Isolation and Characterization of Two Isozymes of Myosin Heavy Chain from Canine Atrium*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The Amerian Soiety of Biologial Chemists, In Vol. 261, No. 10, Issue of April 5, pp , 1986 Printed in U.S.A. Isolation and Charaterization of Two Isozymes of Myosin Heavy Chain from Canine Atrium (Reeived for publiation, June 21,1985) Issei Komuro, Hidetsugu Tsuhimohi, Seigo Ueda, Masahiko Kurabayashi, Yoshinori Seko, Fumimaro Takaku, and Yoshio Yazaki From the Third Department of Internal Mediine, Faulty of Mediine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan To larify the harateristis of myosin isozymes in the atrium, we frationated two isoforms of myosin heavy hain (HC), atrial HCa (A-HCa) and HCP (A- HCP), from the anine heart by affinity hromatography, using monolonal antibodies speifi for HCa (CMA19) and HCP (HMC50), respetively, and then omparedtheirpeptideompositionandenzymati properties with those of ventriular HCa (V-HCa) and HCP (V-HCP). The reativity of these isozymes with three monolonal antibodies revealed that there are at least three different epitopes between A-HCa and A- HCP. Differenes in the primary struture of A-HCa and A-HCj3 were onfirmed by one- and two-dimensional gel eletrophoreti analyses of these peptides, produed by digestion with a-hymotrypsin and yanogen bromide (CNBr). A-HCa and V-HCa were indis- tinguishable proteins, and A-HCB was also very similar to V-HCP. Furthermore,there were differenes be- human atrium and theiredistribution in adaptation to prestween A-HCa and A-HCP in their Ca2+-ativated ATP- sure overload by immunohistohemial studies using monoase ativities. The ATPase ativity of A-HCP was lower lonal antibodies (19, 20). In this study, we isolated two than that of A-HCa and was similar to that of V-HCB. myosin heavy hain isozymes from anine atrium by affinity We onluded that there are two different isozymes of hromatography using the monolonal antibodies, HMC50 myosin heavy hain in the atrium (A-HCa and A-HCB), and CMA19, speifi for HCP and HCa, respetively, and as well as in the ventrile (V-HCa and V-HCP), and determined their harateristis with referene to those of that A-HCB is very similar to V-HCP, the predominant ventriular myosin isozymes. form of ventriular myosin, in its moleular struture The properties of the isozymes were assessed by three and enzymati ativity. approahes: (a) ross-reativity studies with monolonal antibodies speifi for either HCa and HCP; (b) one- and twodimensional peptide mapping after proteolyti leavage; and (e) The heterogeneity of ardia myosins has been revealed by Ca2+- and KC-EDTA-ativated ATPase ativities. By these several different experimental approahes. Enzymati (1-7), analyses, it was onfirmed that there are two different myosin eletrophoreti (8-14), and immunohemial (15-21) analyses heavy hain isozymes (atrial HCa and HCP) in the anine have shown the existene of two distint ardia myosin heavy atrium and that atrial HCP was indistinguishable from venhains, HCa and HCP.l It has also been shown by eletrophotriular HCP. resis under nondenaturing onditions that ardia ventriular EXPERIMENTAL PROCEDURES myosin is omposed of three different isoenzymes, referred to as V1, V2, and V3 (8, 12, 13). VI and V3 orrespond to Animal Models-Mongrel dogs weighing kg were anesthehomodimers of HCa and HCP, respetively, while V2 is a tized with sodium pentobarbital (50 mg/kg, intravenously), and fresh heterodimer of HCa and HCP. The distributions of these heart musle tissue was obtained. Right and left atria, disseted free from the ventriles, were used for the preparation of atrial myosin. isozymes are hanged by developmental (8, 12-14, 16,22, 23) Ventriular myosin was extrated from the free wall of the left and hormonal state (8, 10, 12, 14, 16, 24-26, 28) and ardia overload (15, 18-20, 27-29). This investigation was supported by a grant-in-aid for sientifi researh ( , ) from the Ministry of Eduation, Siene and Culture, and a grant for ardiomyopathy from the Ministry of Health and Welfare, Japan. The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore he hereby marked advertisement in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. The abbreviations used are: HC(s), heavy hain(s); LC(s), light hain(s); CNBr, yanogen bromide; EDTA, etbylenediaminetetraaeti