The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The Levels of Creatine Kinase and Adenylate Kinase in the Plasma of Dystrophic Chickens Reflect the Rates of Loss of These Enzymes from the Circulation H.

DAVID

Department qf Biochemistry.

HUSK

AND

CLARENCE

H.

SUELTER

Michigun Stutr University. East Lansing, Michigan 48824 Received

June

16. 1982

Increased plasma levels of some muscle-associated enzymes are often used in the early diagnosis of individuals affected with muscular diseases and in the identification of carriers of genetically transmitted muscular diseases. Several muscle enzymes including creatine kinase (I), pyruvate kinase (I .2), aldolase (3), and glutamic-oxaloacetate transaminase (4) are elevated in plasma of patients with Duchenne muscular dystrophy. However, the levels of several other abundant muscle proteins including myoglobin (5), phosphofructokinase (5), and AMP aminohydrolase (6) are not increased. Serum adenylate kinase levels are increased in some individuals with the disease (7,8) but not to the extent of the other muscle enzymes noted above. Several explanations for the differences in the plasma activities of abundant muscle proteins are suggested in reviews by Pennington (9) and Rowland (10): (a) the sarcolemma may be differentially permeable to muscle proteins allowing some to pass while retaining others in a manner that may be dependent on size and charge; (b) muscle proteins complexed with intracellular structures may be retained in the muscle cell; (c) differences in the rates of inactivation or clearance of proteins from the circulation would regulate the levels in the blood plasma. A combination of these factors may regulate the circulating levels of muscle proteins in both normal and pathological states. Previously we compared rates of loss of some muscle enzyme activities from the blood plasma of line 413 (dystrophic) chickens to line 412 (normal) chickens (11). The rate of clearance of intravenously injected chicken muscle AMP aminohydrolase from the circulation of both lines of chickens was rapid with a half-life of only 3.3 min. On the other hand, the loss of pyruvate kinase was relatively slow with half-lives for the 318

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2’ 1983 by Academic Prea. Inc of reproduction in any form re\ervsd

CREATINEANDADENYLATE

KINASE LEVELS

319

biphasic loss of 110 and 710 min. We now report that plasma levels of adenylate kinase are not elevated in 4-week-old line 413 dystrophic chickens compared to line 412 chickens and that intravenously injected adenylate kinase is lost rapidly from the blood plasma. Comparison of enzyme activities in muscle press juices to activities in muscle crude homogenates indicates that insufficient adenylate kinase is associated with insoluble intracellular components to prevent the release of the enzyme from muscle cells. When creatine kinase, which is elevated in plasma of line 413 chickens compared to line 412 chickens (11,12), is injected intravenously, its activity is lost relatively slowly and at about the same rate from plasma of both line 412 and 413 chickens. These results and those reported previously (11) support our suggestion that the rates of loss of muscle enzyme activities from the circulation are important in determining the levels of muscle proteins in the blood plasma of normal and dystrophic animals. MATERIALS

AND METHODS

Materials Animals and tissues. Frozen chicken breast muscle was obtained from Pel-Freez Biologicals, Rogers, Arkansas. Line 4 12 and 4 13 chickens were raised from fertile eggs obtained from the Department of Avian Sciences, University of California at Davis, Davis, California. All chickens used in these experiments were between 25 and 3.5 days of age. Reagents. All salt solutions were prepared with reagent-grade chemicals in distilled, deionized water. Phosphocellulose was from Whatman Inc., Clifton, New Jersey. Reagentgrade (NH&SO, from Schwarz-Mann. Inc., Spring Valley, New York was recrystallized from water before use. Aquacide III was from Calbiochem-Behring Corporation, La Jolla, California. Sephadex G-100 was from Pharmacia Fine Chemicals, Uppsula, Sweden. ‘%Na was from Amersham Corporation, Arlington Heights, Illinois. All substrates and coupling enzymes were from Sigma Chemical Company, St. Louis, Missouri except glucose which was from Mallinckrodt Chemical Works, St. Louis, Missouri. Methods Enzyme assays. Adenylate kinase activity was determined by coupling ATP production from ADP to NADP reduction with hexokinase and glucose-6-phosphate dehydrogenase, essentially as described by Oliver (13). The assay mixture contained 3 mM ADP, 5 mM glucose, 5 mM MgCl,, 0.7 mM NADP, 50 mM Tris, pH 7.6, 1.5 unit/ml yeast hexokinase, 1.5 unit/ml yeast glucose-6-phosphate dehydrogenase, and 0.1 mg/ml bovine serum albumin. The rate of increase in absorbance at 340 nm was monitored. Creatine kinase activity was measured by the procedure of Rosalki (14)

