Adenylate Kinase of Porcine Heart

Eur. J . Biochem. 48, 325-331 (1974)

Adenylate Kinase of Porcine Heart Shuichiro KUBO and Lafayette H. NODA Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire

(Received April 3 /June 11, 1974)

The activity of adenylate kinase (ATP : AMP-phosphotransferase, EC 2.7.4.3) in porcine heart is about one-sixth that in porcine skeletal muscle on a wet weight basis. Essentially all of the activity of the heart preparations, 70 - 80 units/g tissue, was present in the soluble fraction. On the basis of acid stability at pH 3.5 and 0 "C and behavior on isoelectrofocusing, there appear to be at least two adenylate kinases in heart, one acid-labile type having an isoelectric point between 4.7 - 7.5 and the other an acid-stable type having an isoelectric point at 9.3. The acid-stable adenylate kinase was purified approximately 1300-fold from heart homogenate. The purified enzyme had a molecular weight of 21 500 and migrated in gel electrophoresis as a single band. A mixture prepared together with purified muscle enzyme also gave a single narrow band in gel electrophoresis. Amino acid composition was found to be Asx13, ThrI4, ser,,, Glxzs, Pro,, Gly,,, Ala,, Val,,, Met,, Ile,, Leul8, Tyr,, Phe,, His,, Lys,, , Arg,, , Cys, (as cysteine) and no tryptophan. Fingerprinting showed correspondence of all major spots of the tryptic peptides from heart and skeletal muscle enzymes preparations. The total amino acid sequence of the skeletal muscle enzyme is accounted for by the peptides found on fingerprinting. These results strongly suggest that the major acid-stable adenylate kinase from porcine heart is identical to porcine skeletal muscle adenylate kinase.

Adenylate kinase (ATP : AMP-phosphotransferase) has been demonstrated to be present in multiple forins in various animal tissues [I -41. On the basis of sulfhydryl reactivity and antibody inhibition studies, Khoo and Russell [5] have shown from data on human and rabbit isozymes from various tissues that there are at least two sets of isozymes with one set (muscle type) in skeletal muscle, erythrocytes and brain, and another (liver type) in liver, kidney and heart.

Adenylate kinase in muscle possesses remarkable acid stability [6] and this fact has been utilized in the purification of adenylate kinase from muscle of various animals [7-91. On the other hand, Chiga and Plaut [lo] have shown that the adenylate kinase in porcine liver is sensitive to acid and easily loses its activity. This instability in acid may represent a significant difference in the nature of the different forms of adenylate kinase. In this paper porcine heart extract was analyzed for multiple forms of adenylate

Enzymes. Adenylate kinase (EC 2.7.4.3); hexokinase (EC 2.7.1.1); pyruvate kinase (EC 2.7.1.40); lactic dehydro- geriase (EC 1.1.3.27); trypsin (EC 3.4.21.4); creatine kinase (EC 2.7.3.2).

kinase by the use of acid stability and isoelectrofocusing. The acid-stable heart adenylate kinase has been purified and crystallized and a comparison of this enzyme to that from porcine skeletal muscle is made.

EXPERIMENTAL PROCEDURE

Materials

ADP, AMP and iinidazole were obtained from Sigma. Hexokinase was prepared from yeast by the method of Darrow and Colowick [ll]. Phospho- cellulose (lot 2122 and 2136) were obtained from Schleicher and Schuell (Keene, N.H. 10962 U.S.A.) and cycled by the method of Peterson and Sober [12]. Celite 501 from Johns-Manville, Co. was twice washed with 2 N HC1 at 80°C, thoroughly washed with distilled water, and dried. Sephadex G-75 was from Pharmacia Fine Chemicals, Inc. and ammonium sulfate (enzyme grade) from Schwarz-Mann (Orange- burg, N.Y.). Electrofocusing columns of 110 ml and 440 ml capacity and carrier ampholytes (40 w/v) of pH 3 - 10 and 7- 10 were obtained from LKB Instruments, Inc. Pyruvate kinase (rabbit muscle),

Eur. J. Biochem. 48 (1974)