aid; SDS, sodium dodeyl sulfate. Atrial HC and ventriular HC are denoted as A-HC and V-HC, respetively The type of myosin isozyme present appears to be an important determinant of the ontratile ativity of ardia musle. The Ca2+- and atin-ativated ATPase ativities of V1 are 2-3 times higher than those of V3, and the hange from V1 to V3 is orrelated with a derease in speed of musle ontration and an inrease in eonomy of fore generation (26,27, 30-32). Isoenzymati hanges from V1 to V3 indued by ardia overload are onsidered to be of great importane in adaptation of heart musle to a demand for inreased ardia work. However, previous studies have been restrited to ventriular myosin beause nondissoiating eletrophoreti gels fail to separate atrial myosin isozymes. The presene and distribution of myosinisozymeswas not onfirmed until quite reently in atrial myoardium. We have reently demonstrated the existene of isoforms of myosin heavy hain in ventrile. Calf ventriular musles were extrated in the same way to determine the harateristis of eah monolonal antibody. Myosin Preparation-Myosin was prepared by a dilution tehnique as previously reported (1). All proedures were arried out at 4 C, and all solutions ontained 1 mm EDTA. The light hains (LCs) were isolated by guanidine denaturation as desribed elsewhere (33). Hybridoma Proedures-The proedures of immunization and preparations of hybridomas were previously desribed (19,20), essentially by the protool of Kohler and Milstein (34). Briefly, BALB/ mie (male, 6 weeks) were immunized by five intraperitoneal injetions at 2-week intervals with ml of 1 mg/ml solution of alf atrial myosin or human ventriular myosin. Mie were killed 3 days after the intravenous boost injetion, and their isolated spleen ells were fused with the myeloma ells (P3X63Ag8Ul).

2 Sreening Assay for Anti-myosin Antibodies-Anti-myosin ativity in a medium from hybridoma olonies was sreened quantitatively by enzyme-linked immunosorbent assays, using goat biotinylated antimouse immunoglobulin and avidin D-peroxidase (Vetor Labs, In.) as desribed previously (19) essentially aording to Guesdon et al. (35). Results were expressed as the perentage of maximum absorbane (at 540 nm) (Fig. 1). Antibodies from 3 hybridoma lines, HMC48, HMC50, and CMA19 speifi for HCP and HCa, respetively, were seleted for use in this study. Preparation of Affinity Columns and Purifiation of Myosin by Affinity Chromatography-Monolonal antibodies (HMC50 and CMA19) were refined by ammonium sulfate frationation (50% saturation) and by their appliation to a DEAE 52 ellulose (Whatman) olumn. After dialyzation against a oupling buffer (0.1 M NaHC03, ph 8.3, ontaining 0.5 M NaCl), anti-myosin antibodies were added to yanogen bromide-ativated Sepharose 4B (Pharmaia), and preparation of an affinity olumn was arried out aording to the proedure of Chizzonite et al. (11). About 1 mg of myosin was applied to the olumn in 1 ml of olumn buffer (20 mm Tris-HC1 ontaining 0.5 M KCl, ph 7.5) and inubated for 3 h at 4 "C. The unbound myosin was removed by washing with olumn buffer until the absorbane at 280 nm of the effluent was 0.01 or less. The bound myosin was eluted with 4 M guanidine HCl in olumn buffer, ph 7.5, and the frations were olleted until the absorbane at 280 nm was less than The bound myosin was dialyzed against olumn buffer, onentrated immediately, and applied to another affinity olumn again to exlude heterodimer (HCaP); frationated HColol or HCPP with HCap was applied again to the affinity olumn with anti-hcp or anti-hca antibody, respetively. It was reyled 10 times over the olumn for 6 h at 4 "C. The unbound myosin was olleted by washing with olumn buffer until the absorbane at 280 nm of the effluent was 0.1, aording to the proedure of Everett et al. (36). The myosin purified in this manner was very pure, as sodium dodeyl sulfate-polyarylamide gel eletrophoresis had determined that it did not ontain any atin or regulatory proteins (Fig. 2). Transfer of Myosins to Nitroellulose Sheets and Immunologial Staining-After being eletrophoresed in 7.5% polyarylamide gels by Laemmli's buffer system (37), myosin was transferred from the slab gels to the nitroellulose sheets (Bio-Rad) with a 200-mA onstant urrent for 16 h at 4 "C. After the transfer, the sheets were ut into vertial strips