320

HUSIC ANDSUELTEK

using the assay system obtained from Sigma Chemical Company. Pyruvate kinase was assayed by coupling pyruvate production from phosphoenolpyruvate to NADH oxidation with lactate dehydrogenase as previously described by Bucher and P!Ieiderer (15). The rate of decrease in absorbance at 340 nm was monitored. AMP aminohydrolase was assayed by following the increase in absorbance at 290 nm as AMP is deaminated to form IMP (16). All assays were at 30°C and were initiated by the addition of 5 ~1 of enzyme to the 1.0-ml assay mixture. Enzyme purzjicatiuns. Adenylate kinase was purified from frozen chicken breast muscle by a modification of the procedure of Schirmer et al. ( 17). Thawed muscle tissue, 480 g, was homogenized in 1 liter 10 mM KCI. Centrifugation, pH fractionation, zinc acetate precipitation, and ammonium sulfate fractionation were as described previously (17). The precipitate obtained from the ammonium sulfate fractionation was extensively dialyzed against 10 mM imidazole, 10 mM /3-mercaptoethanol, pH 7.5, applied to a 8 x 2-cm column of phosphocellulose, and washed with the dialysis buffer until the A180 of the eluent was 0.06. Adenylate kinase was eluted with a linear gradient of O-15 mM potassium pyrophosphate in 10 mM imidazole, 10 mM /I-mercaptoethanol, pH 7.5. Each side of the gradient had a volume of 200 ml. Fractions with a specific activity greater than 400 units/mg protein were pooled and concentrated by precipitation of the enzyme by adding solid (NH,),SO, to 90% saturation (calculated at 0°C). The enzyme was resuspended and dialyzed against 0. I M imidazole, 10 mM /3-mercaptoethanol, pH 7.5, concentrated with solid Aquacide III, applied to an 80 x 2-cm Sephadex G-100 column and eluted with 0. I M imidazole, 10 mM fi-mercaptoethanol, pH 7.5. Peak fractions were concentrated by precipitating the enzyme with the addition of solid (NHJ)2S04 to 90% saturation. Adenylate kinase was stored at -20°C as a slurry in 50% saturated (NH,),SO, and 50% glycerol containing 10 mM imidazole, pH 7.5, and 10 mM p-mercaptoethanol. The enzyme was homogenous as judged by sodium dodecylsulfate-polyacrylamide gel electrophoresis and had a specific activity of 1630 ~molelminimg protein. Creatine kinase was purified to homogeneity from frozen chicken breast muscle by the procedure of Eppenberger et ~1. (18). Radioiodination of enzymes. Adenylate kinase and creatine kinase were radioiodinated as described previously by Martin et al. (19). The specific activities of “‘1-adenylate kinase and “?-creatine kinase were 52.4 and 14.2 &i/mg, respectively. Radiolabeled adenylate kinase and creatine kinase had 60 and 75%. respectively, of the initial enzymatic activity. Preparation of muscle crude extracts and blood samples. Muscle extracts and blood samples were collected and prepared as described previously (II). Samples were immediately chilled on ice and assayed for enzyme activities within 2 hr of collection.

CREATINE

AND

ADENYLATE

KINASE

LEVELS

321

Rates of loss of enzymes from the circulation. Prior to injection enzymes were dialyzed twice against 200 vol of phosphate-buffered saline (0.2 g/ liter KCl, 8 g/liter NaCl, 0.2 g/liter KH2P04, and 0.78 g/liter Na,HPO,). The rate of loss of creatine kinase was determined after injecting 0.50 ml of a solution containing 245 units/ml and 9.2 x 10’ cpm/ml ‘*‘I-creatine kinase per 200 g body weight. The rate of loss of adenylate kinase was determined after injecting 0.50 ml of a solution containing 71.0 units/ml and 8.6 x 10’ cpm/ml ‘251-adenylate kinase per 200 g body weight. Enzyme solutions were injected into the brachial vein of the wing and blood samples of about 0.25 ml were collected in heparinized tubes from the brachial vein of the wing opposite to that injected. Blood samples were collected before injection and at the time intervals after injection given in the Results. The samples were assayed for enzyme activity after brief low-speed centrifugation to remove red blood cells. Aliquots (50 ~1) were counted in a Beckman Biogamma counter. Rate constants for loss of enzyme activity and ‘*‘I following injection were calculated using a nonlinear curve-fitting computer program as described previously (11). Measurement

of the inactivation

of enzyme activity

in serum in vitro.

