adenosine triphosphate-adenosine-5' …8.6) in a corning cassette electrophoresis cell at 90 v...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 21, Issue of November 10, pp. 13120-13126, 1982 Printed m U.S.A.

Adenosine Triphosphate-Adenosine-5'-monophosphate Phosphotransferase from Normal Human Liver Mitochondria ISOLATION, CHEMICAL PROPERTIES, AND IMMUNOCHEMICAL COMPARISON WITH DUCHENNE DYSTROPHIC SERUM ABERRANT ADENYLATE KINASE*

(Received for publication, May 6, 1982)

Minoru HamadaSg, Michihiro Sumidan, Hiromichi Okudan, Tsutomu WatanabeS, and Motoo Nojimall From the Departments of $Hygiene, IBiochemistry, a n d IIOrthopedics, Ehime University School of Medicine, Shigenobu-cho, Onsen-gun, Ehime 791-02 J a p a n

Stephen A. KubygSS From the Laboratory for the Study of Hereditary a n d Metabolic Disorders a n d the Departments of Biological Chemistry and Medicine, University of Utah, Salt Lake City, Utah 84132

Adenylate kinase has been purified approximately 1360-fold to a final specific activity of 280 pmol of ATP formed min-'*mg" of protein at 30 "C from normal human liver mitochondria. The purity of the final preparation was evaluated by studies with polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide, gel electrophoresis and by sedimentation studies. The purified enzyme catalyzes transphospho- rylation reactions between adenosine triphosphate and adenosine monophosphate, ATP and adenosine-5"thiophosphate, ATP and adenosine monophosphate-3'-pyrophosphate, adenosine-5'-(3-thio)triphosphate and AMP. The nearly constant ratios of these activities throughout the purification scheme suggest that all are catlayzed by the same enzyme.

The purified enzyme has a molecular weight of 25,200 by sedimentation equilibrium with the use of a partial specific volume of 0.73 ml-g" calculated from amino acid analysis. This purified enzyme was also found to be a single polypeptide with a molecular weight of 26,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From amino acid analysis, a calculated minimum molecular weight of 26,349 was obtained.

Initial velocity studies revealed a narrow specificity for adenine nucleotides. The Kc values for MgATP2- and MgATP2-yS' were 0.12 and 0.57 p~ with vzKard values of 1.04 (20.04) X lo3 and 7.02 X lo2 pmol- min" mg", respectively. For the monophosphate acceptor, Kc values of 0.56 and 186 p~ were measured for 5'-AMP2- and AMP2-&, respectively. The K d , for MgADP" and ADP3- were 0.53 and 0.17 p~ with a CE"" of 6.40 (k0.03) X lo2 pmol*min"*mg" of protein. The steady state kinetics, a t pH 7.4, 30 "C, and essentially fixed r / 2 of 0.16-0.18, of this enzyme seem

* This work was supported by the Scientific Research Fund of the Ministry of Welfare and Education of Japan and by a grant from the Ehime Prefectural Medical Fund Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertise- ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

9 To whom reprint requests should be addressed. $$Supported by grants from the National Institutes of Health,

United States Public Health Service and the Muscular Dystrophy Association.

' The abbreviations used are: ATPyS, adenosine-5"(3-thio)triphosphate, AMPaS, adenosine-5'-thiophosphate. ADPo, for example, imp ies total concentrations of adenine nucleotide.

to be adequately expressed by a random quasi-equilibrium type of mechanism with a rate-limiting step largely at the interconversion of the ternary complexes, as shown in rabbit muscle, calf muscle, and calf liver adenylate kinase (Hamada, M., and Kuby, S . A. (1978) Arch. Biochem Biophys. 190,772-792). It would appear that normal human liver mitochondrial adenylate kinase largely favors the forward reaction (ADP formation). A specific anti-liver enzyme antibody obtained from rabbit serum inhibited the purified liver mitochondrial enzyme activity, but not the purified human muscle enzyme, nor the aberrant adenylate kinase from Duchenne dystrophic serum (Hamada, M., Okuda, H., Watanabe, T., Ueda, K., Nojima, M., Kuby, S. A., Man- ship, M., Tyler, F. H., and Ziter, F. A. (1981) Biochim Biophys. Acta 660,227-237).

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme which occurs a t high activity levels in many living cells, including the human erythrocyte (1). The enzyme catalyzes an interconversion of the adenine nucleotides, thus providing a unique buffering role against rapid concentration changes of any one component of this pool. The enzyme also serves as a primary regulating agent of those reactions in which the adenine nucleotides may partic- ipate as a substrate, activator, or inhibitor. Adenylate kinases have been prepared in either crystalline or apparently homogeneous form from rabbit (2,3), porcine (4), and rat (5) muscle, erythrocyte (6 ) , porcine heart (7) , and bovine (8), calf (3), and rat (5, 9) liver. Studies on the intracellular distribution of adenylate kinase showed it to be present in the nuclear, mitochondrial, microsomal, and supernatant fractions, al- though the major fractions of the total activity of liver tissues appears to be associated with the mitochondrial fraction.

Adenylate kinase from rabbit skeletal muscle was first crys- tallized by Noda and Kuby (2), who showed it to have a molecular weight of approximately 21,000 and to contain two free sulfhydryl groups, an observation which is consistent with the sensitivity of this enzyme to such reagents as silver and mercury.

Chiga and Plaut (10) described the partial purification of adenylate kinase from aqueous extracts of swine liver and noted marked differences from the skeletal muscle enzyme in substrate specificity as well as its insensitivity to sulfhydryl reagents. These differences in the catalytic properties of skel-

13120

Adenylate Kinase from Human Liver Mitochondria 13121

etal muscle and liver adenylate kinase suggested the possible occurrence of tissue-specific forms of this enzyme and led to a detailed examination of the molecular properties of the bovine liver mitochondrial enzyme (11).

The presence of an aberrant adenylate kinase in Duchenne dystrophic serum (12) and its similarity t o the normal liver enzyme prompted this study.

This report describes the isolation of a highly purified, apparently homogeneous form of adenylate kinase from normal human liver mitochondria. Its amino acid composition and sedimentation and kinetic properties have been determined. Some specific properties of this enzyme and a compar- ative s tudy with t h e aberrant enzyme from Duchenne dystrophic serum are also given in this report. A preliminary report on some aspects given below has been presented (38).