and one/sample was stained with Amido Blak. Three other strips were immunologially stained, essentially aord- ing to the proedure of Towbin et al. (38). At first, they were saturated by inubation for 1 h at 37 "C in 3% gelatin in Tris-saline (20 mm Tris-HC1, 0.5 M NaCl, ph 7.5). After three washings in rinsing solution (Tris-saline ontaining 0.05% Tween 20, ph 7.5), 100 pl of anti-myosin antibodies in Tris-saline were overlaid on eah strip. After a 1-h inubation at 37 "C, the strips were washed again and were soaked in Tris-saline ontaining a goat anti-mouse IgG horseradish peroxidase onjugate (Bio-Rad) for 1 h at 37 "C. After exess antibody was washed as derihed above, olor was developed by 60 mg of N-hloronaphthol dissolved in 20 ml of ie-old methanol and 60 pl of 30% H,02 in 100 ml of Tris-saline. Preparative Eletrophoreti Elution and Proteolyti Digestion of Myosin Heavy Chain-Myosin (500 pg) was eletrophoresed in pre- FIG. 1. Reations of eah monolonal antibody with anine and alf myosin by enzyme-linked immunosorbent assays. Enzyme-linked immunosorbent assay was performed as desribed under "Experimental Proedures.'' Anti-myosin antibodies (CMA- 19, HMC48, and HMC50) were reated with anine ventriular myosin (0), anine atrial myosin (O), anine ventriular light hains (O), and bovine ventriular myosin (A). Results are expressed as the perentage of maximum absorbane (O.D.) at 540 nm. Charaterization of Atrial Myosin Heavy Chain Isozymes n E 0 25 W d 0 parative gels, whih were stained with Coomassie Brilliant Blue for 1 min, and the bands of myosin HC were ut from the gel. Myosin HC was eluted by eletrophoresis overnight into a dialysis bag and then dialyzed against olumn buffer. Limited proteolyti leavage of myosin HC with a-hymotrypsin (Worthington CDI-1450) was performed aording to the method of Cleveland et al. (39). Briefly, myosin (100 pg/ml) was leaved with a-hymotrypsin in the presene of SDS at a myosin to protease ratio of 4:l for 30 min and 81 for 30, 60, and 120 min at 37 "C. The reation was stopped by boiling for 3 min in the presene of 2% SDS and 6% meraptoethanol. The samples were eletrophoresed on 15% arylamide slab gels, using the Laemmli's buffer system at 10 ma onstant urrent for 12 h at 10 "C. Polypeptides were stained with silver aording to the method of Morrissey (40). For CNBr leavage, lyophilized myosin was dissolved in 70% formi aid and treated with solid CNBr in 200-fold exess over methionine residues for 18 h at 25 "C. The CNBr peptides were diluted with water and lyophilized. Amino aid analysis indiated a 94-96% ompletion after the digestion. Two-dimensional gels were run aording to the method of O'Farrell (41). In brief, myosin peptides (20 pg) were separated in the first dimension on isoeletri gels of 4% polyarylamide ontaining 2% Ampholine arrier ampholytes (LKB), onsisting of 1.6% of ph range 4-6 and 0.4% of ph range Gels were eletrophoresed at 350 V for 16 h. The seond dimension slab gel was omposed of 15% arylamide and 0.4% bisarylamide and was eletrophoresed at a onstant urrent of20 ma for 6 h. The gels were stained with silver as above. Determination of ATPase Ativities-Assays of atrial and ventriular myosin ATPase ativities were performed as previously desribed (1). In brief, Caz+-ativated ATPase ativities were assayed in 10 mm CaClZ, 5 mm ATP, and 0.05 M Tris-HC1 (ph 7.5), and K+-EDTAativated ATPase ativities were assayed in 0.6 M KCl, 1 mm EDTA, 5 mm ATP, and 0.05 M Tris-HC1 (ph 7.5). About 100 pg of myosin in 0.1-ml olumn buffer was given to 1.9 ml of the above reation mixture and inubated for 5 min at 25 "C. The reation was stopped by adding 1 mlof 20% trihloroaeti aid, and the preipitated protein was removed by entrifugation at 4 "C. ATPase ativities were determined by measuring the liberation of inorgani phosphate aording to the method of Youngberg and Youngberg (42). Protein was determined by the method of Lowry et al. (43) using rystalline bovine serum albumin as a standard. When the ATPase ativities of myosin isozymes were determined, 1.0 ml of immunoadsorbent Sepharose with myosins was mixed with 1.9 ml of the above reation mixture after washing with olumn buffer. The reation was terminated as above, and Sepharose was removed by entrifugation. The myosin ontent adsorbed with Sepharose was determined by measuring the dissoiated ontent. It had been onfirmed that the monolonal anti-myosin antibodies did not interfere with the ATPase ativities of myosin, beause there were no hanges in Ca2+- or K+-EDTA- ativated ATPase ativities when myosin was inubated with the immunoadsorbent Sepharose binding the monolonal antibodies. RESULTS 0 HMC 48 \\ MONOCLONAL ANTIBODY DILUTION Immunologial Charaterization of Atrial and Ventriular Myosin Isozymes-As illustrated in Fig. 1, HMC48 and