Blood was collected from chickens and allowed to clot 1 hr at room temperature. The clot was separated from the serum by centrifugation. and the serum placed on ice. To 200 ~1 serum in 1.5-ml polypropylene tubes, was added 5 ~1 1 M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), pH 7.5, and adenylate kinase or creatine kinase in phosphatebuffered saline to give final concentrations of enzyme similar to that expected in the plasma after intravenous injection experiments in vivo. Samples (5 ~1) were assayed before warming the tubes (t = O), then the tubes were incubated at 41°C (chicken body temperature) in a water bath and 5-~1 aliquots assayed at the time intervals given in the Results. Determination of the tissue distribution of 12’1-adenylate kinase. Thirty minutes after the injection of ‘251-adenylate kinase as described above, the chicken was decapitated. Tissues were weighed and aliquots were counted in a Beckman Biogamma Counter. Preparation of muscle press juices and muscle crude extracts. Press juices of normal and dystrophic muscle were obtained by placing the entire pectoralis muscle in a centrifuge tube, and centrifuging at 40,OOOg for 4 hr at 4°C. Crude extracts of the opposite pectoralis muscle were prepared in a high-ionic-strength buffer as described previously (11). Enzyme activities in the press juices and crude extracts were determined as described above and protein was determined by the method of Lowry et al. (20). The water content of normal and dystrophic muscle was determined from the difference in weight before and after lyophilization of muscle samples. Normal muscle is 77.3% water by weight and dystrophic muscle is 79.6% water by weight.

322

HUSlC AND SUELTER

RESULTS

Adenylate

Kinase Cleurancr

and Inactiwtion

The data in Table 1 show that adenylate kinase activity is not significantly elevated in the plasma of line 413 dystrophic chickens compared to line 412 normal chickens. These results suggest that either the enzyme is rapidly inactivated or cleared from the blood plasma, or that the enzyme is not released from the dystrophic muscle tissue. To determine whether a rapid loss of enzymatic activity from the circulation could account for the data in Table I, the rates of loss of chicken muscle adenylate kinase activity from the circulation of line 412 normal and line 413 dystrophic chickens were determined after intravenous injection of the homogeneous chicken breast muscle enzyme. Figure 1 shows a rapid loss of adenylate kinase activity from the blood plasma in a monophasic exponential decay with half-lives of 4.6 and 5.3 min in line 412 and 413 chickens, respectively (Table 2). To determine whether inactivation and/or clearance are responsible for the rapid loss of adenylate kinase, we measured the rates of inactivation of adenylate kinase in serum in vitro and the loss of “‘I-adenylate kinase after intravenous injection in line 412 and 413 chickens. Figure 2 (solid circles) shows a significant rate of loss of adenylate kinase activity when incubated in serum in vitro at 41°C. This rate of loss was decreased significantly when 10 mM dithiothreitol was added to the serum (Fig. 2, open circles). Thus, the loss of adenylate kinase activity in the circulation after injection is due at least in part to the oxidation of sulfhydryl groups essential for catalytic activity, and is consistent with reports that show human (21.22). porcine (23). and rabbit (24) muscle adenylate kinases are inactivated by agents which modify sulthydryl groups. The loss of intravenously injected “‘1-adenylate kinase from the cirTABLE ACTIVITIES

Plasmah (units/ml)

Chicken line 412 (Normal) 413 (Dystrophic) Pd

I

OF ADENYLATE KINASE IN THE BLOOD PLASMA AND BREAST MUSCLE ~-WEEK-OLD DYSTROPHIC CHICKENS”

0.13 0.14

k 0.07 (8) k 0.07 (7) >0.5

Breast muscle” (units/g) 13.9 k 0.8 (4) 11.5 i 1.2 (4) <0.025

OF

Percentage of total activity in plasma’ 0.62 0.81

" 0.18 k 0.30 co.2

” The results are expressed as the average r S.D. ’ The number of chickens tested is in parentheses. ’ These values were calculated assuming a plasma volume equal to 6.0% of the total body weight using measured values of body and breast muscle weight. ’ Determined by Student’s f test.

CREATINE

AND ADENYLATE

KlNASE

323

LEVELS

FIG. 1. The loss of intravenously injected adenylate kinase activity from the blood plasma of normal line 412 (0) and dystrophic line 413 (A) chickens as described in Methods. Adenylate kinase in the plasma prior to injection was subtracted from all values before plotting. The curve was calculated from the average of the parameters given in Table 2 using the equation: units/ml = Ae- 9 where A is the units/ml enzyme activity in the plasma expected at the time of injection.

TABLE 2 RATES OF DISAPPEARANCEOF ADENYLATE KINASE ACTIVITY AND “‘1-ADENYLATE KINASE FROMTHE BLOOD PLASMA OF NORMAL AND DYSTROPHICCHICKENS

[r112Onin)

k

96 Lost in rapid phase

0.15 ? 0.03

-

100

-

100

tliZ > 1 hr

87 2 I

I,,: > 1 hr

9024

k,

Enzyme Adenylate kinase

Chicken type” 412 Normal (6)

(min-‘)’

14.61 Adenylate kinase

413 Dystrophic (5)

‘*‘I-Adenylate kinase 12’1-Adenylate kinase

412 Normal (4)

0.13 * 0.02 f5.31 0.33 2 0.17

v.11 413 Dystrophic (3)

0.24 2 0.06

WI

a The number of replications is given in parentheses. A separate chicken was used for each experiment. b Rate constants were calculated by fitting the data to an exponential decay function with a nonlinear curve-fitting computer program as described previously (1 I).