EXPERIMENTAL PROCEDURES

Materials-Reagent-grade chemicals were purchased from Sigma, Nakarai Pharmaceutical Co. (Kyoto), Boehringer Mannheim, Ya- manouchi (Tokyo), and Wako Chemical Works (Osaka). Enzymes used in the coupled enzyme tests (glucose-6-phosphate dehydrogenase, hexokinase, pyruvate kinase, and lactate dehydrogenase) and substrates (ADP, AMP, ATP, NADP', NADH, phosphoenolpyruvate, and glucose) were obtained from P-L Biochemicals Inc., Sigma, and Boehringer Mannheim. ATPyS and AMPaS were purchased from Boehringer and adenosine monophosphate-3"pyrophosphate from Sanraku-Ocean Co., Ltd. (Tokyo). Molecular weight marker proteins used were acquired from Sigma and ICN Nutritional Bio- chemicals. Acrylamide, Bisacrylamide, and N4-tetramethylethylene- diamine were purchased from Nakarai Pharmaceutical Co. (Kyoto). High capacity phosphocellulose P-11 was obtained from Whatman; Sephacryl S-300 and Sephadex G-100 were obtained from Pharmacia. Human tissues were obtained at autopsy and were kept in an ice-bath after removal.

Triple distilled water was used in all experimental work after degassing by boiling or by flushing with NS gas.

Methods-Phosphocellulose cycled by the method of Peterson and Sober (13) was equilibrated with 0.01 M Tris-C1- buffer, pH 7.4, before column packing. Sephadex G-100 was allowed to swell in 0.01 M Tris- CI- buffer, pH 7.4, for 24 h; it was then washed repeatedly with the equilibrating buffer until the supernatant liquid was completely clear. Sephacryl S-300 was equilibrated with the same equilibrating buffer and columns were packed in 5-cm sections under pressure (4 p.s.i.) and washed with IO-column volumes of the equilibrating buffer.

Enzyme Assay a n d Kinetic Studies-The initial velocity of the forward reaction (MgATP*- + AMP2- + MgADP" + ADP3-) was measured by a spectrophotometric procedure for ADP0 employing pyruvate kinase and lactate dehydrogenase. The reaction mixture (1.0 ml) consisted of 10 mM Tris-C1-, pH 7.4, a t 30 "c , 150 mM NaCI, 5 mM dithioerythritol, 0.1 mM EDTA, 0.25 mM phosphoenolpyruvate for the liver enzyme (but 5 mM for the muscle enzyme), 20 mM KCI, 0.5 mg.rn1-I of bovine serum albumin, 1 mM MgC12, 0.2 mM NADH, 20 units of lactate dehydrogenase, 10 units of pyruvate kinase, plus varying and calculated amounts of MgAcz, AMPo, and ATPo for the kinetic studies. Calculations employed a fixed concentration of free magnesium of 1 mM Mg"+ for the forward reaction. The reaction was initiated by the addition of suitably diluted adenylate kinase (dilutions in ice-cold 1 mM dithioerythritol, 1 mM EDTA, pH 7.5, with 1 mg- ml" of albumin). The final ionic strength averaged approximately 0.18. Measurements of the decrease in absorbance at 340 nm were made with a Cary 219 spectrophotometer thermostatted at 30 "C. In this case, the activity was expressed as the rate of ATP0 disappearance where % X (~L43~0/6.22) = pmol of ATPo disappearance.

Measurements of the initial velocity of the reverse direction (MgADPI- + ADP3- + MgATP2- + AMP2-) were made by a coupled enzyme assay for ATPo. The reaction mixture contained 10 mM Tris- Cl-, pH 7.4, a t 30 "C, 150 mM NaCI, 4 mM dithioerythritol, 6.6 mM glucose, 0.67 mM NADP', 0.5 mg.ml" of bovine serum albumin, 1 mM MgCL, 0.1 mM EDTA, 10 units of hexokinase, 5 units of glucose- 6-phosphate dehydrogenase, and varying and calculated amounts of MgAca and ADPo. The final ionic strength averaged approximately 0.16. The reaction was initiated by the addition of the enzyme, and the initial velocity in this case was expressed as (Ua4,,/6.22) = pmol of ATP0 formation. Protein was determined by the Biuret method (15). Reconcentration of protein samples was accomplished by ultra-

filtration through collodion bags obtained from Sartorius-Mem- branefilter, GmbH (Gottingen).

Electrophoresis-Polyacrylamide gel electrophoresis under non- denaturing conditions was performed according to the procedure of Davies and Stark (16), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried by the method of Weber and Osborn (17). Agarose gel thin layer electrophoresis of adenylate kinase isoenzymes was conducted on 1% (w/v) agarose gel film (Corning, containing 5% (w/v) sucrose, 0.035% (w/v) EDTA, and 0.065 M barbital buffer, pH 8.6) in a Corning cassette electrophoresis cell a t 90 V for 45 min at room temperature. The electrophoresis buffer employed was Corn- ing's 0.05 M barbital, pH 8.6, containing 0.035% EDTA, and the coupled enzyme dye stain for ATP, as described by Shaw and Koen (18), was used to visualize the bands of adenylate kinase activity.

Amino Acid Analysis-Amino acid analysis of 0.125 mg of the purified enzyme was performed with a Shimadzu high pressure liquid chomatograph employed as an amino acid analyzer after 6 N HCl hydrolysis a t 110 "C for 48 and 72 h. Tryptophan and tyrosine were determined by the method of Goodwin and Morton (19). Cysteine was determined with 4,4'-dithiodipyridine under acidic conditions by the method of Grassetti and Murray (20); 5,5'-dithiobis-(2-nitrobenzoic acid) and p-chloromercuribenzoate were also used to determine thiol groups (21).

Sedimentation Equilibrium a n d Sedimentation Velocity Experi- ments-Sedimentation equilibrium experiments were performed in a RA72T rotor in a Hitachi Model 227 ultracentrifuge at 12 "C by using UV absorption at 280 nm according to Schachman (22), employing a double sector standard cell run at 12,000 rpm. Sedimentation velocity experiments were performed in an RAGOHC rotor a t 60,000 rpm and 20 "C by employing a double sector cell. Samples of the purified enzyme were extensively dialyzed against the appropriate buffer prior to ultracentrifugation. Photographs were taken at 8- or 16-min intervals a t an angle of 50'. For the calculation, a partial specific volume of 0.73 was used from the amino acid analysis according to the method of Schachman (23) using a T159 program (Texas Instruments) (24).

