THE JOURNAL OF BIOLOQICAL CHEMIBTRY Vol. 250, No. 17, Issue of September 10, pp. 6653-6658, 1975

Printed in U.S.A.

Rat Muscle S-Adenylic Acid Aminohydrolase

I. PURIFICATION AND SUBUNIT STRUCTURE*

(Received for publication, March 17, 1975)

CAROLE J. COFFEES AND W. ANDREW KOFKE

From the Department of Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

SUMMARY

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) has been purified to apparent homogeneity from rat muscle. The preparation exhibits a single polypeptide band with a molecular weight of 60,000 on polyacrylamide gel electro- phoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 11.3 S. Analysis by sedimentation equilibrium techniques showed the native enzyme to have a molecular weight of 238,000, whereas the enzyme, when analyzed in 6 M guanidine hydrochloride and 10 mM Z-mercaptoethanol, had a molecular weight of only 59,500. The amino acid composition of the enzyme was determined and peptide mapping was performed on a tryptic digest of S-carboxymethylated enzyme. NH&erminal analysis by both the dansylation and cyanate procedures failed to identify a free NH2 terminus. Treatment of the enzyme with car- boxypeptidase A resulted in the release of approximately 0.5 mol each of valine and leucine per 60,000 g of enzyme. The data presented indicate that the native enzyme has a tetra- merit structure consisting of four polypeptide chains each having a molecular weight of 60,000. The COOH-terminal analysis can be interpreted either as an indication of subunit heterogeneity or as a result of incomplete digestion of a -X-Leu-Val sequence at the end of a single type of polypep- tide chain. Tryptic peptide maps strongly support the latter interpretation and suggest that the subunits are essentially identical.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) cata- lyzes the hydrolytic deamination of AMP to generate IYIP and NH,. The enzyme is widely distributed in animal tissues. How- ever, a considerably higher concentration is found in skeletal muscle than in any other tissues, including cardiac and smooth muscle (1). Although the physiological role of AMP deaminase remains obscure, the relatively high concentration of the enzyme in muscle tissue, together with the fact that it is found tightly complexed to myosin (2, 3), implies that it may be significant in

* This work was supported by Grant AM lG728 from the Na- tional Institutes of Health.

$ To whom all correspondence should be directed.

directing the energy flow required for muscle contraction. It has recently been proposed by Chapman and Atkinson (4) that the role of AMP deaminase in liver is to stabilize the adenylate energy charge. Because of the similarities in the properties of the liver and muscle enzyme, it is likely that the enzyme functions in the same way in muscle tissue. Even though the precise role of the enzyme remains to be clearly elucidated, it is of considerable interest because of the marked reduction in activity observed in the dystrophic mouse (5), human Duchenne dystrophy (6), and in patients with hypokaliemic periodic paralysis (7).

The kinetic properties of AMP deaminase from a number of tissues have been examined in several laboratories, and there is general agreement that the enzyme is subject to very stringent regulation. Llonovalent cations, especially potassium, are potent activators of enzymatic activity, and nucleoside triphosphates, ADP, creatine phosphate, and inorganic phosphate, at concentra- tions near or lower than those found in muscle, greatly modulate AMP deaminase activity from all sources examined (8).

This paper reports a purification procedure and a detailed analysis of the subunit structure of AMP deaminase from rat muscle. This, we feel, is an initial effort in the direction of correlating the catalytic, regulatory, and molecular properties of the enzyme.

MATERIALS AND METHODS

Materials-Substrate was obtained from Sigma Chemical Co. Trypsin, treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone, and carboxypeptidase A were bought from Worthington Biochemical Corp. Cellulose phosphate and DEAE-cellulose were purchased from Whatman. Bio-Gel was obtained from Bio-Rad Laboratories. White rats were purchased from Zivic-Miller. Iodo- acetic acid was recrystallized from cold petroleum ether before use.

Enzyme Assay-Enzyme activity was measured at 285 nm, using a Gilford model 240 spectrophotometer. The amount of AMP converted to IMP was calculated using a AS,M of 0.23 (9). The standard assay was performed in cuvettes of l-cm light path with a volume of 1.0 ml containing 50 mM imidazole-HCl, pH 6.5, 100 mM KCl, and 2 mM AMP. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the deamination of 1 pmol of AMP per min at 20” under the conditions described above. Specific activity is reported as micromoles of AMP deaminated per min per mg of protein.