324

HUSIC AND SUELTER

time (mid FIG. 2. The loss of adenylate kinase activity in chicken serum in vitro at 41°C. One unit of adenylate kinase is incubated in 200 /.d normal chicken serum containing 10 mM Hepes, pH 7.5. and IO mM dithiothreitol (0). or containing 10 mM Hepes, pH 7.5 (0).

culation is shown in Fig. 3. The loss of ‘251-adenylate kinase is biphasic; the half-lives for loss of 12?-adenylate kinase during the first phase are 2.1 and 2.9 min in line 412 and 413 chickens, respectively. About 10% of the ‘*‘I is cleared very slowly with a half-life greater than 1 hr. The tissue distribution of ‘251, 30 min after injection of ‘*‘I-adenylate kinase, is shown in Table 3. The largest portion of the “‘1 is recovered in muscle tissue; however, the spleen and liver have the highest concentration. Despite the high concentration of ‘*‘I in the spleen and liver, no increase

FIG. 3. The loss of intravenously injected “%adenylate kinase from the blood plasma of normal line 412 (0) and dystrophic line 413 (A) chickens as described in Me&hods. The curve drawn is calculated from the average of the parameters given in Table 2 using the equation cpm = Ae-*f’ + Be-“2’ where A and B are the amounts of radioactivity lost in the rapid and slow phases, respectively.

CREATINE

AND ADENYLATE TABLE

TISSUE DISTRIBUTION

OF “‘I-ADENYLATE

KINASE

325

LEVELS

3

KINASE

30 MIN AFTER INTRAVENOUS

Tissue

Percentage of injected radioactivity recovered

Spleen Liver Gizzard Kidneys Lungs Leg, back, and breast muscles Leg, back, and breast bones Excrement All other tissues Total

1.3 15.9 2.2 2.9 2.3 28.1 16.6 8.5 20.5” 98.3

INJECTION

Percentage of injected radioactivity recovered per gram tissue 3.4 2.3 1.5 1.4 1.1 0.6 0.6

-

~0.6

’ No other single tissue contained more than 0.6% of the injected radioactivity per gram.

in adenylate kinase enzymatic activity was observed in homogenates of these tissues 30 min after injection (data not shown). Thus adenylate kinase is both inactivated and cleared from the circulation. Creatine Kinase Clearance

The loss of intravenously injected creatine kinase activity from the circulation of line 412 and 413 chickens is shown in Fig. 4. The clearance of creatine kinase activity fits a biphasic exponential loss with half-lives of 1.4 and 12 hr for the two phases in line 412 chickens, and 0.69 and 9.9 hr in line 413 chickens (Table 4). These half-lives for clearance are much longer than the half-life of about 5 min observed for the loss of adenylate kinase activity from the circulation of line 412 and 413 chickens (Table 2). Creatine kinase is cleared at similar, or slightly more rapid rates in line 413 chickens compared to line 412 chickens. Therefore, elevated creatine kinase activity in the plasma of line 413 chickens compared to line 412 chickens is not due to a decreased rate of clearance or inactivation of the enzyme in the circulation of the line 413 chickens compared to line 412 chickens. When creatine kinase is incubated at 41°C in vitro in serum from line 412 or 413 chickens at concentrations similar to those obtained after intravenous injection, 17% of the activity is lost in line 412 serum, and 27% of the activity is lost in line 413 serum after 22 hr. Of the endogenous creatine kinase activity 21% is lost in line 413 serum after 22 hr at 41°C. These in vitro rates of inactivation of the enzyme are relatively slow

326

I-WSIC AND SCELfEK

-- -~_..~-.

r---

I

200

I

400

I

I

600

800

I

looo

I

I200

TIME: AFTER fNJECTloN MfW FIG. 4. The ioss of intravenously injected creatine kinase activity from the blood plasma of normal line 412 (i)) and dystrophic line 413 (A) chickens as described in Methods. Creatine kinase activity in the plasma prior to injection is subtracted from all values before plotting. The curve was calculated from the average of the parameters given in Table 4 using the equation units/ml = Aem’+ + Be-Lz1 where A and 8 are the amounts of the enzyme activity lost in the rapid and slow phases, respectively.

