOLOGY, VOL. LI

All rightsof reproductionin any formreserved.

ISBN 0-12-181951-5

[65]

A M P DEAMINASE FROM RAT SKELETAL MUSCLE

491

i s o z y m e s o f A M P d e a m i n a s e m a y be found in red and white muscle fiber types, a T h e e n z y m e in skeletal muscle is tightly associated with actomyosin 4 and can be dissociated either b y heat 5 or by inorganic phosphate.6 The e n z y m e has b e e n d e m o n s t r a t e d to participate in the purine nucleotide cycle, TM and the reaction catalyzed b y A M P deaminase is the major source of a m m o n i a in skeletal muscle, a'l° Highly purified preparations of the e n z y m e h a v e b e e n r e p o r t e d f r o m skeletal muscle o f rabbit, I~ hen, TMfish, ~3 and rat. ~4 Assay Method Principle. The e n z y m e m a y be a s s a y e d either indirectly b y monitoring NH3 production by the microdiffusion technique 15 or directly b y the m o r e convenient s p e c t r o p h o t o m e t r i c method developed b y Kalckar. TM A b s o r b a n c y changes resulting f r o m the enzymic hydrolysis of the purines or purine derivatives h a v e b e e n tabulated by Zielke and Seulter. 17 Routinely, the activity of the e n z y m e is m e a s u r e d by monitoring the change in absorption at either 265 or 285 nm which a c c o m p a n i e s the conversion of A M P to I M P . T h e a m o u n t of A M P c o n v e r t e d to I M P is calculated using A~m~t values o f 8.86 and 0.23 at 265 and 285 nm, respectively. Stock Reagents

Imidazole-HC1, 0.5 M , p H 6.5 Potassium chloride, 1 M AMP, 20 raM, p H 6.5 P r o c e d u r e . 14 An a s s a y mix sufficient for 100 assays is p r e p a r e d from the a b o v e stock reagents as follows: I m i d a z o l e - H C l (10 ml), KC1 (10 ml),

a A. Raggi, C. Bergamini, and G. Ronca, FEBS Lett. 58, 19 (1975). 4 E. W. Byrnes and C. H. Suelter, Biochem. Biophys. Res. Commun. 20, 422 (1965). Y. P. Lee, J. Biol. Chem. 227, 987 (1957). 6 R. D. Currie and H. L. Webster, Biochim. Biophys. Acta 64, 30 (1962). 7 j. M. Lowenstein and K. Tornheim, Science 171, 397 (1971). 8 K. Tornheim and J. M. Lowenstein, J. Biol. Chem. 247, 162 (1972). 9 G. Embden and H. Wassermeyer, Hoppe-Seyler's Z. Physiol. Chem. 179, 226 (1928). l0 j. K. Parnas, Biochem. Z. 206, 16 (1929). li K. L. Smiley, A. J. Berry, and C. H. Suelter, J. Biol. Chem. 242, 2502 (1967). 12H. Henry and O. P. Chilson, Comp. Biochem. Physiol. 29, 301 (1969). 13W. Makarewicz, Comp. Biochem. Physiol. 29, 1 (1969). 14C. J. Coffee and W. A. Kofke, J. Biol. Chem. 250, 6653 (1975). 15G. Schmidt, Hoppe-Seyler's Z. Physiol. Chem. 179, 243 (1928). ~6H. M. Kalckar, J. Biol. Chem. 167, 461 (1947). iT C. L. Zielke and C. H. Suelter, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 4, p. 47. Academic Press, New York, 1971.