326 Adenylate Kinase of Porcine Heart

lactic dehydrogenase (bovine heart) and cytochrome c (horse heart) from Sigma; serum albumin (bovine) from Mann ; trypsin (bovine pancreas) from Worthing- ton; and creatine kinase from rabbit muscle by procedure B [ 131 and adenylate kinase (porcine muscle) [9] were used as molecular weight markers. Acryl- amide, methylenebisacrylamide, N,N,N',N'-tetrame- thylethylenediamine and 2-mercaptoethanol were obtained from Eastman. Coomassie brilliant blue and bromphenol blue were obtained from Mann. All other chemicals were analytical grade. Fresh porcine hearts were obtained from a local slaughter house and frozen hearts shipped in dry ice were obtained from a Philadelphia slaughter house.

Preparation of Heart Extract Frozen porcine hearts thawed in a cold room,

were trimmed to remove fat and ground in a motor- driven meat grinder. Cold 0.01 M KCI equal to 2.5 times the weight of ground heart was added, and the mixture homogenized in a Waring blender for 1 min. The homogenate was centrifuged for 30 min at lo5 x g in a Beckman Spinco centrifuge, Model L, and a clear solution, free of congealed fat, was obtained from the supernatant fluid by passage through cheese cloth.

Preparations of Crude Adenylate Kinase front Mitochondria and Cytosol

Mitochondria were prepared from 2.5 kg of fresh porcine heart by the method of Hogeboom [14]. Mitochondria, collected as a sediment was stored at - 20 "C. Crude mitochondria1 adenylate kinase was prepared by the method of Markland and Wadkins [15] and the supernatant fraction containing the cytosol adenylate kinase was retained.

Enzyme Assay and Protein Determination

Adenylate kinase activity was determined by the titrimetric pH-stat method of Mahowald et al. [16] using a Radiometer TTTla equipped with a scale expander and SBR2b titrigraph as described previously [7] and modified for a total incubation volume of 1.5 ml. Protein concentrations in crude fractions were determined by the biuret method of Gornall e ta / . [17] with bovine serum albumin as a standard. The concentrations of purified adenylate kinase were determined spectrophotometrically using A&" x 2.25 = mg of the enzyme per ml.

Isoelectrofocusing

Isoelectric fractionation by pH gradient stabilized by a density gradient was performed as described by

Vesterberg [18]. After constant current was achieved fractions of 2 ml or 3 ml were collected and monitored for activity, absorbance and pH. Measurements of pH were made at 0 "C with use of a Radiometer pH meter 26.

Dodecylsulfate - Polyacrylamide-Gel Electrophoresis

Electrophoresis in 10% acrylamide at pH 7.0 was carried out essentially according to the procedure of Weber and Osborn [19]. The gels were stained with Coomassie brilliant blue and electrophoretically destained in 75 % acetic acid - 5 % methanol.

Amino -Acid Analyses

Samples of approximately 0.2 mg of enzyme were hydrolyzed for 24, 48, 72, 96 and 120h at 106°C with constant-boiling glass-distilled HCl in sealed evacuated tubes. Analyses were carried out on a Beckman Spinco analyzer, model 120C, by the method of Spackman et al. [20]. Half-cystine was determined as cysteic acid after oxidation with performic acid

Tryptic Digestion and Peptide Maps

Tryptic digestion in 0.5 % ammonium bicarbonate and the fingerprinting were carried out as previously described [9].

Reaction of Adenylate Kinase with 5,5'-Dithiobis- (2-nitrohenzoic acid)

described by Ellman [22]. Titration of -SH groups was carried out as

RESULTS Eflect o f p H on Heart Extract

The stability of enzymatic activity of extract from heart at 30 "C for 1 h was investigated over the range of 1 to 12. Adenylate kinase activity of extract was stable in the pH range 5 - 9 as shown in Fig. 1. In the low range, irreversible loss of activity was maximal near pH 3.2 and at lower pH values stability was greater under the conditions of the test.