TABLE 4 RATES OF DLSAPPEARANCE OF CREATINE KINASE ACTIWY AND '251-C~~~~~~~ KINASE FROM THE BLOOD PLASMA OF NORMAL AND DYSTROPUKC CNICKENS~ -._l_l.--_____-l____---“~” - -kz (hr-“1 Chicken type’ k, (hr-‘) % Lost in Enzyme rapid phase ftti2 (hr)l [tlil (W ~~._..._l_---l-~.l.-.-.___ .-.0.057 It 0.025 Creatine 412 Normal (9) OS1 z 0.22 Sl + 18 kinase I121 11.41 Creatine 1.0 ? 0.9 44 + 21 4I3 Dystrophic (71 0.070 r 5.03I kinase lO.691 f9.91 “‘I-Creatine 412 Normal (4) 1.58 + O,fS 0.14 f 0.01 74 i 2 kinase f5.441 IS.51 ’WZreatine 413 Dystrophic (3) 1.67 k 0.54 0.12 rt 0.01 77 I 4 kinase [0.42] IS.81

-~

-.---

“lll”,l.ll-.-,- ~------

’ The rate constants are expressed as the average t standard deviation as described in footnote a of Table 2. * The number of chickens tested is in parentheses.

CREATINE

AND ADENYLATE

KINASE

LEVELS

327

compared to the loss of 88% of the injected creatine kinase activity 22 hr after intravenous injections (Fig. 4). The loss of lz51-creatine kinase after intravenous injection is shown in Fig. 5. These data also fit a biphasic exponential loss with half-lives of 0.44 and 5.0 hr in line 412 chickens and half-lives of 0.42 and 5.8 hr in line 413 chickens (Table 4). The loss of ‘251-creatine kinase was more rapid than the loss of creatine kinase enzymatic activity in both line 412 and 413 chickens, but was still much slower than the loss of “‘1-adenylate kinase or adenylate kinase activity. Enzyme Activities

in Muscle Press Juices

To estimate the relative extent of association of several muscle enzymes with intracellular structures, we measured the activities of these enzymes in breast muscle press juices and compared them to the total enzyme activities in high-ionic-strength crude breast muscle extracts (Table 5). The centrifugation method for the preparation of muscle press juices (25) results in the disruption of the sarcolemma, and the resulting press juice presumably contains proteins free in the sarcoplasm. Myofibrillar proteins, and those proteins associated with the myofibrils, are retained in the muscle tissue. On the other hand, muscle crude extracts prepared by

TIME AFTER INJECTKIN(MINI FIG. 5. The loss of intravenously injected %creatine kinase from the blood plasma of normal line 412 (0) and dystrophic line 413 (A) chickens as described in Methods. The curve is calculated from the average of the parameters given in Table 4 using the equation cpm = A~-‘I’ + Be-% where A and B are the amounts of radioactivity lost in the rapid and slow phases, respectively.

OF Press

Jrww

Chicken tine .~-.l^“-” ,... 412 (5) 413 (4) 412 (51 413 (4) 412 (5) 413 14) 412 (5) 413 (4)

AND PROTJSFI CONTENT ._____.-.--_ ;I

TABLE

5

mgiml press juice

AND DYSTRCJPHK

mglml muscle H@ (muscle homogenate)

Units/ml muscle H,O tmuscle homogenates - ..-_*--_. _ _..--... .-.___-, 107 z!z 31 111 t 25 2080 2 310 1890 z!z 556 783 I? 229 398 rt 175 263 i: 92 looz!z 27

EXTRACTS FROM NORMAL

Unitsimf press juice .-- --,_ --. _. IS4 rt 26 161 ic 41 1930 2 190 1730 -‘- loo 1420 r 430 448 F 128 104 + 36 24.9 rt: 3.4

AND CRUDE

CHKKEN

mgjmi press juice mglml muscle H,O tmuscie homogenate)

Units;ml press juice unitsiml muscle HZU rmuscte homogenates ,, 1.6 2 0.4 I.5 t 0.3 I.0 ir 0.2 I.0 f 0.3 2.0 i 0.3 1.3 i 0.7 0.43 i 0.12 0.19 2 0.09

BREAST MUSCLE” ..-_

103 rf: 11 72.1 z+z 8.0 1.43 I 0.28 69.2 r 16.2 56.1 2 13.3 1.30 f 0.47 ..--. I__. .-.._-..-.l_ . _.. .-.-.-. .. number of chickens in parentheses. The ratios of the levels of component\ in the for each chicken and averaged to give the values shoun: occasionally these ratiu\r average activities in the press juice and musde given in this table.