3 Charaterization of Atrial Myosin HeavyC h i n Ioszymes 4506 HC CMAlg HMC48 HMC50 ABCD A B C D A B C D FIG.3. Immunologial haraterizationof myosin isozymes by immunoblot tehnique.purified myosin isozymes wereeletrophoresed, transferred from the polyarylamide gels to nitroellulose sheets, and stained immunologially with monolonal antibodies AL2 (CMA19, HMC48, and HMC50), as desribed under "Experimental Proedures." Samples are: A, atrial HCn; R, atrial HCB; C, ventriular HCn; D,ventriular HCj3. Heavy hains of myosin are indiated by FIG.2. Analysis of purified myosin isozymes by SDS-poly- HC. arylamide gel eletrophoresis. About 5 pg of eah myosin isozyme, ventriular HCn ( A ) and HCP ( E ), atrial HCn (C) and HCB ( D ), was applied on a 7.5% slab gel, eletrophoresed a t a 10-mA r 3 -.onstant urrent for 8 h, and stained with silver as desribed under " r l "Experimental Proedures." The positions of the heavy hain subd units (HC)and of light hain subunits of ventriular (VLCZ and VLC2) and atrial (ALCI and ALC2) myosins are indiated. V L C l ALCl vl2 A B C D reated with anine ventriular myosinspeifially, and CMA19 reated with anine atrial myosin seletively. They did not appear to reat with light hains. The redued amount of ross-reativity between HMC48 or HMC50 and atrial myosin and between CMA19 and ventriular myosinsuggested the existene of a small amount of HCB in the atrium and HCa in the ventrile, respetively. On the other hand, HMC50 reated with alf ventriular myosin, but HMC48 did not. This result means that the antigeni determinant of HMC50 is different from that of HMC48, and, of ourse, CMAl9 reognizes a different epitope from the other two antibodies. Therefore, we used immunoblotting tehniques in order to haraterize the antigeni differenes between atrial HCa and atrial HCB. Purified myosin heavy hain isozymes, A-HCa, A-HCj3, VHCa, V-HCp, were eletrophoresed in the presene of SDS, transferred from the polyarylamidegels to nitroellulose sheets, and stained as desribed under "Experimental Proedures." Heavy and light hains of myosin were identified by Amido Blak staining, butwith immunologial staining, only A-HCj3 and V-HCp showed reativity with HMC48 and -50, while A-HCa and V-HCa reated with CMA19 but not with HMC48 nor HMC50. This result meant that A-HCB and VHCB ontained only HCB and that A-HCa and V-HCa were pure isozymes omposed of HCa (Fig. 3). Based on the reativity of the isozymes with HMC48 and -50, and CMA19, AHCP and V-HCp differed immunologially from A-HCa and V-HCa in at least three of their determinants, and A-HCB and V-HCj3 shared at least two antigeni determinants. Polypeptide Mapping-At least three strutures were demonstrated to be different, between HCa and HCj3 by immunologial omparisons. However, we further ompared the polypeptide fragments produed by proteolyti digestion of eahisozyme.fig. 4 shows representative one-dimensional peptide maps produed by a-hymotrypti digestion of four -- t I I FIG.4. One-dimensional gel analysisof partial hymotrypti digests of myosin isozymes.affinity-purified myosin isozymes. ventriular HCn ( A ),atrial HCn ( B ).atrial HCfi ( C ).and ventriular were leaved with a-hymotrypsin at a myosin to protease HCj3 (D), ratio of 4:l for 30 min and eletrophoresed at a 10-mA onstant urrent for 12 h as desribed under "Experimental Proedures." The asterisks and arrows indiate HCn-speifi peptides and HCP-speifi peptides, respetively. myosin isozymes. The peptide maps of A-HCa and V-HCa were indistinguishable as previously reported (12, 14, 17). Again, those of A-HCj3 and V-HCB were also very similar. The asterisks and arrows indiate HCa and HCB speifi peptides, respetively. There are several harateristi and reproduible differenes between HCa and HCB. To visualize a variety of peptides, time ourses for eah digestion were run (Fig. 5). The peptides of A-HCB were indistinguishable from