Protein Determination-The protein concentration was deter- mined at various stages of purification by both the biuret method (lo), using bovine serum albumin as a standard, and by the spec- trophotomet,ric method of Warburg and Christian (11). Variations between the two methods were less than 8%.

Gel Electrophoresis-Sodium dodecyl sulfate polyacrylamide gel

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electrophoresis was performed by the procedure of Weber and Osborn (12).

Analytical Ultracentrifugation-Centrifugation was performed using a Spinco model E ultracentrifuge equipped with an electronic speed control and a temperature control unit. Sedimentation velocity experiments were performed at 52,000 rpm and 20” using schlieren optics. The protein concentration for sedimentation velocity experiments was approximately 4 mg/ml.

Weight average molecular weights were determined at 20” by the meniscus depletion method of Yphantis (13). A multichannel centerpiece was used, and three different protein concentrations ranging from 0.4 to 1.2 mg/ml were examined simultaneously. Equilibrium was achieved by centrifugation at 12,000 rpm for 24 hours for the native enzyme, and at 26,000 rpm for 41 hours for samples in guanidine hydrochloride. Rayleigh interference fringes were analyzed on a Nikon model 6 Shadowgraph microcomparator. The solutions were dialyzed for 48 hours at 4” prior to ultracentrif- ugation. Partial specific volumes were calculated from the amino acid composition and the values of ii for each amino acid (14).

S-Carboxymethylation-Protein was dissolved in 0.1 M Tris buffer at pH 9.3 containing 6 M guanidine hydrochloride and re- duced in a nitrogen atmosphere in the presence of 0.1% P-mer- captoethanol for4 hours. After adjusting the pH to 8.0, a neutral solution containing a 5.fold excess of iodoacetic acid was added. Alkylation was allowed to proceed for 5 min at room temperature. The reaction was terminated by the addition of a lo-fold molar excess of 2-mercaptoethanol. After dialysis against six changes of deionized water for 48 hours, the S-carboxymethyl protein was recovered by lyophilization.

Amino Acid Analysis-Analyses were performed with a Beck- man model 120 amino acid analyzer according to the procedure of Spackman et al. (15). Samples were hydrolyzed with 6 N HCl in evacuated sealed tubes for 22 to 72 hours at 110”. Half-cystine was determined both as S-carboxymethylcysteine after reduction and alkylation, and as cysteic acid following performic acid oxidation by the procedure of Moore (16). Tryptophan was determined spectrophotometrically by the method of Bencze and Schmidt (17), and by amino acid analysis after hydrolysis with p-toluene sulfonic acid (18).

Enzymatic Digestions-Tryptic digestion of S-carboxymethyl- ated protein was performed at pH 8 and 37” for 10 hours. The amount of trypsinwas 3y0 by weight of the protein. The digestion was terminated bv the addition of acetic acid to pH 2.0. For digestion with carboxypeptidase A, a modified procedure of Light (19) was used in which sodium dodecyl sulfate in 0.05 M NHJIC03 buffer at pH 8 was substituted for sodium lauryl sulfate in Verona1 buffer.

Peptide IMapping-Two-dimensional peptide maps were made on tryptic digests from approximately 1 mg of S-carboxymethylated protein. Electrophoresis at pH 1.9 (formic-acetic acid) at 3000 volts for 30 min was used for the first dimension. Descending chromatography in I-butanollpyridinelacetic acid/water (15/ 10/3/12) for 16 hours was employed for the second dimension. Whatman No. 3 MM paper was used. Peptides were located by dipping the paper in ninhydrin-collidine reagent (20). Trypto- phan-containing peptides were located with the Ehrlich stain for indoles (21). Peptides containing arginine were visualized with the alkaline phenanthrenequinone reagent of Yamada and Itano (22).