,_

412 (5) 413 (4) _-.---. .--_l_lll ““”.-. .--^._ --._--._ . ” The results are expressed as the average i SD for the press juice to that in the muscle were calculated separately differ slightly from the ratios obtained if calculated from the

Protein

AMP aminohydrolase

Pyruvate kinase

Creatine kinase

Adenylate kinase

Component

--

ENZYME ACTIVITIES _--- .-_--“.-~

g t: cn c m t-.A r-l+ ic

CREATINEANDADENYLATEKINASE

LEVELS

329

the homogenization of tissue in a high-ionic-strength buffer solubilizes myofib~lar and associatedproteins. Therefore, we assumethat an enzyme with a low ratio of enzyme activity in the press juice to that in the highionic-strength crude muscle extract is associatedwith intracellular structures to a greater extent than an enzyme with a higher ratio. These ratios are calculated as the ratio of the units/ml in the press juice to the units/ml muscle water in the crude extract. Examination of the ratios allows a direct comparison of the relative extent of intracellular association of different muscle enzymes to ultrastructural components in the crude extract. A 4 hr centrifugation of intact muscle tissue results in about 100 &I press juice per gram breast muscle. Amberson et al. (25) obtained 250-300 ~1 press juice per gram white muscle only after 20-24 hr of cent~fugation, and this quantity is only about 40% of the total muscle water. The complete removal of water from muscle cannot be achieved by this procedure, so we have avoided long centrifugation times to minimize the potential changes in the extent of intracellular association of one enzyme with time after the dissection of the muscle from the chickens. Because of the inability to remove all water from the muscle by this procedure we expressed the activities of muscle enzymes in crude muscle extracts per gram muscle water. if the enzymes are accessible to all water in the muscle, then the ratio of units/ml press juice to units/ml muscle water is an estimate of the fraction of the enzyme associated with intracellular components, and should be one for an enzyme which is not appreciably bound to ultrastructural components. Yet several of the values in Table 5 are greater than one. Values greater than one suggest that the enzyme is compartmentalized in the water released by centrifugation. This suggestion is supported by the results in Table 5 which show that the protein content of the press juice is 1.43 and 1.30 times that in the crude homogenate in line 412 and 413 chickens, respectively, when the protein content in the crude homogenate is expressed as mg protein/ml muscle water. The ratios of adenylate kinase activity in the press juice to the activity in muscle tissue are as high, or higher than the ratios for pyruvate kinase and creatine kinase, enzymes which are elevated in the plasma of dystrophic chickens compared to normal chickens (11,12,26). The ratio of the activity in the press juice to that in the muscle for creatine kinase is lower than for pyruvate kinase or adenylate kinase, suggesting that a greater percentage of the total creatine kinase is associated with intracellular structures. Of all the enzymes examined AMP aminohydrolase is associated with intracellular structures to the greatest extent. DISCUSSION Adenylate kinase activity, like AMP aminohydrolase activity (II), is cleared more rapidly than creatine kinase and pyruvate kinase (1I) activity

330

HUSIC‘

AND

SUELTF,K

from the circulation of both normal and dystrophic chickens following intravenous injection. These results are consistent with the observation that creatine kinase is elevated in the plasma of line 413 (dystrophic) chickens compared to line 412 (normal) chickens ( I I, 12). and that adenylate kinase is not elevated (Table I ). These data are consistent with our thesis that the levels of muscle enzyme activities in the plasma of dystrophic chickens inversely reflect the rates of loss of these enzymes from the circulation. The results presented here and previously (11) demonstrate the capacity of chickens to rapidly remove adenylate kinase and AMP aminohydrolase from the circulation after intravenous injection of purified enzymes. However, elevated levels of these enzymes are not observed in the plasma of dystrophic chickens, and thus we have no direct evidence that these enzymes are released from dystrophic chicken muscle at rates comparable to creatine kinase and pyruvate kinase. The mechanism by which muscle proteins are released into the blood plasma of dystrophic animals is not known. If the loss of enzymes into the blood is the result of loss from necrotic muscle fibers, then all soluble muscle constituents would be released into the blood at similar rates. On the other hand, the efflux of proteins through abnormally permeable muscle membranes into the circulation could be regulated by the size and charge of the protein as well as by the extent of association of the protein with intracellular components, However, as reviewed by Rowland (IO), there is no apparent correlation between molecular size and plasma levels of muscle enzymes in human dystrophies. Our results with normal and dystrophic chickens offer an explanation for this lack of a correlation. The rapid rate of clearance of both muscle adenylate kinase with a molecular weight of about 21,000 (27) and AMP aminohydrolase with a molecular weight of 276,000 (28) reduces the steady-state levels of both of these enzymes in the plasma of dystrophic chickens to low levels. Creatine kinase with a molecular weight of 80.000 (29) and pyruvate kinase with a molecular weight of 212,000 (30) are cleared relatively slowly from the circulation, and the activities of these enzymes are markedly elevated in the plasma of dystrophic chickens compared to normal chickens (I 1,12,26). These observations show no apparent correlation between molecular size and plasma levels or clearance rates in dystrophic chickens. That dystrophic muscle has an increased permeability to all soluble muscle constituents is supported by the data of Dawson (31) showing the same rates of release of several enzymes including creatine kinase and adenylate kinase from dissected chicken breast muscle into physiological saline; the rate of efflux of all enzymes from dystrophic muscle is larger than the rate from normal muscle. These results are consistent