492

PURINE METABOLIZING ENZYMES

[65]

AMP (10 ml), and H~O (60 ml) are mixed and equilibrated in a constanttemperature bath at 20 °. An aliquot of 0.9 ml of this mixture is pipetted into a quartz cuvette with a 1-cm pathlength. The reaction is initiated by the addition of 0.1 ml of appropriately diluted enzyme solution. The increase in absorbance at 285 nm is recorded as a function of time. Best results are obtained using an expanded-scale recorder with a full-range scale of 0.2 optical density (OD) units. The final concentration of AMP in the standard assay mixture is 2.0 mM. In order to measure the activity of the enzyme at levels of AMP -< 0.15 mM, the decrease in absorbance at 265 nm is recorded. Units. One unit of enzyme activity is the amount of enzyme required to catalyze the deamination of 1 ~mole of AMP per minute at 20 ° under the standard assay conditions described above.

Purification Procedure Preparation of Crude Extract. Leg and back muscles (1 kg) from rats are excised, sliced into small pieces, and passed through a meat grinder. The ground muscle is mixed with 3.3 volumes (w/v) of 0.1 M potassium phosphate buffer (pH 6.5) containing 0.18 M KCI and 2 mM/3-mercaptoethanol and homogenized in a Waring Blendor for 1 min at high speed. The resulting slurry is stirred at room temperature for 1 hr and then centrifuged at 20,000 g for 15 min. The supernatant fluid is filtered through cheesecloth. Fractionation on Cellulose-Phosphate. Cellulose-phosphate (Whatman P-11) is prewashed successively with 0.5 N KOH, 1-120, 0.5 N HC1, H20, 5 mM EDTA, H~O, and 0.1 M potassium phosphate buffer (pH 6.5) containing 0.18 M KC1 and 2 mM/3-mercaptoethanol. For each liter of crude extract, 25 ml of cellulose-phosphate are added, and the slurry is stirred at room temperature for 30 min. Approximately 90% of the enzyme activity is absorbed to the cellulose-phosphate under these conditions. The slurry is centrifuged at 10,000 g for 10 min, and the supernatant fluid is discarded. The resin is washed 3 times with 4 volumes of extraction buffer each time and recovered by centrifugation. The slurry is transferred to a 2 × 50 cm glass column and washed with 0.45M KC1-2 mM /3-mercaptoethanol (adjusted to pH 7.0 with K2HPO4) until the effluent has an absorbance at 280 nm of -< 0.01. A linear gradient, consisting of 150 ml of 0.45 M KCI-2 mM/~-mercaptoethanol (pH 8.0) and 150 ml of 1.5 M KC1-2 mM fl-mercaptoethanol (pH 8.0), is applied to the column. The enzyme elutes between 100-170 ml.

[65]

A M P DEAMINASE FROM RAT SKELETAL MUSCLE

493

The yield of enzyme at this step is about 80%, and it is estimated to be approximately 50% pure by the criteria of SDS gel electrophoresis. 34 Affinity Chromatography on 5'-AMP Sepharose. The preparation and purification of N6-(6-aminohexyl)-5'-AMP is carded out as described in exquisite detail by Craven et al. 38In order to couple the ligand to Sepharose, 10 mg of N e-(6-aminohexyl)-5'-AMP are dissolved in 5 ml of 0.1 M NaHCOa (pH 10) and mixed with 10 g (moist weight) of cyanogen-bromide-activated Sepharose 4B (Sigma) which had been prewashed with 500 ml of ice-cold 0.1 M NaHCOa (pH 10). The suspension is mixed gently by rotation overnight at 4 °. The gel is washed free of excess ligand by successive washings with 500 ml each of 0.1 M NaHCO3 (pH 10), H~O, 1 M KCI, and H20. The gel was stored at 4 ° in 0.02% sodium azide when not in use. Prior to fractionation of AMP deaminase on the AMP-Sepharose gel, the pool of enzyme obtained from the previous cellulose-phosphate column was dialyzed against 0.02 M potassium phosphate buffer (pH 7.5). Dialysis was performed for approximately 24 hr against 3 changes of buffer (20 volumes each). The AMP-Sepharose gel is poured into a glass column and equilibrated with 0.02 M potassium phosphate (pH 7.5). The enzyme solution is applied to the column at a flow rate of approximately 10 ml/hr. Following application of the sample, the column is washed with the same buffer until no more protein can be detected in the effluent (Azs0 <- 0.01). The column is then eluted with 0.02 M potassium phosphate--0.1 M KC1-2 mM/3-mercaptoethanol. The elution profile obtained in this step is shown in Fig. 1.39 Approximately half of the total protein applied to the column is unabsorbed and comes off in the wash. However, all of the enzyme activity remains absorbed and is eluted with 0.1 M KC1 in 20 mM potassium phosphate (pH 7.5). Comments on the Purification Procedure. A summary of the purification is shown in the tablefl ° The procedure is rapid, easily performed, and results in a high-yield preparation (50--60%) which is homogeneous by the criteria of sedimentation velocity and equilibrium analyses, 34 standard polyacrylamide gel electrophoresis, 23 and SDS polyacrylamide gel electrophoresis, x4The purification scheme reported here is a simplification of a procedure previously described by Coffee and Kofke. 14In the 18 D. B. Craven, M. J. Harvey, C. R. Lowe, and P. D. G. Dean, Eur. J. Biochem. 41,329 (1974). ~9 p. Bohlen, S. Stein, W. Dairman, and S. Udenfriend, Arch. Biochern. Biophys. 155, 213