RESULTS

Purijication of AMP Deaminase

The initial stages of the purification, through the cellulose phosphate step, were performed essentially according to the procedure of Ronca-Testoni et al. (23). Since the enzyme ob- tained by this method was not homogeneous, either by the

for 30 s at high speed in a Waring Blendor. The slurry was sitrred at room temperature for 1 hour and allowed to stand at 4” overnight. The homogenate was centrifuged at 20,000 x g for 15 min. The supernatant was filtered through cheesecloth. The total volume of the supernatant from 40 rats was 2,500 ml and con- tained 9.76 x lo4 units of enzyme activity. The specific activity of the crude extract was 3.3 units per mg.

Cellulose-Phosphate Chromatography-Cellulose phosphate (1.5

g dry weight of cellulose phosphate per liter of crude extract) which had been prewashed successively with 0.5 IV KOH, HzO, 0.5 N HCl, H,O, 5 mM EDTA, and extraction buffer was added to the crude extract. The slurry was stirred at room temperature for 30 min. More than 90% of the enzyme activity was bound to the cellulose phosphate. The slurry was centrifuged at 10,000 x g for 10 min, the supernatant was discarded, and the cellulose phosphate transferred to a sintered glass column (2 x 50 cm). The column was washed with 0.45 M KCI, adjusted to pH 7.0 with KzHP& and containing 2 mM 2-mercaptoethanol, until the efauent had an absorbance at 280 nm of <O.Ol. A linear gradient, consisting of 150 ml of 0.45 M KC1/2 mM mercaptoethanol, pH 8.0, and 150 ml of 1.0 M KC1/2 mM mercaptoethanol, pH 8.0, was applied to the column. The elution profile obtained from this fractionation is shown in Fig. 1. The major portion of the enzyme activit,y coeluted wit,h the major protein peak and had a specific activity comparable to that previously reported (23). The frac- tions of highest specific activity were pooled as indicated in Fig. 1.

DEAE-cellulose Chromatography-The pool of enzyme ob- tained from the cellulose phosphate column was dialyzed against 0.045 M potassium phosphate/2 mM mercaptoethanol, pH 7.2, and applied to a DEAE-cellulose column which had been pre- equilibrated with the same buffer. The column was washed with an additional 100 ml of buffer prior to elution with a linear gradient consisting of 150 ml each of 0.045 M potassium phos- phate, and 0.45 M potassium phosphate, both of which contain 2 mM mercaptoethanol at pH 7.2. The results of this chroma- tographic step are shown in Fig. 2. Although only 40% of the total protein loaded onto the column could be accounted for, approximately 85% of the enzyme activity was recovered, and the specific activity was increased by about 2-fold. The enzyme was pooIed as shown in Fig. 2, placed in a dialysis bag and concentrated to approximately 5 ml by covering the dialysis tubing with Sephadex G-150.

criterion of sedimentation velocity or Na dodecyl-S04-gel FRACTION NUMBER

electrophoresis, it was necessary to introduce new steps into the purification procedure.

FIG. 1. Chromatography on cellulose phosphate. Crude extract was applied to a column (1.0 X 60 cm) and washed with 0.45 M

Homogenization-Leg and back muscles from rats were excised, k-Cl/2 InM 2-mercaptoethanol, pH 7.0, until no more protein came

cut into small pieces, and passed through a meat grinder. To the off. A linear gradient containing 150 ml each of 0.45 M KCl/2 mM

ground muscle was added 3.3 volumes (v/w) of extraction buffer 2-mercaptoethanol, pH 8.0, and 1.0 M KCl/2 mM 2-mercapto-

containing 0.18 M KCI, 0.054 M KH2P0.+ 0.035 M K2HP04, and ethanol, pH 8.0, was applied. The column was eluted at 20 ml per h our, and fractions of 2 ml each were collected and monitored for

2 mM 2-mercaptoethanol, pH 6.5. Homogenization was performed total protein at 280 nm (0) and AMP deaminase activity (A).

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FIG. 2. DEAE-cellulose chromatography. The pool of enzyme from the cellulose phosphate column was fractionated on a column (2 X 25 cm) of DEAE-cellulose. Elution was achieved with a linear gradient containing 150 ml each of 0.045 M potassium phosphate/2 mM 2-mercaptoethanol, pH 7.2, and 0.45 M potassium phosphate/2 mM 2-mercaptoethanol, pH 7.2. Flow rate was 40 ml per hour and fractions of 2.0 ml each were collected and assayed for AMP deaminase activity (A) and protein at 280 nm (0).