Preparation of Antisera-Antisera to highly purified liver adenylate kinase and to crystalline porcine muscle adenylate kinase were prepared in New Zealand White rabbits. Adenylate kinase (8 mg in 0.5 ml of equilibrating buffer) with an equal volume of Freund's complete adjuvant. Two aliquots (0.1 ml) of the emulsion were injected into two footpads, and the remainder was injected intrader- mally a t multiple sites. The rabbits received three additional intra- dermal injections of adenylate kinase (1 mg) in incomplete adjuvant a t 10-day intervals. Seven days after the final injection, anti-enzyme antibody titers reached maximal levels which were maintained for 3 weeks. Serum collected 7 to 20 days after the last injection was used in all the experiments described below.

Ouchterlony Immunodiffusion-Immunodiffusion (25) was carried out on microscope slides coated with 1 mm of 1% agarose containing 50 m~ sodium phosphate, pH 7.4, and 0.1 mM EDTA. The slides were incubated at 4 "C for 3 days in a humid atmosphere until precipitin lines became clearly visible. The slides were washed by gentle swirling in 50 mM potassium phosphate, pH 7.4, and 0.3 M NaCl for 3 days (the buffer was changed twice daily) to remove unprecipitated serum and antigen. The slides were stained for 1 h in 7% acetic acid containing 0.1% naphthol blue black and washed with 7% acetic acid to remove unbound stain.

Anti-enzyme Assays-Aliquots of the enzyme sample (reduced with 2 mM dithioerythritol) containing 0.1-0.2 units of activity were added to two test tubes (10 X 75 mm) containing 1100 1.11 (total) of Tris-C1- buffer ( r / 2 = 0.20), pH 7.4,0.15 M NaC1,l rng.rn1-l of bovine serum albumin, 2 mM dithioerythritol, 2 mM EDTA and, in one tube, 2.5 anti-enzyme units of purified anti-human liver or partially purified anti-porcine muscle adenylate kinase; the second tube was without anti-enzyme activity. After a minimum of 3 h at 25 "C, an aliquot from each tube was added to each of two adenylate kinase assay cuvettes, and the percentage inhibition of the adenylate kinase by the anti-liver or muscle type was calculated from the relative activities to yield the percentage liver or muscle adenylate kinase.

RESULTS AND DISCUSSION

All operations were carr ied out at 2-4 "C unless otherwise stated. Purification procedures were a modification of the method of Kuby et al. (3). The concentrations of (NH4)2S04 and the required amoun t s of solid (NH&S04 were estimated b y the formulas given in Refs. 2 and 39.

13122 Adenylate Kinase from Human Liver Mitochondria

Preparation of Mitochondria-Mitochondria from 2-3 kg of normal human liver were prepared by a slight modification of the method of Johnson and Lardy (26). The livers were chilled immediately after removal from the autopsy materials and were subsequently defatted and sliced into small pieces. Two hundred grams of liver were homogenized in 500 ml of cold 0.25 M sucrose for one min in a Waring Blendor run at 60% of line voltage. The homogenate was filtered through four thicknesses of cheesecloth, and the nuclei and cell debris were removed by centrifugation at 1700 x g for 12 min in a No. 8 head of a Kubota KR 20000 centrifuge. The mitochondria present in the supernatant fraction were sedimented by acid treatment with cold 1 N acetic acid to pH 5.6, followed by a similar centrifugation at 2500 X g for 25 min in a No. 3 rotor, and the supernatant liquid was discarded. Special care was taken to remove the fluffy layer found directly above the mitochondria. The precipitate was washed by centrifugation with the same sucrose solution.

Finally, the mitochondria were frozen at -20 "C (the enzyme retained full activity for many months under these conditions) and were subsequently thawed and extracted as required.

Fractionation with Zinc Acetate-To the mitochondrial extract of the fraction I supernatant, an aliquot of cold 2 N NaOH was added to adjust the pH to 6.3, and an amount of 1 M Zn(Ac)* (freshly prepared) was added equal to 0.008 volume of fraction I supernatant (final concentration of Zn(Ac)* is 7.93 mM) while maintaining the pH at 6.3 with cold 2 N NaOH. The enzyme solution was slowly stirred for 10 min at 0 "C and centrifuged at 2500 rpm for 30 min. To the supernatant liquid obtained (fraction II), a volume of 1 M Zn(Ac)2 was added equal to 0.060 volume of fraction I1 (final calculated concentration of zinc acetate is 0.06584 M). The pH of the mixture was raised to 8.3 with 2 N NaOH with rapid stirring, and, during the next 30 min, stirring was continued slowly while monitoring the pH and maintaining it at pH 8.3. The mixture was centrifuged at 2500 rpm for 30 min at 0 "C. The pellet obtained was dissolved in %sth volume of fraction I using 0.25 M citric acid, pH 7.6, and titrated to pH 7.6 with concentrated NH,OH (fraction 111). To fraction I11 solid ammonium sulfate was added to give 90% saturation at pH 7.6, and, after slow stirring for 20 min, it was allowed to stand overnight in a refrigerator. The resultant precipitate obtained was centrifuged at 9500 rpm for 40 min and dissolved in 930th volume of fraction I using 0.1 M citrate ammonia at pH 7.6 (fraction IV).

Ammonium Sulfate Fractionation-To the fraction IV solution, solid ammonium sulfate was added with stirring to give 0.40 saturation. After 30 min at 0 "C, the resultant precipitate was centrifuged down at 9500 rpm for 40 min. To the supernatant, liquid solid ammonium sulfate was then added with stirring to 0.80 saturation. After 15 min, it was centrifuged at 9500 rpm for 40 min and the pellets dissolved in %wth volume of fraction I using 0.03 M citrate ammonia, pH 7.6 (fraction VI).