(1973). R. F. Itzhaki and D. M. Gill, Anal. Biochem. 9, 401 (1964). 21 C. J. Coffee and C. Solano, in preparation. 2o

494

PURINE METABOLIZING ENZYMES

[65]

J2 "_o

0

x

I

0

io

I00

w o

z bJ o u) laJ nO ._l u. txl

80

0.1 M KCI

60

>_

o/Ox °

/\

40

N

6

<

w o

/ \

.J

8

4

N m z

o.~a:$:gUl.,_,

.8:Q:ti

-o-o-e-o-o-

I

20

I0

" "

7,

30

FRACTION

Flo. 1. Fractionation of AMP deaminase on AMP-Sepharose-4B. A solution of AMP deaminase in 20 mM potassium phosphate buffer (pH 7.5) was applied to a 1 × 10 cm column of Nr-(6-aminohexyl)-5'-AMP at a flow rate of 10 ml/hr, and the column was washed with 20 mM potassium phosphate (pH 7.5) until no more protein emerged. Elution was achieved with 0.1 M KC1 in 20 mM potassium phosphate (pH 7.5). Fractions of 4 ml each were collected. Aliquots were assayed for protein by the fluorescamine procedure, la and for enzyme activity as described in the text. p r e v i o u s m e t h o d , p u r i f i c a t i o n to h o m o g e n e i t y w a s a c h i e v e d b y s u c c e s sive chromatographic fractionation on cellulose-phosphate, DEAE-cellulose, and Bio-Gel A-5m. In the simplified procedure described here, the final t w o c h r o m a t o g r a p h i c s t e p s o n D E A E - c e l l u l o s e a n d B i o - G e l h a v e b e e n r e p l a c e d b y a single s t e p u t i l i z i n g a n a f f i n i t y c o l u m n o f A M P S e p h a r o s e . T h e m o d i f i e d p r o c e d u r e h a s t h e a d v a n t a g e t h a t it is b o t h m o r e r a p i d a n d it r e s u l t s in a h i g h e r y i e l d . PURIFICATION OF A M P - D E A M I N A S E FROM 1 KG OF RAT SKELETAL MUSCLE

Purification step Crude extract Cellulose-phosphate 5'-AMP-Sepharose-4B

Total Specific protein Total activity activity Yield (mg) a (x 10-a units) (units/rag) (%) 38,700 116 47

a Determined by the microbiruet method.S°

130 104 74

3.3 896 1580

I00 80 57

[65]