600 -

500-

;i A 400-

2 300-

:, k z 200 - 3

100 -

20 40 60 *Cl 100 120 140 160 160 200

FRACTION NUMBER

FIG. 3. Gel filtration. The pool of enzyme from the DEAE- cellulose column was concentrated and chromatographed on a column (2.5 X 90 cm) of Bio-Gel A-5 m. The column was eluted with a buffer containing 0.18 M KC1/0.45 M KHtP04/0.03 M

KzHPOa/2 mM 2-mercaptoethanol, pH 6.5, at a rate of 20 ml per hour. Fractions of 2.5 ml were collected and monitored for protein (---) and AMP deaminase activity (A--A)

TABLE I Purification of rat muscle AMP deaminase

step Total protein Total enzyme

Specific activity Yield

mg ulds units/mg %

Crude extract 29 ) 900 97,650 3.28 100 Phosphocellulose 72.1 78,120 1089 80 DEAE-cellulose 29.8 74,411 2497 76 Bio-Gel A-5m 24.6 20,240 823 21

Chromatography on Bio-Gel A-6m-The enzyme recovered from DEAE-cellulose was loaded onto a column (2.5 X 90 cm) of Bio-Gel and eluted with buffer consisting of 0.18 M KC1/0.045 M KHzP04/0.03 M KzHP04/2 KkM mercaptoethanol, pH 6.5. All of the activity eluted as a single symmetrical peak (Fig. 3)) and is clearly resolved from two minor protein components which are of lower molecular weight than the enzyme.

The results of the purification procedure are summarized in Table I. Although the over-all yield is 21%, a yield of 76% was obtained until the last step in the purification. The low yield achieved during the Bio-Gel step reflects a precipitous loss in specific activity. A characteristic feature of the enzyme after

FIG. 4. Na dodecyl-Sod-gel electrophoretic patterns of AMP- deaminase at different stages of purification. Left, cellulose phos- phate pool; center, DEAE-cellulose pool; right, Bio-Gel pool.

filtration on Bio-Gel appears to be its instability. The reason for this increased lability is currently being investigated.

Purity of Enzyme

At each step in the purification procedure (Figs. 1 to 3), the specific activity across the peak of enzyme activity was constant. Although this is often a useful criterion of homogeneity, it is clear that in this case it is insufficient in defining a pure prepara- tion. The results of gel electrophoresis performed in the presence of sodium dodecyl sulfate at various steps in the purification, are shown in Fig. 4. Following chromatography on cellulose phosphate one major band and several minor bands of protein were observed. Subsequent fractionation on DEAE-cellulose, which resulted in enzyme with the highest specific activity ob- tained throughout the preparation, eliminated several of the minor protein species; Na dodecylLS04 gel electrophoresis at this step, however, revealed three protein bands two of which were smaller and present in amounts less than that of the major component. The minor bands were eliminated in the final step of the purification, and the enzyme obtained from the Bio-Gel column electrophoreses as a single narrow band, indicative of homogeneity.

Sedimentation velocity experiments on the final preparation revealed a single symmetrical boundary with no evidence of heterogeneity (Fig. 5). The sedimentation coefficient, calculated from these data and corrected to water at 20”, is 11.3 S.

Attempts to observe the behavior of the native enzyme on standard polyacrylamide gels have been unsuccessful. The

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FIG. 5. Sedimentation velocity of purified AMP deaminase at a protein concentration of 4.0 mg/ml in 0.18 M KC1/0.45 M KHsPOJ 0.03 M KzHP0*/2 mM 2-mercaptoethanol buffer, pH 6.5. The experiment was conducted at 20” with a speed of 52,000 rpm and a bar angle of 75”. The photograph shown was taken at 16 min.