Acid Denaturation of Inert Protein-Fraction VI suspension was stirred and the pH lowered rapidly to 2.6 with 2 N HC1 and kept at pH 2.6 for 5 min with stirring at 0 "C and the pH readjusted back to pH 7.6 more slowly with 2 N NaOH. After 30 min of stirring at 0 "C, the suspension was centrifuged at 9500 rpm for 45 min and the clarified supernatant liquid (fraction VII) retained. Fraction VI1 supernatant was concentrated with 90% saturation ammonium sulfate with gentle stirring for 20 min, and the pellets obtained were dissolved in %oth volume of fraction I using 0.02 M citrate ammonia, pH 7.6, and dialyzed overnight against three changes in a 50-fold volume of the suspension with 0.02 M citrate ammonia, pH 7.6

(fraction VIII). Sephacryl S-300 Gel Filtration-Sephacryl S-300 column

(2.5 X 100 cm) was previously equilibrated with 0.01 M Tris- C1-, pH 7.6, containing 10 mM 2-mercaptoethanol and 1 mM EDTA. The sample (874.0 mg of specific activity = 0.63 units mg") was applied to the column and displaced with the above equilibrating buffer with use of a 68-cm height of hydrostatic head at a flow rate of 8.0 ml/h. 12-ml fractions were collected, and the active fractions were pooled and concentrated with 90% saturation ammonium sulfate. A typical gel fitration profiie was shown in Fig. 1; almost 100% yield was recovered in the middle main peak having a specific activity of 1.66 pmol. min"-mg".

High Capacity Phosphocellulose Column Chromatogra- phy-The packed column of activated phosphocellulose (Whatman P-11) (2.6 X 50 cm, 265.3 ml) was equilibrated with 0.1 M Tris-CL- buffer, pH 7.2, containing 10 mM mercaptoethanol, 0.1 M NaC1, and 0.1 mM EDTA. The enzyme solution (34.2 ml, 235.6 mg of protein) was applied to the column, then washed with 2-column volumes of the same buffer until the optical density of the washings decreased to less than 0.01. At this point, the linear gradient elution was initiated with 2 M NaCl added to the equilibrating buffer in the reservoir and 500 ml of equilibrating buffer in the mixing chamber. A flow rate of 12.5 ml/h at 3.5 "C was maintained by a 60-cm hydrostatic head, and 10-ml fractions were collected.

The active fractions were pooled and concentrated by the addition of solid ammonium sulfate to 0.95 saturation. The resulting precipitate was spun down at 25,000 X g for 20 min at 0 "C, dissolved in minimum volume of the equilibrating buffer, and dialyzed overnight with stirring against three changes in 1 liter of equilibrating buffer a t 3.5 "C.

Agarose-Hexane ATP Affinity Chromatography-The final step of this purification procedure was carried out with the use of an agarose-hexane ATP affinity chromatography column (1.0 x 2.5 cm, 2 ml) which was equilibrated with 0.01 M Tris-C1- buffer, pH 7.4, containing 10 mM mercaptoethanol and 0.1 mM EDTA. The concentrated enzyme solution was applied to the column, washed with 5 ml of equilibrating buffer, and eluted with a 0-1.0 M sodium chloride linear gradient, using 50 ml of equilibrating buffer in the mixing chamber. The total enzyme units were recovered in excess of 82% yield between 0.1-0.3 M NaCI. The active fractions were pooled and concentrated with saturated ammonium sulfate. The resulting precipitate was centrifuged off and dialyzed

t

Fraction Number (12-ml Fraction)

FIG. 1. Sephacryl S-300 gel filtration of normal human liver adenylate kinase of fraction VIII. The enzyme solution (550.6 units and 874.0 mg of protein) was applied to a column of Sephacryl S-300 (97 X 2.5 cm). Each fraction was 12 ml. 0, absorbance at 280 nm; 0, adenylate kinase activity.


extensively against the same buffer to eliminate any bound nucleotide which had been released from the gel, and concentrated with the aid of a collodion bag. A typical elution profile is shown in Fig. 2.

The purification summary is shown in Table I. The isolated enzyme was purified 1600-fold from the mitochondrial extract in 5.6% yield (for the first preparation; after the second preparation, the yield was up to 27%), and its final specific activity was 280 pmol of ATP formed min" .mg" of protein. The specific activity was similar to that of the crystalline calf liver enzyme (3); however, it was 20% less than that of the rat (5, 27) and bovine (8) liver enzymes. The purified enzyme obtained from the final step of Table I can be stored at -20 "C for periods up to several weeks and thawed with no detectable loss of activity. After 3 months, the loss of enzyme activity ranges from 5-10%. The enzyme is stable to storage at 3 "C for a month and much less stable in dilute solutions (0.2 mg. ml"), but can be stabilized by the addition of dithioerythritol (4 mM) and bovine serum albumin (1 mg-ml"). For this reason, enzyme solutions were routinely diluted in 0.05 M Tris- C1- buffer, pH 7.4, containing 1 mg.ml" of bovine serum albumin and 4 mM dithioerythritol prior to assay or storage. The enzymatic activity is quite stable to dialysis in the presence of 4 mM dithioerythritol, and at low (pH 2.0) or high (pH 11.5) pH exposure a t 0 "C for 1 h (data not shown).

Spectral Properties of the Normal Human Liver Adenylate Kinase-The solutions of normal human liver adenylate kinase were colorless, and the ultraviolet absorbance spectrum of this enzyme is presented in Fig. 3. The faint shoulder in the absorption spectrum a t 290 nm indicated the presence in the protein of small amounts of tryptophan and tyrosine, and this was confirmed by the spectrophotometric analysis (according

1.0 1 A

?

E -

0.5 - 0 0

0 - 1

Fractlon Number (2.3-ml Fractlon)

FIG. 2. Affinity chromatography of normal human liver adenylate kinase on ATP-hexane agarose (2-ml column bed volume). Chromatography was carried out on 347.4 units of enzyme (12.9 mg of protein). Gradient elution was identical (0-1.0 M NaCI) with that described for the final step of the purification procedure under "Results and Discussion." Enzyme protein concentration was calculated from absorbance measurements at 280 nm.

to Ref. 19) of 2 tryptophan and 4 tyrosine residues in alkaline solution, and these values are close to those estimated from the amino acid analysis. The protein has an A28o/A260 ratio of 1.72, indicating the absence of significant contamination by nucleic acids or nucleotides a t neutral pH. The measured absorbance coefficient of the enzyme (E:;;'') was found to be 0.64 at 278 nm at a protein concentration of 1.00 mg.rn1-I in 0.01 M Tris-C1-, pH 7.40, containing 10 mM mercaptoethanol and 0.1 mM EDTA (r /2 = 0.17) a t 30 "C.