4 Charaterization of Atrial Myosin Heavy Chain Isozymes 120' 60' 4507 TABLE I 30' Comparison of ATPase atiuities of myosin isozymes A B C A B C A B C The assay solutions for Ca2+-and K+-EDTA-ativatedATPaae are those desribed under "Experimental Proedures." Valuesare means f S.E. for eight experiments. ATPane ativities Cay K'-EDTA pmol PJmg/rnin Atrial myosin ' 0.65 f 0.05 Ventriular myosin 0.35 f 0.03" 0.59 f 0.03 A-HCn 0.77 f 0.03" 0.61 f 0.06 A-HCB 0.30 f 0.06" V-HCa 0.75 f 0.06" 0.58 f 0.05 V-HCB 0.34 f 0.OT 0.54 f 0.05 Atrial myosin uersua ventriular myosin, A-HCa uersus A-HCB, and V-HCa uersua V-HCB;p < kinds of animals. However, there is a heterogeneity of ATPase ativity level, evenin the atrial myosins (Table I). DISCUSSION FIG. 5. Kinetistudy of partialhymotryptidigests of myosin isozymes. Peptides of myosin isozymes were digested by ahymotrypsin at a myosin to protease ratio of 8:l for 30, 60, and 120 minandwereeletrophoresed as desribedunder"experimental Proedures."Samples are:a, atrial HCa;B, atrial HCB; C, ventriular HC& The asterisks and arrows indiated HCa-speifi peptides and HCP-speifi peptides, respetively. V-HCB, while there were onsistent differenes between the A-HCa and A-HCB peptides. In two-dimensional gel analysis of peptides produed by omplete CNBr digestion, A-HCB and V-HCB werealso very similar, if not idential, while the map of A-HCa was different from those of A-HCB and V-HCB (Fig. 6). Thus, both the one- and two-dimensional peptide maps onfirmed the immunologial data that A-HCB and V-HCB wereindistinguishable proteins and that they were different from A-HCa. ATPase Ativities of Eah Isozyme-To know the enzymati properties of eah isozyme, we assayed Ca2+-and K+-EDTAativated ATPase ativities. A high level of Ca2+-ativated ATPase ativity was observed in myosin isozymes omposed of HCa; their ativity levels (A-HCa, 0.77 f 0.03; V-HCa, 0.75 f 0.06 pmol of P,/mg/min) were about twie as great as those of the HCB isozymes (A-HCB,0.30 k 0.06; V-HCB, pmol/mg/min) ( p < 0.001). However, there were lose similarities in ATPase ativity level between A-HCa and VHCa and between A-HCB and V-HCB, respetively. It has been demonstrated that the ATPase ativity levels of atrial myosins are higher than those of ventriular myosins in many FIG. 6. Two-dimensional gel analysis of peptidesproduedby omplete CNBr digestion. Peptides of eah myosin isozyme, atrialhca ( A ), atrial HCP ( E ),and ventriularhcb (C), were isoeletrophoresed at first (left to right) and eletrophoresed in the presene of SDS (top to bottom). The asterisks indiate HCa-speifi peptides and arrows indiate HCO-speifipeptides. In this study, we learly showedthe presene of two types of myosin heavy hains in the anine atrium (A-HCa and AHCB) and the Similaritiesbetween A-HCB and V-HCB in peptide omposition and enzymati ativities. We purified four isozymes of myosin heavy hains (A-HCa, A-HCB, VHCa, and V-HCB) from the anine atrium and ventrile by affinity hromatography using monolonal antibodies and haraterized these isozymes. Byimmunologialomparisons, A-HCB was different from A-HCa in at least three epitopes, and A-HCB and V-HCB shared at least two antigeni determinants. Strutural differenes were also demonstrated by one- and two-dimensional peptide mapping, produedby partial andomplete digestion usinga-hymotrypsin and CNBr, respetively. There were onsistently different peptides between A-HCa and A-HCB, while A-HCB was indistinguishable from V-HCB. In theassay of Ca2+-ativatedATPase ativities, A-HCB and A-HCa showed different enzymati properties. Thus, it was demonstrated that two different isozymes of myosin heavyhains (A-HCa anda-hcb) exist in the atrium as well as in the ventrile (V-HCa and V-HCB) and that AHCB, whih might inrease in the pressure-overloaded atria, is indistinguishable from V-HCB, the predominant form of ventriular myosin, in ita moleular struture and ATPase ativity. Muh information has been aumulated about ventriular myosin isozymes. There are two isozymes, HCa and HCB, in the ventrile, and they are differentially expressed during development, in response to thyroid hormones and ardia pressure overload. The differenes between them have been