Time Course of the Activity Loss

In Fig. 2 are shown the time course of the activity loss in heart extract during incubation at pH 3.5 and 0 "C plotted logarithmically. Loss of about 20 % of the original activity occurred rapidly during the initial


S. Kubo and L. H. Noda 327

100 I 1

PH Fig. 1. Eflect o j p H on loss o j enzymatic activity of heart extract. Heart extract was diluted with buffer to 1.59 mg/ml and incubated at the pH indicated at 30 "C for 1 h. Samples were added to the pH-stat assay mixture and the pH was immediately adjusted to 8.00

! '. ! ! ! '. '. !

0 20 40 60 80 100 120 140 160 1 Time (min 1

Fig. 2. Time course o j loss of activity at p H 3.5 and 0 "C. Heart extract was diluted with 0.05 M glycine-HCI buffer, pH 3.5, and held at 0 "C. The control sample was 2 units/ml and was held at pH 6.5 and 0 "C. Protein concentration was 1.59 mg/ml. Samples were withdrawn for assay at times shown and assayed by the hexokinase pH-stat method. The upper curve shows decline of total activity which may be separated into a labile and more stable components. The nearly linear decrease in activity at times after 80 min represents the loss of activity of the more stable component. The lower line, obtained by a plot of the activity remaining when the slow loss of activity is subtracted from the total activity, represents loss of activity by the more acid-labile component

13

12

11

10

9

6 I a

7

6

5

A- -- Tube number

Fig. 3. Isoelectrofocusing profile of adenylate kinase of cytosol extract. The enzyme solution of 837 units was applied to a column of 110 ml capacity. Tsoelectricfocusing was continued for 72 h 2-~nl fractions were collected. (0) pH; (0) adenylate kinase activity (Ujml)

period of incubation, thereafter the losses increased slowly. Allowing for the slow loss of activity in the initial period the loss of activity of the more labile component is indicated in Fig. 2 by the dotted line. The ratio of rate of loss of activity by the more labile (k = 42 x l op3 min-l) to more stable component (k = 1.3 x min-') is about 32: 1. These results suggest the possible existence of two kinds of adenylate kinase in porcine heart extract, an acid-stable and an acid-labile enzyme.

Isoelectrofocusing of Adenylate Kinase from Cytosol and from Mitochondria1 Fraction

Markland and Wadkins [ 151 purified adenylate kinase from bovine liver mitochondria and studied its properties. Criss [23] showed the existence of different forms of adenylate kinase in various sub- cellular fractions of rat liver. Adenylate kinase activity of heart cytosol and mitochondria1 extract was examined by isoelectrofocusing and by comparing the distribution of enzymatic activity (Fig. 3 and 4). The activity in cytosol focused as a broad peak from pH 4.7 to 7.5 (fraction A collected from tube numbers 22 to 39) and a sharp peak at pH 9.3 (fraction B, collected from tube numbers 40 to 53). The distribution of the activity was 13% and 87% in fraction A and B, respectively, and the sum of the activities, fraction A (67 units) and B (776 units), was equal to the units applied to the column. The isoelectrofocusing



Tube number

Fig. 4. Isoelectrojocusing profile of adenylate kinase in rnito- chondrial extract. The enzyme solution of 71 units was applied to a column of 1 I0 ml capacity. Isoelectrofocusing was continued for 72 h 2-ml fractions were collected. (0) pH; (0) adenylate kinase activity (U/ml)

of the enzyme in mitochondrial extract showed almost the same profile as that of cytosol; however, the distribution of the activity was 38 in fraction A (27 units, tube numbers 22 to 29) and 62 % in fraction B (44 units, tube numbers 41 to 52). The recovery of activity was equal to 100 % of the activity originally applied to the column.