Optimal pH for the Normal Human Liver Adenylate Ki- nase Reaction-In the forward direction, the optimal pH was found to be approximately 7.4, as shown in Fig. 4; however, in the reverse direction, it shifted slightly to 7.9 at 30 "C (r/2 = 0.16). These data resemble those adenylate kinases obtained from other mammalian tissues.

Molecular Weight and Subunit Structure of the Normal Human Liver Adenylate Kinase-On agarose gel electrophoresis a t pH 8.9, the native enzyme migrated as a single band (data not shown); and, on calibrated sodium dodecyl sulfate- polyacrylamide gels in the phosphate buffer sodium dodecyl sulfate gel system of Weber and Osborn (17) (Fig. 5), the protein also migrated as a single band in a position correspond- ing to a molecular weight of 26,500 compared to bovine serum albumin (68,000), y-globulin, heavy chain (50,000), ovalbumin (43,000), chymotrypsinogen (25,700), y-globulin light chain (23,400), apoferritin (18,500), and cytochrome c (11,700) as standards. This subunit molecular weight is very similar to that reported for the calf liver adenylate kinase (3). A molecular weight of this protein was estimated to be 25,200 by the sedimentation equilibrium method at pH 7.4 and r / 2 = 0.16 (Fig. 6A) with use of a partial specific volume (6 = 0.73) of the enzyme estimated from its amino acid composition (23, 24). The protein sedimented as a single component under the same conditions and yielded an szo,u. = 2.52 S extrapolated to zero protein concentration (Fig. 6B). These data are in excellent agreement with that obtained by the method of Weber and Osborn (17), and the native normal human liver adenylate kinase is a monomer of 26,500 daltons. However, the mitochondrial enzyme seems to be easily aggregated to the dimer and possibly the tetramer (compare the rat enzyme (27)). In one preparation, an Szo,w of 4.85 S was obtained by sedimentation velocity, with little or no concentration dependency, and, when compared to a sedimentation equilibrium run, an aW of 52,800 was obtained, with evidence of still higher aggregates near the bottom of the cell.

Amino Acid Analysis-The weight of the protein in the sample to be used was estimated by the Biuret method (15). The enzyme samples were exhaustively dialyzed against 0.15 M ammonium bicarbonate and lyophilized. The samples were then hydrolyzed with twice distilled 6 N hydrochloric acid at 110 "C in sealed evacuated tubes that had been degassed by

TABLE I Purification of normal human liver mitochondrial adenylate kinase

Fraction" Volume Total protein activity Total Specific Purification Recovery activity of activity

ml mg units b units/mg protein w

I. Mitochondrial extract 4,500 29,370 6,050.2 0.206 590

1 100 11. Zn fractionation 2,668 877.7 0.329

874.0 1.59

550.6 14.5

0.630 3.06 9.1 111. Acid denaturation of inert protein followed by precipita- 19.0

IV. Sephacryl S-300 gel filtration 34.2 235.6 391.5 1.66 8.06 6.5 V. Phosphocellulose chromatography followed by precipita- 1.6 12.9 347.4 26.93 130.7 5.7

VI. Agarose-hexane ATP affinity chromatography followed 1.5 1.2 336.3 280.19 1,360.1 5.6

tion with 90% saturation (NH,),SO, and dialysis

tion with saturated (NH&SO,

by precipitation with saturated (NH4)2S04 a Initially, 3.5 kg of human liver. * One unit = 1 pmol/min by spectrophotometric (coupled-enzyme) procedure. See Kuby et al. (3).


2.0 t I

0 1 2 4 3 2 6 0 2 8 0 3 0 0 p o 3 4 0 3 6 0 3 8 0 4 0 0

Wave Length (nrn)

FIG. 3. Ultraviolet absorption spectra of normal human liver adenylate kinase, 2.068 mg/ml, in 10 mM Tris-C1-, pH 7.4, 0.138 M NaCL, 1.0 m~ EDTA, 6 m~ MgCl, @'/2 = 0.16) at 25 "C.

.- 5 5.0 6.0 7.0 8.0 9.0 10.0 pH

FIG. 4. Effect of pH on purified normal human liver adenylate kinase activity. 0, forward reaction (ATP disappearance); 0, reverse reaction (ATP formation). The following buffers (10 mM) were used: acetate (pH 5.0-6.0); potassium phosphate (pH 6.5-7.0); Tris-C1- (pH 7.4-8.5); and glycine-NaOH (pH 9.0-10.0).

the method of Crestfield et al. (28). Amino acid analyses were carried out with a Shimadzu (ISC09/S2504 (4 mm X 25 cm) with a step gradient mobile phase at 55 "C) high pressure liquid chromatograph analyzer using postlabeling with o- pthalaldehyde (29). The amino acid composition of the normal human liver adenylate kinase protein is given in Table I1 and was calculated on the assumption that the protein has a molecular weight of 26,500. The protein is rich in acidic residues and sparse in aromatic and sulfur-containing amino acids. The analysis is compared to that reported for bovine liver enzyme (8) and given in Table 11. Determination of tyrosine and tryptophan by the spectral method of Goodwin and Morton (19) yielded 4 Tyr and 2 Try residues/mol of protein. The method of Grassetti and Murray (20) was used for titration of sulfhydryl groups under acidic conditions uti- lizing the reaction with the disulfide 4,4'-dithiodipyridine. By cysteic acid analyses, there are 4 half-cystine residues/subunit. There was no significant optical density change in the titration of the native human normal liver adenylate kinase with p - chloromercuribenzoate (21) and 5-5'-dithiobis-(2-nitrobenzoic acid), and, accordingly, the enzyme appears to contain no accessible sulfhydryl groups in the native state; but under acidic conditions, four SH groups are made available for reaction with 4,4'-dithiodipyridine. Moreover, the native enzyme is not inhibited by 5-5'-dithiobis-(2-nitrobenzoic acid) nor by p-chloromercuribenzoate, and in this respect, is in agreement with the early observations of Chiga and Plaut (10). Considerable similarity exists between the two enzymes with respect to their content of histidine, methionine, tryptophan, aspartate, and proline, and only small differences are

noted in others such as arginine, tyrosine, glutamate, threonine, valine, and lysine. Substantial differences were detected in serine, glycine, alanine, leucine, isoleucine, and phenylalanine and also in the sum of the amino acid residues, for both enzymes.