A M P DEAMINASE FROM RAT SKELETAL MUSCLE

495

Properties of the E n z y m e Stability. The enzyme can be stored in 0.1 M potassium phosphate1.0 M KC1-2 mM fl-mercaptoethanol (pH 6.5) at 4 ° for several weeks without a significant decrease in specific activity. 14 However, rapid loss of enzyme activity is observed at pH values higher than 6.8 or lower than 5.6. 22 Freezing the enzyme at - 7 0 ° results in inactivation which is associated with the formation of insoluble aggregates. 22'23 The enzyme activity is decreased by a number of chelating agents which is likely a reflection of the fact that the enzyme contains zinc. 24 Molecular Weight and Subunit Structure. The molecular weight of the homogeneous enzyme has been estimated to be 238,000 by sedimentation equilibrium analysis. 14 A somewhat higher value of 290,000 obtained by sucrose density centrifugation and with a less pure preparation has been reported. 22 In the presence of denaturing agents such as guanidine hydrochloride or sodium dodecyl sulfate, the enzyme dissociates into polypeptides having a weight average molecular weight of approximately 60,000. These data indicate that the enzyme is composed of four subunits which are identical with respect to size. A tetrameric structure of the native enzyme is supported by tryptic peptide mapping, and the maps further indicate that the primary structure of the individual subunits is either identical or very similar.14 Amino Acid Composition. Amino acid analysis of the enzyme indicated the presence of 530 amino acids per subunit. 14 The tryptophan content was observed to be 4 residues per subunit. The absorption spectrum of the enzyme is that expected for a tryptophan-containing protein with no ultraviolet-absorbing cofactors. For the native enzyme, the absorbance maximum is at 280 nm ,~2so c~l% nm = 9.8) and the A28o/A26o = 1.85. 22 Catalytic Properties. The fundamental enzymology of AMP deaminase has been reviewed earlier by Ziekle and Suelter, lr and here only selected basic properties are repeated along with information resulting from more recent studies. Although the enzyme requires zinc 24 and monovalent cations for activity, 22'25"26no other cofactors have been identified. Potassium is the most effective monovalent cation and the one considered to be physiolz2 S. Ronca-Testoni, M. Ranieri, and G. Ronca, ltal. J. Biochem. 19, 262 (1970).

zzC. Coffee,unpublishedobservations. z4A. Raggi, M. Ranieri, G. Taponeco, S. Ronca-Testoni, G. Ronca, and C. A. Rossi, FEBS Lett. 10, 101 (1970). z~G. Ronca, A. Raggi, and S. Ronca-Testoni,Biochim. Biophys, Acta 167, 626 (1968). 26C. J. Coffeeand C. Sonalo,J. Biol. Chem. 252, 606 (1977).

496

PURINE METABOLIZING ENZYMES

[65]