TABLE II

Amino acid composition of AMP deaminase

Amino acid Residues per subunita N~~s~$~C~~~~ Rabbit muscle subunit AMP-deaminax?

mo1/60,000 g

Lysine 43.2 f 2.3 43 45.4 Histidine 18.8 f 1.1 19 18.3 Arginine 25.7 f 1.0 26 29.2 Aspartic acid 53.6 & 1.1 54 53.9 Threonine” 25.1 f 1.8 25 26.9 Serine” 36.2 f 2.3 36 36.7 Glutamic acid 54.9 f 1.2 55 53.2 Proline 31.8 & 2.3 32 29.8 Glycine 25.3 f 1.0 25 23.8 Alanine 30.6 zt 1.7 31 31.2 Half-cystined 7.7 f 0.5 8 7.4 Valinee 29.0 f 1.4 29 23.5 Methionine 15.4 f 1.3 15 15.5 Isoleucinee 23.6 zt 0.7 24 24.1 Leucine 49.5 f 1.2 50 50.2 Tyrosine 24.7 f 1.1 25 27.7 Phenylalanine 28.5 f 0.4 29 27.8 Tryptophanf 3.8 4 4.5

0 Average of values obtained from triplicate samples and calcu- lated for a subunit molecular weight of 60,000.

b Data of Wolfenden et al. (24), recalculated to moles per 60,000 g.

c Corrected for decomposition by extrapolation of values ob- tained from 24-, 48-, and 72-hour hydrolysates.

d Determined as cysteic acid after performic acid oxidation. e Values obtained from 72-hour hydrolysate. f Determined spectrophotometrically.

enzyme has remained as a tight band at the point of application on 10, 7.5, and 5% polyacrylamide gels run in Tris-HCl (pH 8.9).

Amino Acid Analysis

The amino acid composition of the enzyme is given in Table II. Numbers of residues were calculated for a subunit molecular weight of 60,000 (see below). The results are given as the averages of three analyses performed on separate hydrolysates.

The half-cystine content of the enzyme was determined as cysteic acid on samples which had been oxidized with performic acid. The thiol content was also determined as S-carboxymethyl- cysteine following reduction and alkylation. Both methods indi- cate that there are 8 mol of half-cystine per 60,000 g.

Tryptophan was determined spectrophotometrically (17) and a value of 3.8 mol per 60,000 g of enzyme was found.

I 1 I I I I 50.4 5006 50.8 51,O 51.2

X2 (cm12

FIG. 6. Sedimentation equilibrium data of purified AMP deaminase. The protein concentration was 0.4 mg/ml in 0.18 M KC1/0.45 M KHtPOd/0.03 M KzHP01/2 mM 2-mercaptoethanol, pH 6.5 (Plot A) or 6 M guanidine hydrochloride/l0 mM 2-mercapto- ethanol, pH 6.5 (Plot B). Sedimentation was at 20” and 12,000 rpm for 24 hours for the native enzyme (A) and at 20” and 26,000 rpm for 48 hours for the denatured enzyme (B)

The amino acid composition of AMP deaminase from rabbit muscle, as reported by Wolfenden et al. (24) and recalculated as residues per 60,000 g, is included in Table II. The composition of the enzyme from rat and rabbit skeletal muscle is remarkably similar, suggesting that they are closely related.

Molecular Weight

Molecular weight determinations of AMP deaminase were determined by sedimentation equilibrium analysis using the meniscus depletion technique (13). The results of a typical experi- ment are shown in Fig. 6A. A plot of t.he In concentration, represented by fringe displacement, versus the square ofthe radial distance gave a straight line for three different concentrations of enzyme ranging from 0.4 to 1.2 mg/ml. These results are indicative of homogeneity and suggest that the size of the native enzyme is not dependent on the protein concentration, at least within the concentration range examined. Calculation of the molecular weight from these data give a weight average molecular weight of 238,000.

Subunit Composition

The subunit size was examined by the meniscus depletion method on samples containing 6 M guanidine hydrochloride and 10 mM 2-mercaptoethanol. Plots of 1nC versus X2 (Fig. 6B) con- formed to straight lines, indicating homogeneity with respect to subunit size. The molecular weight of the enzyme under reducing and denaturing conditions was calculated to be 59,500. These calculations assumed a partial specific volume of 0.72 in 6 M

guanidine hydrochloride (25). Electrophoresis of the enzyme on polyacrylamide gels in the

presence of sodium dodecyl sulfate (Fig. 7) indicated that the dissociated enzyme had a molecular weight of 60,000 f 2,000. These results are in excellent agreement with the values obtained by sedimentation in guanidine hydrochloride.