CREATINE

AND

ADENYLATE

KlNASE

LEVELS

331

with the data in Table 5 showing the lack of a significant association of adenylate kinase with intracellular structures compared to creatine kinase or pyruvate kinase. Since the activities of creatine kinase and pyruvate kinase are elevated in dystrophic chicken plasma compared to normal chicken plasma, the low activity of adenylate kinase in the plasma of dystrophic chickens compared to the activities of creatine kinase and pyruvate kinase cannot be explained by extensive association of the enzyme with intracellular components. The observation that creatine kinase is associated to a greater extent than adenylate kinase or pyruvate kinase is consistent with the association of creatine kinase with myofibrillar proteins reported by Wallimann et al. (32). Pyruvate kinase is also associated with myofibrillar proteins in low-ionic-strength extracts of muscle tissue (33), though our results suggest the extent of this interaction in viva is small. On the other hand, AMP aminohydrolase appears to be associated with intracellular components to a significant extent in viva. This is consistent with the binding of AMP aminohydrolase to subfragment2 of myosin (34), and throughout the A bands in muscle (35). This association, as well as the rapid circulatory clearance rate of AMP aminohydrolase (1 l), may contribute to low activities of this enzyme in the plasma of dystrophic chickens. The rapid clearance of adenylate kinase, the dithiothreitol-inhibited inactivation of adenylate kinase in serum in r&o, and the inability to observe increased adenylate kinase enzymatic activity in the liver and spleen shortly after injection, suggest that the enzyme is either inactivated by oxidation of sulfhydryls essential for catalytic activity and is then cleared, or that the enzyme is rapidly inactivated after clearance from the circulation. We examined the rates of clearance of radioiodinated enzymes as well as the clearance of catalytic activity to assess the roles of circulatory clearance and inactivation in the circulation on the loss of intravenously injected enzymatic activity. The data in Tables 2 and 4 show that the radioiodinated enzymes are cleared at similar, but significantly more rapid rates than the rates of loss of catalytic activity for both adenylate kinase and creatine kinase. If the radiolabeled enzymes are cleared in a manner identical to the loss of catalytic activity, then the rates of clearance of radioiodinated enzymes should not exceed the rates of clearance of catalytic activity. Apparently the iodination procedure modified the enzymes in such a way that the enzymes are more rapidly cleared. The physical properties of the radioiodinated enzymes were not examined extensively, however, both adenylate kinase and creatine kinase lost some enzymatic activity as a result of the iodination procedure (see Methods). Whether the mechanism for the clearance of the radioiodinated enzyme differs from that responsible for the loss of catalytic activity from the circulation

332

HCISI<' AND SUEL-I'EK

is not known. If the mechanism of clearance is different, then the tissue distribution of cleared ““I-adenylate kinase given in Table 3 may not be an accurate reflection of the clearance of unmodified adenylate kinase. Furthermore, because of the questionable validity of the radiolabeledenzyme clearance data the significance of circulatory clearance and inactivation on the rates of loss of catalytic activity after intravenous injection remains unclear. Despite the rapid loss of muscle adenylate kinase following intravenous injection, the ratio of adenylate kinase activities in the plasma to that in muscle in both normal and dystrophic chickens (Table I), are comparable to those of slowly cleared muscle proteins in normal chickens (I I). Perhaps adenylate kinase activity in normal and dystrophic chicken plasma is due to adenylate kinase isozymes from tissues other than muscle which are more slowly inactivated and/or cleared. Adenylate kinase isozymes from nonmuscle tissues in humans are not inactivated by sulfhydryl-modifying agents (21,22). Hamada rt al. (8) reported that the increased adenylate kinase activity in the serum of patients with Duchenne dystrophy is due to an “aberrant” form of the muscle isozyme which is not inactivated by sulfhydryl-modifying agents but is precipitated by antibodies to muscle adenylate kinase. The rate of loss of chicken muscle creatine kinase from chicken blood is more rapid than the loss of dog muscle creatine kinase from the circulation after intravenous injection into dogs (36). Kotuku rt al. (36) reported a half-life for the rapid phase of loss of 4.5 hr. The rate of the slower phase was not determined. Dog brain creatine kinase activity was lost more rapidly with a half-life of 1.5 hr. The loss of creatine kinase activity from the circulation in humans following myocardial infarction has a half-life of about I4 hr (37); however, the primary creatine kinase isozyme in human heart is different from the skeletal muscle isozyme (38) and may be cleared at a different rate. Furthermore, rates of loss following myocardial infarction are difficult to interpret because the precise time at which enzyme ceases to be released from the tissue cannot be determined. The direct measurement of plasma levels and circulatory clearance rates for muscle creatine kinase activity reported here provide the parameters necessary to estimate the rate of efflux of creatine kinase from dystrophic chicken breast muscle. The reasoning and assumptions necessary to calculate the rate of efflux are discussed below. The biphasic behavior of the loss of creatine kinase activity from the circulation following intravenous injection can be discussed in terms of the scheme shown in Fig. 6 which was previously suggested by Boyd (39). For most enzymes the rate of loss of enzyme activity following intravenous injection follows a biphasic exponential model (40). Presum-