In Fig. 5 is shown the time course of the activity loss during incubation at pH 3.5 and 0 "C for each fraction isolated by isoelectrofocusing. Fraction B obtained from cytosol and from mitochondrial extract had the same high stability under acidic conditions. However, the loss of activity by fraction A obtained from mitochondria and cytosol occurred rapidly during incubation and at 60 min only 10 % of the activity of the control was observed

Isolation and Characterization of Acid-Stable Enzyme

The purification and isolation of acid-stable enzyme was performed with frozen heart by a modifica- tion of the method used with porcine muscle [9] up to the crystallization step. Variations were that in the acid treatment step the denatured proteins were removed after adjusting the pH to 7.0 then to pH 5.0

1 I 1 I 1

20 LO 60 80 100 120 Timelmin)

Fig. 5. Time course of activity loss ojjractions A and B of cytosol and of mitochondria1 adenylate kinase. The conditions and the methods were the same as in Fig. 2 except the protein concentration in the incubation mixture. The protein concentration of fraction A and B in cytosol enzyme was 1.0 mg/ml and 2.0 mg/ml, respectively, and that of fraction A and B in mitochondrial enzyme was 1 .0 mg/ml and 2.0 mg/ml, respectively. Cytosol : (A) isoelectrofocusing component A ; (0) component B. Mitochondria: ( x ) isoelectrofocusing component A ; (0) component B

to 0.2. Elution and collection of activity as a paste precipitated by 90 ammonium sulfate was as described [9]. Fig. 6 shows the profile for isoelectrofocusing of fraction IV. The activities were collected as fraction V and stored in a freezer.

The result of this purification procedure is sum- marized in Table 1. The heart adenylate kinase purified by isoelectrofocusing was stable to acid treatment and the time course of activity loss at pH 3.5 and 0 "C was similar to that of fraction B shown in Fig. 5 .

Molecular Weight

The purified acid-stable heart adenylate kinase was found to give a single band in dodecylsulfate gel electrophoresis [19]. In addition, a mixture of the adenylate kinase prepared from heart and from skeletal muscle migrated as a single band. For the determination of the molecular weight a plot of electrophoretic mobilities of marker proteins against the logarithm of their molecular weights gave a molecular weight of 21 500 f 800 for the acid-stable adenylate kinase isolated from porcine heart as shown in Fig. 7 .

Crystalline Heart Adenylute Kinuse instead of at pH 7.0. The phosphocellulose column was washed with 2 column volumes of water, 15 column volumes of 0.05 M imidazole-HCI pH 7.0 and then with 0.1 M (not 0.07 M) imidazole-HCI pH 7.0 until the absorbance at 280 nm decreased

Crystallization was carried out with fraction IV (Table 1) at a protein concentration of 30 mg/ml in 0.01 M phosphate buffer pH 6.0 and 62 % ammonium


S. Kubo and L. H. Noda

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$ 0, 1.0 N -

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2000 $. I

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Fig. 15. Zsoelectrofocusing profile of fraction ZV. The enzyme solution of 1.6 x lo5 units and 100 mg protein was applied to a column of 440 ml capacity. Isoelectrofocusing was

continued for 144 h 3-ml fractions were collected. (0) Ab- sorbance at 280 nm; (0) pH; ( x ) adenylate kinase activity; (A) specific activity

5 E .5 4 - 3 ol

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Tabl'e 1. Purijication of acid-stable adenylate kinase from porcine heart Total ground porcine heart used for starting material was 30.18 kg and the activity was 75 unit per g wet weight

1

Fraction

Cytochrome c (11700) \ I I

Total Total Total Yield Specific Purification volume protein activity activity

units/mg ml mg units % I. Extract of cytosol 86130 1 390 000 2250000 100 1.6 1.0 11. Acid treatment 82 620 492 000 1 640 000 12.9 3.3 2.1 111. Phosphocellulose

chromatography 1530 3 175 532 000 23.7 168 105 IV. Sephadex G-75 gel

filtration 730 311 406 000 18.0 1301 813 V. Isoelectrofocusing 143 155 323 000 14.4 2083 1302

sulfate saturation. After two weeks, during which the amrnonium sulfate saturation was slightly increased and turbidity of precipitated proteins was removed by centrifugation, rhombic crystals similar to those of porcine muscle enzyme were obtained. Crystal shape was improved by a second crystallization. By isoelectrofocusing, the twice-crystallized enzyme sho,wed two peaks, in which the minor peak corre- sponding to 20% of the total enzyme focused at pH 9.3 and the major peak focused at pH 8.80. Since the purified enzyme (fraction IV, Table 1) showed a single peak in electrofocusing it is clear that a major portion of the adenylate kinase had shifted in isoelectric point from 9.3 to 8.8 very probably by deamidation of carboxamide groups.