The most significant difference thus far noted is the number of half-cystine residues and the presence of tryptophan in the liver enzyme compared to the rabbit muscle enzyme which

ChymdrypYnogen $ r-Globulin (L) a Norm4 Human Liver Milochondriol -late K m a s e I f l , S 2 6 , 5 f l O )

ChymdrypYnogen Norm4 Human Liver Milochondriol

-late K m a s e I f l , S 2 6 , 5 f l O )

LIpoferritin

Cylochrorne c

W t i v e Mobilily

FIG. 5. Molecular weight analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified normal human liver adenylate kinase. Disruption was conducted at 40 "C for 2 h in 4 M urea, 2% sodium dodecyl sulfate, 2% 2-mercaptoethanol, 0.1 mM EDTA, 10 mM sodium phosphate, pH 7.0 (25 "C). Standard 10% gels were used for the electrophoresis by the method of Weber and Osborn (17) with 0.1% sodium dodecyl sulfate and 0.1 M sodium phosphate, pH 7.0. Samples of 5 pg of the enzyme were analyzed electrophoretically with a constant current of 8 mA/gel at 25 "C for 4 h. Each gel had been subjected to pre-electrophoresis with 30 pl of 0.1 M mercaptosuccinate in 0.1 M sodium phosphate, pH 7, prior to the electrophoresis of the standards and sample. The gels were stained with 0.25% Coomassie brilliant blue (R-250) (17) and destained by diffusion. Molecular weights selected for the polypeptide chains used as reference standards in the gel electrophoresis were summarized in Ref. 3.

B

FIG. 6. Sedimentation equilibrium and sedimentation velocity measurements of normal human liver adenylate kinase. A, a molecular weight determination of normal human liver mitochondrial adenylate kinase from absorbance scanning experiments expressed in terms of a plot of (-log c, c ( A ) ) uersus X' (cm') (square of the distance, in centimeters squared, from axis of rotation). The initial concentration of protein was 0.55 mg/ml in 0.15 M NaCL, 0.01 M Tris, 0.001 M EDTA, pH 7.4, a t 4 "C. The protein was sedimented at 14,000 rpm for 7.5 h. B, sedimentation coefficients of normal human liver mitochondrial adenylate kinase as a function of protein concentration at 25.2 "C in 0.15 M NaCl, 0.01 M Tris, 0.001 M EDTA, pH 7.4. y = -0.0998 (X) + 2.5198. The protein was sedimented at 60,000 rpm for 2 h. The bar schlieren diaphragm (phase plate) angle was set at 60".


TABLE I1 Amino acid composition of normal human liver mitochondrial

adenylate kinase The purified enzyme was hydrolyzed for 48 and 72 h. Threonine

and serine were obtained by extrapolation of the results of 48- and 72-h hydrolysates to zero time.

Nearest internal number of residues Amino acid

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Half-cystine Lysine Histidine Arginine Tryptophan

Total

Human per 26,500 g

protein 18 12 20 22 13 23 22 15 5 6

15 4b 4 4'

12 4

12 2b

Bovine" per 21,500 g

protein 18 9

11 20 13 15 17 13 5 9

20 5 7 4

15 4 10 2

213 197

Integral number of residues X mo- 26,369 21,744 lecular weight ' I Markland and Wadkins (8).

' Based on the determination of cysteic acid. Calculated by the spectral method of Goodwin and Morton (19).

contains two exposed sulfhydryl groups and no tryptophan. From these and other molecular properties, it appears that the liver enzyme, compared to the muscle enzyme, may exist in a less flexible structure to allow for the necessary binding and required juxtaposition of the binding sites already partially fixed in some preferred conformation so as to facilitate phosphoryl transfer to AMP2-. This may be its important role in the mitochondria since VLaX << V i , , in the human liver mitochondrial enzyme (see below) and in the calf liver enzyme (14, 30).

Catalytic Properties-In Table 111, a summary is given of the substrate specificities of the normal human liver mitochondrial adenylate kinase compared to the human muscle adenylate kinase. Identical substrate specificity patterns were observed for both isoenzymes; however, there are some differences in the degree of the specificity between the liver mitochondrial and the normal human muscle enzyme data. In addition to adenine nucleotide, catalytic activity was also observed in the presence of adenosine-5"(3-thio)triphosphate, adenosine-5'-thiophosphate, and adenosine monophosphate- 3'-pyrophosphate. UTP, CTP, GTP, and ITP with AMP were essentially unreactive; adenosine-5"(3-thio)triphosphate plus adenosine-5'-thiophosphate proved also to be essentially unreactive, which indicates that there is little, if any, contamination by GTP-AMP transphosphorylase (31).

Adenosine-5'-0-( 1-thiodiphosphate) should be a good phosphate acceptor in the presence of pyruvate kinase and phosphoenolpyruvate (Scheme 1). Also, in Scheme 1, the struc- tures of ATP@ and adenosine monophosphate-3'-pyrophosphate are presented.

Treatment of Data and Kinetic Analyses-Calculations of MgATP2- and AMP2- for the forward reaction were adequately made over the range of concentrations explored by employing the set of approximate conservation equations:

TABLE I11 Substrate .specificity patterns of the normal human liver

mitochondrial adenylate kinase and the normal human muscle adenylate kinase (AKase)

Purified enzyme protein was 0.02 pg/assay unless specified otherwise. Nucleoside monophosphates were added at 1.0 mM and nucleoside triphosphates at 3.0 mM (2.1 mM nucleoside diphosphate, 2.1 mM MgSO, in the reverse reaction). Reactions were carried out at pH 7.4 for forward reaction and pH 8.1 for reverse reaction in the presence of 1.0 mM magnesium concentration, and the results obtained were described as relative activities.