End Group Analysis

Attempts to identify an NH&erminal amino acid residue have been unsuccessful. With the cyanate method (26), only trace

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MOBILITY

FIG. 7. Determination of the subunit molecular weight of AMP deaminase on 10% polyacrylamide gels in Na dodecyl-SOa accord- ing to Weber and Osborn (12). Protein standards used were bovine serum albumin (HA), type IgG immunoglobulin (ZGG-heavy chain and ZGG-light chain), lactate dehydrogenase (LDH), lyso- zyme, and carp muscle calcium binding protein (MCBP). The molecular weight of AMP deaminase is indicated as 60,000 (A).

amounts of amino acids, at levels that were essentially the same as those found in control samples lacking either cyanate or enzyme, were found. Treatment of the enzyme for 8 hours with carboxypeptidase A in 0.05 M NH,HCO, at pH 8.0 resulted in the release of 0.49 mol of valine and 0.45 mol of leucine per 60,000 g of protein. These half-stoichiometric results can be interpreted either as arising from approximately equimolar amounts of chemically distinct subunits, or from microheterogeneity at the COOH terminus of very similar, if not identical subunits, or from incomplete digestion of identical subunits having -X-Leu-Val at the COOH terminus. In order to distinguish between essentially identical and chemically distinct subunits, tryptic hydrolysates were mapped.

Tryptic Peptide Maps

A typical tryptic peptide map performed on a tryptic digest of S-carboxymethylated enzyme is shown in Fig. 8. It, should be noted that after tryptic hydrolysis, all of the peptides were completely soluble, and thus, the tryptic maps are representative of the entire starting material. Upon repeated mapping experi- ments, between 59 and 62 ninhydrin-posit.ive spots of varying intensity could be detected. In all these experiments four spots were very strongly stained with Ehrlich’s reagent and another faintly positive spot was sometimes found. When the maps were sprayed with alkaline phenanthrenequinone to locate arginine- containing peptides, 22 positive fluorescent spots were identified.

A summary of the tryptic peptide data is given in Table III. From the amino acid analysis, there are a total of 276 lysine and arginine residues per molecule of enzyme (240,000 molecular weight). I f there are two different types of subunits, there should be approximately 141 peptides generated by digestion with trypsin. If, on the other hand, the subunits are identical, only 70 peptides would be produced by tryptic hydrolysis. The total number of peptides resolved in Fig. 8 was between 59 and 62. Additional evidence for identical subunits is provided by the number of tryptophan and arginine-containing peptides found. Both values are very close to those predicted on the assumption of chemical identity of subunits.

DISCUSSION

The purification of AMP deaminase from rat muscle, as de- scribed here, is an extension of the procedure previously reported

7 m -9, ELECTROPHORESIS. pH I.9

FIG. 8. Tryptic digest map of carboxymethylated AMP deami- nase. Approximately 1 mg of digested enzyme was applied to What- man No. 3 MM paper. Electrophoresis was performed in the first dimension at pH 1.9 and 3000 volts for 30 min. Descending chro- matography was performed for 16 hours in 1-butanol/pyridine/ acetic acid/water (15/10/3/12). Peptides were detected with ninhydrin. Peptides containing tryptophan (shaded spots) were located with Ehrlich’s reagent (21).

TABLE III

Tryptic peptide mapping of AMP-deaminase

Peptide characteristics Predicted peptides

Nonidentical Identical subunitsa subunits*

Peptides observed

Ninhydrin-positive peptides Tryptophan-containing pep-

tides” Arginine-containing peptidesd

141 70 59-62

8 4 4-5

52 26 22

a Predicted on the assumption of equal molar amounts of non- identical subunit,s having molecular weights of 60,000.

* Based on molecular weight of 60,000. c Peptides having a positive reaction to Ehrlich’s reagent (21). d Detected as fluorescent spots by the procedure of Yamada

and Itano (22).

by Ronca-Testoni et al. (23). Although these authors report a homogeneous preparation, their preparation, in our hands, gives an enzyme exhibiting significant heterogeneity 011 Na dodecyl- SO1 gels, although the specific activity is the same as that re- ported. The additional steps incorporated into the purification procedure described here result in an enzyme which has a specific activity more than 2-fold greater than previously reported.