Serum albumin (68000)

Pyruvate kinase ( 57000)

\

Trypsin ( 2 3 3 0 0 ) W A d e n y l a t e kinase

121500)

\*

\ Amino-Acid Composition

The amino acid composition of porcine heart adenylate kinase is presented in Table 2 together with that of porcine muscle enzyme [9] for comparison.

0 0.2 0.4 0.6 0.8 Relative migration

Fig. 7. Determination of the molecular weight of the ucid-stable adenylate kinase from porcine heart by dodecylsulfate electrophoresis [19]. (0) Marker proteins; ( x ) acid-stable heart adenylate kinase



Table 2. Amino-acid composition of acid-stable adenylate kinase from porcine heart The data was obtained from 24, 48, 72, 96 and 120-h hydro- 1 ysates

Amino acid Amino-acid residues (total 194)

acid-stable heart skeletal muscle adenylate kinase adenylate kinase

Aspartic acid Threonine" Serine" Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine Histidine Lysine Arginine Tryptophan Cysteined

12.8 13.3 11.1 24.9 6.4

18.5 7.6

17.3 6.0 8.4

18.8 6.9 4.9 2.1

20.6 11.1 0 1.7

13.0 13.6 10.7 25.2 6.2

19.2 8.0

17.2 6.0 8.5

18.6 6.8 4.9 1.8

20.9 11.0 0 1.7

a Extrapolated value to 0 time. Values are for the 120-h hydrolysis. Determined by spectrophotometric method of Good-

Determined as cysteic acid. win and Morton [24].

The amino acid analyses for the acid-stable adenylate kinase from porcine heart and the enzyme from skeletal muscle were found to be the same within experimental error. There are per mole of enzyme two each of histidine and cysteine groups and no tryptophan. Tryptophan was determined in the heart enzyme by the spectrophotometric method of Goodwin and Morton [24]. When acid-stable adenylate kinase from heart was treated with 5,5'-dithiobis-(2-nitro- benzoic acid), 1.83 mol sulfhydryl group per 21 500 g enzyme had reacted and the activity was lost complete- ly. This result together with the amino acid analysis data (Table 2) leads to the conclusion that there are two cysteine residues in acid-stable adenylate kinase from heart.

Peptide Map Fingerprinting of the enzyme from heart was

carried out in parallel with that of the enzyme from skeletal muscle. The number and location of all major peptide spots from the heart enzyme on the map were essentially identical to those from the muscle enzyme. The peptide spots of the fingerprint of the enzyme isolated from heart accounted for all the peptide spots identified on the fingerprint of the skeletal

muscle enzyme. For the latter, the peptide spots accounted for the total amino acid sequence which has been determined [9]. Thus it may be concluded that on the basis of acid-stability properties, the isoelectric point, amino acid composition and fingerprinting the major adenylate kinase component of porcine heart is the same as adenylate kinase from skeletal muscle.

DISCUSSION

Acid-stable adenylate kinase was purified approximately 1300-fold from porcine heart homogenate to a final specific activity of 2000 units/mg enzyme. By dodecylsulfate electrophoresis the molecular weight was estimated to be 21 500 and the isoelectric point by isoelectrofocusing was determined to be 9.3. These molecular properties and the amino acid composition are in agreement with those of porcine skeletal muscle enzyme [9]. Unlike the enzymes isolated from porcine liver [lo] and bovine liver [15,25] which were not sensitive to sulfhydryl reagents and which contained tryptophan [15], the acid-stable enzyme from heart was like the skeletal enzyme with regard to these characteristics. The major peptide spots of the finger- prints of the acid-stable enzyme from heart and of skeletal muscle were in the same location. Thus, by agreement of molecular weight, isoelectric point, acid stability, amino acid composition and finger- prints, it may be concluded that the acid-stable enzyme in porcine heart must be the same as adenylate kinase isolated from skeletal muscle.