CREATINE

AND ADENYLATE

KINASE

LEVELS

333

bo

FIG. 6. A hypothetical model for the distribution of enzymes between body fluids (29).

ably, the rapid phase is due to the distribution of the injected enzyme between the intravascular and extravascular spaces (kzj and kjz). For the purpose of this discussion, intravascular space refers to the blood plasma. It is the loss of enzyme from this space that is measured in the experiments reported here. The extravascular space is the sum of all of the spaces where an enzyme can reversibly equilibrate with the intravascular space, and includes equilibration with the interstitial fluids of all tissues and organs, and the reversible binding to these tissues. The second, slower phase is attributed to the rate of irreversible elimination or inactivation of the enzyme (i& and k3,,). The steady-state concentration of an enzyme in the intravascular compartment depends on the difference between the rate of efflux of the enzyme from muscle (k,J and the rate of irreversible loss of enzyme from the intravascular (kzo) and extravascular compartments (k,,). If the sum of k,, and k3,, is large compared to klz, the steady-state concentration of the enzyme in the intravascular and extravascular compartments will be small or negligible depending on the magnitude of the differences. This may be true in the cases of adenylate kinase and AMP aminohydrolase (11) since these proteins are rapidly cleared and plasma elevations of these enzymes are not observed. The increased steady-state concentrations of creatine kinase (11,12) and pyruvate kinase (11,26) in the plasma of dystrophic chickens would then be explained by a rate of inactivation or clearance (kzo and k3”) that is not large compared to the rate of eftlux from muscle tissue (k,,). The values of several of the rate constants shown in Fig. 6 were calculated as suggested by Boyd (39) from the data for the clearance of intravenously injected creatine kinase (Table 6). For this calculation we

334

HUSK

RATE CONSTANTS

Chicken

line”

412 (9) 413 (7) ” h ’ ”

The k,,, kZ, k,,

AND

SUELTtK

TABLE h FOR THE DISTRIRC~TI~N OF CREATINE INJECTION OF CREATINE k,, (hr 0 0

‘)

X,,, (hr

KINASF AC.TIVITY KINASE”

I)”

0. I I ” 0.05 0.16 ? 0.07

XI, thr 0.28 0.52

‘)

+- 0.12 k 0.48

AFTER INTRAVENOUS

X,, (hr 0.18 0.41

I)”

-e 0.12 k 0.40

results are expressed as the average & SD for the number of chickens in parentheses. = k,kz/kz,. = A (k, -Q + kL where A is the fraction of the enzyme lost in the rapid phase. = k, + k, - k,, - k ,,,.

assume that klz is constant and irreversible. Also since the inactivation of creatine kinase incubated in serum is slower than the loss of activity after intravenous injection, we assume kzo = 0. These results suggest that kXO, kZ3, and k32 are increased in the dystrophic chickens when compared to normal chickens, however the error associated with these values is large. Using the assumptions made by Boyd (39). the rate of efflux (kJ of creatine kinase from dystrophic muscle can be estimated from the relation k

I?

= Cz Vz k, kz k 32

where CZ is the steady-state concentration of the enzyme in a blood volume (Vz), and k, , kl, and kj2 are the rate constants given in Tables 4 and 6. If we assume all of the plasma creatine kinase in dystrophic chicken is from breast muscle, using measured values of 4-week-old line 413 chicken breast muscle weight (25 g), muscle creatine kinase levels (1010 units/g), plasma creatine kinase activities of 9.3 units/ml (ll), a plasma volume of 6.0% of the body weight (41), and the rate constants for loss of enzyme activity from the circulation reported here; only 2.0% of the breast muscle creatine kinase would need to be released from muscle to the circulation daily to maintain the observed plasma activity of creatine kinase in dystrophic chickens. The assumption that the plasma creatine kinase is solely from breast muscle is a valid approximation since primarily white-fiber muscle is affected by dystrophy in the chicken (42), and the breast muscle comprises most of the total white-fiber muscle. SUMMARY

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and

CREATINE

AND ADENYLATE

KINASE

LEVELS

335

dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day. ACKNOWLEDGMENTS This work was supported in part by grants from the Muscular Dystrophy Association of America and National Institutes of Health GM 20716. This is Michigan State Agricultural Experiment Station Journal Article 10461. We gratefully acknowledge the assistance of the Department of Animal Science, Michigan State University, particularly Ms. Bridget Grala, Dr. Robert K. Ringer, and Dr. Donald Polin. and the Department of Avian Sciences, University of California at Davis, particularly Mr. Fayne Lantz.

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