5 4508 Charaterization of Atrial Myosin Heavy Chain Ioszymes demonstrated by peptide mapping (9-11, 13), by immunologial properties (11-13,16), by measurement of ATPase ativities (1-7), and by sequene analysis of DNA (44-48). Reently, the two genes oding for the two myosin heavy hains have been isolated and shown to be organized in tandem (47). Furthermore, it has been shown that ventriular HCa is indistinguishable from atrial myosin (14), and the same HC gene is expressed both in the atria and ventriles (46, 47). In ontrast, there has been little experimental evidene onerning the heterogeneity of or hanges in isozymes of atrial myosin heavy hains until quite reently. By gel eletrophoresis of native ardia myosins under nondenaturing onditions, Hoh et ul. (8) found that atrial myosins ould be separated into two omponents, A1 and A2 (8), but using a ross-hybridization tehnique, Chizzonite et al. (14) showed that A1 and A2 are separated as a result of some heterogeneity in atrial myosin light hains and that the heavy hains of these two isozymes are most likely idential (14). Thus, isozymes of atrial myosin heavy hains have not yet been isolated. It is diffiult to separate the myosin isozymes of larger animals, suh as dogs, attle, and humans, by pyrophosphate gel eletrophoresis, so most previous studies of ardia myosin isozymes were restrited to the smaller animals, suh as mie, rats, and rabbits. Sine the predominant isozymes of ventriular myosin heavy hains differ between smaller animals and larger animals, the experimental information about the smaller animals annot thus be applied diretly to the ardia myosin of larger animals. Reently, by immunohistohemial studies, espeially using monolonal antibodies, it was revealed that there are two isozymes of ardia myosin in bovine and human heart and that although HCa and HCP are predominant isozymes in the atrium and ventrile, respetively, in larger animals, HCP and HCa! also o-exist in both the atrium and ventrile. Furthermore, our laboratory revealed that in pressure-overloaded atria, the HCP ontent inreased strikingly while that of HCa showed a orresponding derease (19, 20). But this atrial HCP was the myosin isozyme whih was reognized by the antibodies of HCP in the ventrile. Its moleular strutures and enzymati properties had not previously been known at all. However, in this study, we demonstrated that the HCP in the atrium was similar to HCP in the ventrile in its peptide omposition and enzymati ativities. We demonstrated the differenes in antigeni strutures and proteolyti fragments between A-HCa and A-HCP, but there were no differenes between the LCs of A-HCa and A- HCP in their eletrophoreti pattern in the presene of SDS (Fig. 2). In ontrast, A-HCP was indistinguishable from V- HCP in its moleular struture and enzymati ativities, but the LCs of A-HCP and V-HCP were distintly different. Reently Robert et al. (49) have demonstrated that the myosin LC genes of mouse atrium and ventrile are loated on different hromosomes, the atrial LC gene being on hromosome 11 and the ventriular LC gene being on hromosome 9, and heavy hain genes have been shown to be loated on hro- mosome 11 (50). These results and observations suggested that HCs and LCs might be regulated independently. In a omparative study of ATPase ativities, we assayed the ATPase ativities of isolated myosin isozymes in an immunoadsorbent Sepharose-bound form, sine guanidine HC1 in the elution buffer suppresses ATPase ativities. These monolonal antibodies did not interfere with the ATPase ativities of myosin isozymes, as all but one of the monolonal antibodies (51) do not interfere with the enzymati ativities of myosin alone. Our results demonstrated that the Ca2+- ativated ATPase ativities of the two isozymes of atrial myosin (A-HCa and A-HCB) were quite different and that the ATPase ativity of A-HCB was very similar to that of V- HCP. Barany (52) reported that Ca2+-ativated ATPase ativities of the myosin were generally proportional to the speed of shortening of their respetive musles, so two myosin isozymes that have different ATPase ativities may be regulated to meet the ontratile ativity of ardia musle under onditions of pressure overload. In this study, we onluded that there are two different isozymes of myosin heavy hains in anine atrium and that A-HCP and V-HCP are almost idential in peptide omposition and enzymati ativities. 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