It was observed that crystalline acid-stable heart adenylate kinase prepared by standing in about 62 "/, saturated ammonium sulfate, pH 6.0, in a refrigerator for more than two weeks showed a change in the isoelectrofocusing pattern. The pH 9.3 peak was decreased to about 20 of the total protein and a new broad peak at pH 8.8 appeared containing about 80% of the total protein. The enzyme in the two peaks migrated in dodecylsulfate gel electrophoresis as a single band of molecular weight 21 500. The enzyme having an isoelectric point of 8.8 had full activity and the same acid stability at pH 3.5 and 0 "C as the enzyme having isoelectric point 9.3. Such shifts in isoelectric point have been observed in other cases. Blumenthal and Heinrikson [26] showed that crystalline bovine liver rhodanese prepared by ammonium sulfate and pH precipitation is composed of two fully active components, rhodanese A and B, which are identical with respect to molecular properties and are separable by electrophoresis and by ion-exchange chromatography. They suggested that rhodanese B may arise during the course of purification by deamidation of


S. Kubo and L. H. Noda 331

the Pi form. In studies on the non-enzymatic deamidation of cytochrome c in vitro, Flatmark [27] found that even under conditions sometimes used for the crystallization of cytochrome c (80 % ammonium sulfate saturation, neutral pH and room temperature) there was rapid loss of amide groups and conversion of parent cytochrome c to fractions distinguishable in gel electrophoresis as more negatively charged protieins. It seems reasonable to assume that the decrease in isoelectric point of the main fraction from pH 9.3 to 8.8 without loss of activity can be ascribed to deamidation of glutamine or asparagine residues as has been observed for rhodanese and cytochrome c.

The total adenylate kinase activity in porcine heart was found to be 70 - 80 units per g of tissue (Table 1) which is, on a wet basis, about one sixth that in skeletal muscle [9]. Adenylate kinase from heart could be separated by electrophoresis into an acid- stable and an acid-labile form. Mitochondria isolated by differential centrifugation was found to contain a relatively higher percentage of acid-labile adenylate kinase (38 ‘x ) than the cytosol which was found to contain about 13 acid-labile form. The question may be raised whether the acid-labile form is not peciiliar to mitochondria and the acid-stable form to the cytosol. Cross contamination of mitochondria and cytosol enzymes could possibly explain the relative percentages found. Markland and Wadkins [15] purified from bovine liver mitochondria, which contained 20 of adenylate kinase activity, an acid- stable enzyme which was like the skeletal muscle enzyme in this regard but unlike the muscle enzyme in having tryptophan and cystine rather than cysteine present. Brdiczka et al. [28] showed that in rat liver mitochondria the adenylate kinase is localized between the outer and inner membrane of mitochondria and thus may easily leak out in spite of the use of sucrose solutions of the present experiments. While it may be invalid to assume that adenylate kinase from mitochondria of liver and heart are the same, the question of the identity of the acid-labile adenylate kinase in heart remains an unanswered question.

Khoo and Russell [5] proposed on the basis of antibody and sulfhydryl reactivity studies that in humans and rabbits there are at least two sets of adenylate kinase isozymes. One set (muscle type) is to be found in skeletal muscle, erythrocytes and brain and another set (liver type) is found in liver, kidney and heart. The results reported here indicate that the major adenylate kinase from porcine heart is like

that purified from porcine skeletal muscle with respect to acid stability, isoelectric point, molecular weight, amino acid composition and fingerprinting.

The authors wish to express thanks for the support of this work by United States Public Health Service Grant HL-03599. Grateful acknowledgment is made for the determination of fingerprint patterns by Dr Inge von Zabern.

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S. Kubo’s present address : Laboratory of Biochemistry, Faculty of Veterinary Science, Hokkaido University, Sapporo, Japan

L. H. Noda, Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, U.S.A. 03755


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Adenylate Kinase of Porcine Heart