The enzyme is free of ATPase, myokinase, and adenosine kinase activities. Additional criteria of purity include a single symmetrical boundary observed with sedimentation velocity analysis, linearity of 1nC versus cm2 obtained from sedimenta- tion equilibrium data, and a single band of protein observed on Na dodecyl-SOJ-polyacrylamide gels.

The specific activity of the enzyme is subject to considerable variation during the course of the preparation. The highest specific activity (2500 units per mg) was obtained by fractiona- tion on DEAE-cellulose. The subsequent step which gave a homogeneous enzyme preparation also resulted in a precipitous

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decline in specific activity. The reason for this loss in activity is not clear at this time, but it reflects an enhanced lability of the enzyme as judged by temperature inactivation curves. The half- life of the enzyme at 45” prior to fractionation on Bio-Gel is 80 min, while that for the final preparation is 20 min under the same conditions of buffer, pH and ionic strength.’ Recombination of aliquots of the Bio-Gel enzyme pool with other fractions across the column did not result in increased activity.

The molecular weight of the native enzyme, calculated from sedimentation equilibrium analysis, is 238,000. This is in good agreement with the molecular weight calculated from the amino acid composition. In the latter determination, 99.4$& of the dry weight of the protein could be accounted for in terms of amino acid mass, indicating that the enzyme is composed entirely of proteinaceous material. The molecular weight of 238,000 found in these studies is significantly lower than that of 290,000 reported by Ronca-Testoni et al. (23). These authors also report a sedi- mentation coefficient of 12.2 S, whereas we find an sZo,w value of 11.3 S. The reported molecular weight of 290,000 and sZo,,,, of 12.2 S were determined by sucrose gradient centrifugation on a preparation which was not as pure as that reported here.

The sum of the evidence presented here indicates that the native enzyme is a tetramer composed of identical, or nearly identical, subunits. The enzyme, when treated with either 6 M

guanidine hydrochloride or sodium dodecyl sulfate dissociates into polypeptides of 60,000 molecular weight. The tryptic peptide maps strongly support the identity of subunits, since the number of ninhydrin-positive peptides, as well as the number of peptides containing tryptophan and arginine, are very close to the numbers predicted for identical subunits.

Clearly, both the catalytic and regulatory properties of muscle AMP-deaminase are quite complex. Nucleoside t,riphosphates, ADP, and Pi all profoundly affect the activity of the enzyme (8). Moreover, it appears that all of these metabolites may be signifi- cant in the in vivo regulation of the enzyme. Both ATP and Pi, at physiological concentrations, inhibit the enzyme, whereas ADP serves as an activator, and can reverse the inhibition imposed by ATP and Pi. However, since almost all of the ADP in resting muscle has been shown to be tightly bound to the myofibrils (27), it is likely that AMP deaminase is inhibited in resting muscle, and becomes active under conditions in which free ADP ac- cumulates. It has been suggested (8) that AMP deaminase works in conjunction with muscle myokinase in scavenging the high energy phosphate of ADP during periods of intense muscular activity. In this vein, it is significant that myokinase is inhibited by AMP (28), and a mechanism for relieving the inhibition, such as could be mediated by AlMP deaminase, would be required for maximum energy production.

Although the kinetic properties of the isolated enzyme have been studied in a number of laboratories (4, 8, 29, 30), very little attention has been focused on the molecular properties of the enzyme, nor has an attempt been made to correlate the molecular properties with the catalytic and regulatory properties of the enzyme. The finding that AMP deaminase is a tetramer composed

1 C. Coffee, unpublished observations.

of identical subunits should provide a framework for further experiments directed toward understanding the regulation of the enzyme in terms of molecular structure.

Acknowledgments-We would like to thank Ms. Cynthia Solano for her help in the purification of the enzyme and in performing the amino acid analysis. The expert assistance of Ms. Ingrid Kuo in performing the ultracentrifugal analyses is gratefully acknowl- edged.

1. 2. 3.

4.

5. 6.

7.

8.

9.

10.

11.

12.

13. 14.

15.

16. 17.

18.

19. 20. 21. 22.

23.

24.

25.

26.

27.

28.

29.

30.

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C J Coffee and W A KofkeRat muscle 5'-adenylic acid aminohydrolase. I. Purification and subunit structure.

1975, 250:6653-6658.J. Biol. Chem. 

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