ogically important. The concentration of K + required for maximum activation varies from 25-100 mM depending on the concentration of substrate present. 2~'2~As the substrate concentration approaches saturation, the level of K + required for maximum activation decreases. The pH optimum of the reaction catalyzed by AMP deaminase is approximately 6.5. 2~ The enzyme presents a sigmoidal substrate-velocity curve when assayed at low levels of potassium ion (5 mM). ~5"~6 The sigmoidicity disappears at higher concentrations of KCI (100 mM) or in the presence of ADP. 25-~r The concentration of AMP required for half-maximum velocity is approximately 0.4-0.5 mM. 25"2~'28Hill plots indicate a cooperativity coefficient (nil) of 1.127 to 1.526 when assayed in the presence of 100 mM KCI, whereas at a KCI concentration of 5 mM, the n. is 3.2. z6 Regulatory Properties. The activity of AMP deaminase is affected by a number of biologically important phosphorylated metabolites. Nucleoside triphosphates, creatine phosphate, and Pi, at concentrations comparable to those found in skeletal muscle, all inhibit the enzyme when assayed at physiological concentrations of K ÷ (100-150 mM) and AMP (0.1-0.2 mM). ~5'~6'28 The inhibition by nucleoside triphosphates is sensitive to alterations in pH. For example, the extent of inhibition is significantly higher at pH 7.1 than at pH 6.5. z7 The inhibition observed with nucleoside triphosphates and Pi is reversed by ADP. In addition to this effect, ADP strongly activates the enzyme at low concentrations of KC1. However, at physiological concentrations of KC1, the activation is either very weak 2r or completely absent. 26 The profile of AMP deaminase activity generated in response to variations in the adenylate energy charge shows that within the physiological range of energy charge values (0.75-0.95), 29 the activity increases linearly with decreasing energy charge. Moreover, this response is insensitive to the total adenylate pool size (from 1-10 mM) and the presence of Pi or creatine phosphate. 26 These observations suggest that although Pi and creatine phosphate exert dramatic effects on the enzyme activity in isolated in vitro experiments, they may play an insignificant role in the in vivo regulation of skeletal muscle AMP deaminase. Perhaps in an in vivo situation where creatine phosPhate, ADP, and phosphocreatine kinase are all present, creatine phosphate may play an indirect role in the modulation of AMP deaminase activity by serving as a precursor of ATP. However, it appears that the most important regula~7 S. Ronca-Testoni, A. Raggi, and G. Ronca, Biochim. Biophys. Acta 198, 101 (1972). 3s S. Ronca-Testoni and G. Ronca, J. Biol. Chem. 249, 7723 (1974). 39 A. G. Chapman, L. Fall, and D. E. Atkinson, J. Bacteriol. 108, 1072 (1971).

[66]

A M P DEAMINASE FROM HUMAN ERYTHROCYTES

497

tory factor at the direct level of metabolite-enzyme interaction is the relative concentrations of the three adenine nucleotides. Under conditions similar to those found intraceUularly within skeletal muscle (total adenylate pool sizes of 5-8 mM; energy charge ratios of 0.75-0.95), the activity of AMP deaminase is very stringently regulated as evidenced by the fact that under these conditions, the enzyme is working at only 10-15% of its capacity. ~6 These observations suggest that in resting muscle or in muscle undergoing moderate activity, the enzyme may be inhibited to a great extent. In fact, maximal activity may be approached only under conditions of extreme stress.

[66] A M P

Deaminase

from Human

Erythrocytes

1

By GENE R. NATHANS, DONALD CHANG, and THOMAS F. DEUEL AMP + H20 ~ IMP + NHa

Assay Method

Principle. AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) activity is measured by colorimetric means in an assay adapted from Chaney and Marbach. ~ Ammonia released in the deamination of AMP generates a stable blue color in the catalyzed indophenol reaction. The absorbance is measured at 625 nm and related to a standard curve obtained with ammonium chloride. The method is used when crude extracts are assayed and when assays are performed in the presence of high concentrations of nucleotides other than AMP. AMP deaminase activity may also be measured by a spectrophotometric assay 3 which records either the decrease in optical density at 265 nm as AMP is hydrolyzed or the increase in optical density at 240 nm as IMP is formed. This latter assay has been used to establish both the hydrolysis of AMP and formation of IMP in order to validate the 1This work was supported by Contract EY-76-C-02-0069, awarded to the Franklin McLean Memorial Research Institute (operated by the University of Chicago for the United States Energy Research and Development Administration), and by Grant CA13980 from the National Institutes of Health. Gene R. Nathans was supported by Training Grant 5-T01-CA-05250 from the National Institutes of Health. Thomas F. Deuel holds Faculty Research Award No. 133 from the American Cancer Society. 2 A. L. Chancy and E. P. Marbach, Clin. Chem. (Winston-Salem, N. C.) 8, 130 (1962). H. M. Kalckar, J. Biol. Chem. 169, 445 (1947). METHODS IN ENZYMOLOGY, VOL. LI

Copyright© 1978by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5