Substrate pair Relative activity

AKase AKase Liver Muscle'

[ U d l ATP:AMP UTP:AMP CTP:AMP GTP:AMP 1TP:AMP Adenosine-5'-(3-thio)triphosphate:AMP Adenosine triphosphate-3"pyrophosphate ATP:adenosine-5"thiophosphate ATP:adenosine monophosphate-3'-pyrophos-

Adenosine-5"(3-thio)triphosphate:adenosine- phate

5'-thiophosphate

100 100 0 0 0 0

70 97 0 0

95 56 86 57

0 0

[Vur 1 ADP 100 100 Adenosine-5'-(2-thio)diphosphate 0 0 Adenosine diphosphate-3'-pyrophosphate 0 0

Liver enzyme K M g x r P z = 1.2 X M (V!& = 1.0 X loJ pmoI.min-l.mg-l) R A M P = 5.6 X M K M g A . w ys = 5.71 X M (V',,,, = 7.2 X pmoI.min-".mg-:) K A M w .,S = 1.8ti X M ( V L = 4.1 X lo2 umol.min".me-

A I 5 0 0

I

uo-0-6-0

AK 0- 4 Ado-5 [fnto.] + ATP - AOP.8 + ACP I 1 > 0-6-0-

O H

0 0-6-0-

A J h 5 ' ATP.8 b H

AD2 } "'%= 1 ATP 1 2 1

2Phasphoenalpyr""afe 2Pyr""aTe

SCHEME 1. Ado-B'[B-thioPPP], ATPyS, adenosine-5"(3-thio)triphosphate; Ado-S'P,3'PP, AMP-3'[PP], adenosine monophosphate- 3'-pyrophosphate; Ado-5'[thioP], adenosine-5'-thiophosphate.

ATPo .- MgATP2- + ATP4- + HATP3- + MgHATP-

+ KATPJ- + NaATP'

AMPI, = MgAMP + AMP" + HAMP- + KAMP- + NaAMP-

Mgll .- Mg'+ + MgATP" + MgHATP- + MgAMP

Nal, = Na+ + NaATP" + NaAMP~

KIl = K+ + K ATPJ- + KAMP

where subscript 0 implies total concentrations and appropriate valences are assigned to each of the ionizable or complex species.

For the reverse direction, for calculation of MgADP-, the set of conservation equations could be adequately approxi- mated by the following.

M. Hamada and M. Okuda, unpublished observations.


ADP,, = MgADP- + ADP" + HADP2- + MgHADP + NaADP"

Mgo = Mg" + MgADP- + MgADP- + MgHADP

Nao = Na' + NaADP'-

The values assigned for the chelation and dissociation constants are given in Ref. 14. The method of calculations was similar to that described in detail for the rabbit muscle ATP- creatine transphosphorylase (32) and rabbit muscle, calf muscle, and calf liver adenylate kinase (14). Calculations employed a fixed concentration of free Mg2+ of 1 mM for the kinetic studies on the forward reaction, where either MgATP" or AMP2- were variable substrates, with each substrate varied at fixed concentrations of the complementary substrate. For the reverse reaction, where it proved impossible to maintain a fixed concentration of uncomplexed Mg2+, either MgADP- or ADP"- were the variable substrates, with its paired substrate fiied.

As will be seen, under these conditions of a reasonable fixed ionic strength a t 30 "C and pH 7.4, the steady state kinetics conformed to first degree velocity expressions (33) for two substrate reactions. For the quasi-equilibrium random mechanism similar to the one proposed for calf brain ATP-creatine transphosphorylase (34) and where independent binding was not invoked, the following intrinsic constants may be given as shown in Scheme 2, where MA = MgATP2-, B = AMP2-, MC = MgADP-, and C = ADP3-. For the limiting cases, where the concentrations of products may be set to zero initially, then for the forward and reverse initial velocities, respectively,

where uof and uor denote initial velocities for forward and reverse reactions. For the variable MgATP2- (MA) and fixed AMP2- (B) , e.g. a primary plot of l / d uersus l/(MA) would yield

and, from the appropriate secondary plots of slopes and ordinate intercepts and relation (l), values KI - K4 may be estimated for the forward reaction. Thus,

y intercept = - + -e-. Viax Vkay (B)

1 K , 1

Also, for variable AMP" and fixed MgATP", again all four constants (and Viax) may be estimated for the forward direction. Within the experimental errors and calculation uncer- tainties, the values for the kinetic parameters proved to be the same, and average values will be presented together with some of their ranges of uncertainty. Similarly, appropriate

M A + E + B E . M C ' D M C + E + D

SCHEME 2.

secondary plots and relation (2) yield values for all four constants for the reverse reaction (cf analogous graphical treatments of data by Dalziel(35) and by Florini and Vestling (36)).

Kinetic analyses of the enzyme-catalyzed bimolecular re- versible reaction included initial rate measurements as a function of substrate concentration. In Fig. 7 (A and B ) are shown double reciprocal plots of reaction velocity uersus variable MgATP2- a t constant AMP2- concentrations and variable AMP2- a t constant MgATPZ- concentrations, respectively. The plots resulted, in each case, in a series of straight lines intersecting at a common point. Secondary plots of the data (see insets) allow calculation of V,,, and dissociation constants from the binary and ternary enzyme-substrate complexes. K., (i.e. Kid) values from these data are 1.19 X and 5.60 X for MgATP" and AMP", respectively (see Table IV) .

Initial rate measurements carried out in the direction of MgATP2- formation with ADP"- or MgADP- as the variable substrate are shown in Fig. 7 (C and D). K s values for K A D p

and KM~ADP- were found to be 1.68 X lo-' and 5.25 X lo-$ M, respectively. These data suggested a quasi-equilibrium random mechanism with a rate-limiting step at the interconversion of the ternary complexes, as reported for rabbit muscle, calf muscle, and calf liver adenylate kinase (14). Rut these data alone will not exclude the possibility of a random mechanism in which product release is rate-limiting. Moreover, the data presented in Fig. 7 are consistent with the conclusion of Rhoads and Lowenstein (37), uiz. that any mechanism involv- ing covalent enzyme intermediates is unlikely in view of the sets of intersecting double reciprocal plots. KAMFA- or KAMPI- values (i.e. K A M ~ A - = 2.4 X M and RAMFA- = 5.6 X lo-' M) (see Table IV) are similar to those for the calf enzyme (14). Vi,,/Et and V',,,/Et values are also of similar magnitude for the calf liver adenylate kinase (14), with V & being smaller than Vi,, for the liver enzymes. This observation might be of

-

TABLE IV Values for derived kinetic parameters at p H 7.4, 30 "C, and r/2 = 0.16 for ATP-AMP transphosphorylase from human

liver mitochondria

Defined

rameters

Defined in- kinetic pa- Defined equilibrium ::$::$:: Value

stant M

1.19 x lo-> 2.35 X 10-4 5.6" x

5.00 X 1.6, x 5.25 x 3.18 X 1.02 x lo->

[1.04 (+ 0.4) X io3 wmol min" .mg";

1 1.68 x lo4 mol. min"(mo1

&;n!02 s-I

-ih

" K , s , dissociation constant of the particular substrate from the binary complex. Ks, dissociation constant of the particular substrate from the ternary complex.

Calculated on the basis of molecular weight of the enzyme, 26,400.


/ / I 6 0 5 10

(l/~MgA@I).l& (mdes-lxhter).l@

FIG. 7. Kinetics at pH 7.4, r = 0.16-0.18, and 30 "C of the forward reaction, MgATP2- + AMP2- + MgADP- + ADP3-, and of the reverse reaction, MgADP- + ADP3- + MgATP2- + AMP-, catalyzed by ATP-AMP transphosphorylase from normal human liver. A, primary plots: l/uof uersus 1/MgATP2- a t several fixed values of AMP2-, Mg% fixed at 1.0 mM. Secondary plots: slopes ( O " - O ) and ordinate intercepts (o"--o) from linear extrapolated primary plots uersus 1/AMP2-. Secondary plots fitted to the expressions, slope = (K4/Vfnax) + (KzK4/K,).(1/B); y intercept = (I/V!&) + (K3/Vfna.).(l/B). B, primary plots: (l/ud) uersus (l/AMP") at several fKed values of MgATP2-; M g k fixed a t 1 mM. Secondary plots: slopes (W) and ordinate intercepts (M)

some physiological importance if the mitochondrial adenylate kinase plays any regulatory role in oxidative phosphorylation, for it may indicate that this enzyme is already partially fiied in some preferred configuration so as to facilitate phosphoryl group transfer to AMP", which might be its important role in the liver mitochondria.

Preparation and Properties ofAntibodies Directed against Human Liver Adenylate Kinase-Injection of purified normal human liver adenylate kinase into rabbits led to the produc- tion of antibodies directed against this enzyme. As shown in Fig. 8, serum from injected animals could inactivate the liver enzyme to at least 65%, whereas preimmune serum was inac- tive, and there was no inactivation of the enzyme from normal muscle, the erythrocyte enzyme, and the serum enzymes from normal and Duchenne dystrophics. Furthermore, double immunodiffusion studies indicated that the antibody was capable of forming precipitin bands with the liver enzyme (Fig. 9). Also, sodium dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitates indicated that the antibody prepared from immune serum precipitated the liver adenylate kinase

( 1 / [ ~ ~ ~ 1 ) - 1 0 - ~ ( m ~ e s - ~ r ~ ~ t e r l ~ l O ~ ~

from linear extrapolated primary plots uersus (1/MgATP2-). Second- ary plots fitted to the expressions, slope = (Ks/Kmax) + K&/VfnaX). (I/MA); y intercept = (l/VfnJ + (K4/Vfmar).(1/MA). C, primary plots: (l/uo') uersus (I/MgADP-) at several fixed values of ADP"; Mg;$:+., not fixed. Secondary plots: slopes (M) and y intercepts (M) uersus (1/ADP3-). Secondary plots fitted to the expressions, slope = (K8/VLax) + (K6KB/Vmax).(1/C); y intercept = ( l / V L ) + (Ks/K.,). (l/C). 0, primary plots: (I/VO') uersus (1/ADP3-) at several fixed values of MgADP-; M g k not fixed. Secondary plots: slopes (H) and y intercepts (M) uersus (l/MgADP-). Secondary plots fitted to the expressions, slopes = (K9/VLax) + (K7K9/Vmax). (1/MC); y intercept = (l/K.J + (K8/Vkax). (l/MC).

X Llver AK 5 Ant[-MAb 0 DMD-Ser AKF Ant] -LAb 0 DMD-Ery AK C Antl-LAb

s DMD-Ery AK E Antl-MAb DMD-Ser AK? Anti-

0 0.1 0.2 0.3

s

0 0 0.1 0.2 0.3

"Ab

Antlswum (mO(46.0mg Iml of Protein)

FIG. 8. Titration of adenylate kinases with their specific antibody. Adenylate kinase (2 units) was incubated with various concentrations of antibody in 1 ml of 0.05 M Tris-C1-, 0.163 M NaCl, 1 mM EDTA, 1 mM dithioerythritol, and 1 mg of bovine serum albumin, pH 7.7, for 1 h a t 30 "C. Then 5-2O-pl samples were assayed for enzymatic activity by the enzyme-coupled method. DMD, Duch- enne Muscular Dystrophy; Ser, serum; Ery, erythrocyte; Anti LAb, antinormal human liver adenylate kinase antibody; Anti-MAb, anti- porcine muscle adenylate kinase antibody.


1

FIG. 9. Ouchterlony’s double diffusion analysis. Center well, antinormal human liver adenylate kinase antibody; well 1, porcine muscle adenylate kinase; well 2, normal human serum; well 3, normal human erythrocyte; well 4, rabbit muscle adenylate kinase; well 5, normal human liver adenylate kinase; well 6, normal human muscle adenylate kinase.

from an acid-treated supernatant fraction of serum, whereas antibody prepared from preimmune serum did not show such anti-enzyme activity. The antibody directed against liver adenylate kinase did not cross-react with the muscle-type isoenzyme and the liver-type aberrant adenylate kinase from Du- chenne dystrophic serum. The antibody preparation against each isoenzyme, including aberrant enzyme, should prove extremely useful for further structural studies of adenylate kinase and for examination of its interaction with the isoenzymes from normal, dystrophic, and autosomal recessive muscular dystrophies.

As mentioned in a previous paper (12), the aberrant adenylate kinase from Duchenne dystrophic serum seemed to show similar enzymatic properties as that of normal human liver enzyme; however, it appeared to have different immunochemical properties, and this interesting observation is con- fvmed again in this paper.

Acknowledgments-We wish to express our appreciation to Drs. R. Fukunish, R. Tabei, and H. Mori of the Department of Pathology, Ehime University School of Medicine, for kindly supplying the autopsy material and helpful suggestions, to K. Oka for his dedicated technical assistance during the progress of this research, and to T. Takaku and K. Kameda of the Central Laboratory Analysis Division. We also gratefully acknowledge the assistance in preparation of the manuscript by K. Miyoshi of this laboratory.

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