283
Biochimica et Biophysica Acta, 581 (1979) 283--294 © Elsevier/North-Holland Biomedical Press
BBA 38303
COMPARATIVE ANALYSIS OF CHICKEN ATRIAL AND VENTRICULAR MYOSINS
L. DALLA LIBERA, S. SARTORE and S. SCHIAFFINO
National Research Council Unit for Muscle Biology and Physiopathology, Institute of General Pathology, University of Padova, 35100 Padova (Italy) (Received April 4th, 1979)
Key words: Myosin polymorphism; Atrial myosin; Ventricular myosin; (Chicken heart)
Summary 1. Structural and enzymic properties of myosins from atrial and ventricular cardiac muscle of the chicken were investigated and compared with myosins from the fast skeletal pectoralis and the slow skeletal anterior latissimus dorsi muscle. 2. The Ca2*-ATPase activity, both in function of pH and [K÷], of atrial myosin closely resembled that of the fast pectoralis myosin, whereas the enzymic properties of ventricular myosin were similar to those of slow skeletal myosin. 3. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on gradient gel and two~limensional electrophoresis, involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension, no difference could be demonstrated in the light-chain pattern of atrial and ventricular myosin. Complete identity was also found between anterior latissimus dorsi and cardiac light chains. 4. Electrophoretic analysis of soluble peptides released by tryptic digestion of myosin and electron microscopic study of light meromyosin paracrystals showed significant differences between the heavy chains of atrial and ventricular myosins, as well as between the heavy chains of cardiac and skeletal myosins. 5. The results confirm previous immunochemical findings and provide direct biochemical evidence for the existence of a new, unique type of myosin in the chicken atrial tissue.
Abbreviation: SDS, sodium dodecyl sulphate.
284 Introduction
Cardiac muscle has been generally considered as a homogeneous tissue in biochemical studies concerned with contractile proteins. However, in the last years it has been shown that different types of myosin are present in cardiac muscle cells of mammals and birds [1--3]. In a immunohistochemical study of the chicken heart we found that muscle cells from atrial, ventricular and conduction tissue contain antigenically distinct myosins [2]. In particular atrial myosin was antigenically different from ventricular myosin and showed immunological cross-reactivity with myosin from fast skeletal muscle. We have therefore undertaken a biochemical study to compare the structural and functional properties of chicken atrial and ventricular myosin with fast and slow skeletal myosin. The results show that atrial myosin can be clearly distinguished from all other types of cardiac and skeletal myosins investigated and must therefore represent a new type of myosin. Materials and Methods
Myosin preparation. Freshly excised cardiac and skeletal muscles of adult chicken were used for myosin preparation. Atrial and ventricular myosins were obtained from pooled right and left atria and ventricles, respectively. The skeletal muscles used were the fast pectoralis and the slow anterior latissimus dorsi. Myosin was purified according to previously reported procedures [4,5] as specifically detailed in Dalla Libera et al. [6]. Protein concentration was determined b y the method of Lowry et al. [7]. Myosin ATPase activity. (K÷/EDTA) - or Ca2÷-activated ATPase activity was assayed as described by Balint et al. [8]. The assay mixture for (K÷/EDTA) ATPase contained 0.6 M KC1, 10 mM EDTA, 2.5 mM ATP, 50 mM Tris-HC1, pH 7.6. The assay mixture for Ca2÷-ATPase contained 25 mM KC1, 10 mM CaC12, 2.5 mM ATP, 50 mM Tris-HC1, pH 7.6. Protein concentration was 0.1 mg/ml in a final volume of 1.0 ml. The reaction was carried out for 5 min at 25 ° C. The reaction was started by adding ATP, and the Pi liberated was measured as described by Lindberg and Ernster [ 9]. Alkali lability of the myosin ATPase activity was assayed by preincubating myosin at pH 9.2 for 10 min before the reaction [10]. The actin-stimulated ATPase activity of myosin was determined as described by Barany and Close [4]. Protein concentration of myosin was 0.2 mg/ml. Gel electrophoresis. One
Biochimica et Biophysica Acta, 581 (1979) 283--294 © Elsevier/North-Holland Biomedical Press
BBA 38303
COMPARATIVE ANALYSIS OF CHICKEN ATRIAL AND VENTRICULAR MYOSINS
L. DALLA LIBERA, S. SARTORE and S. SCHIAFFINO
National Research Council Unit for Muscle Biology and Physiopathology, Institute of General Pathology, University of Padova, 35100 Padova (Italy) (Received April 4th, 1979)
Key words: Myosin polymorphism; Atrial myosin; Ventricular myosin; (Chicken heart)
Summary 1. Structural and enzymic properties of myosins from atrial and ventricular cardiac muscle of the chicken were investigated and compared with myosins from the fast skeletal pectoralis and the slow skeletal anterior latissimus dorsi muscle. 2. The Ca2*-ATPase activity, both in function of pH and [K÷], of atrial myosin closely resembled that of the fast pectoralis myosin, whereas the enzymic properties of ventricular myosin were similar to those of slow skeletal myosin. 3. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on gradient gel and two~limensional electrophoresis, involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension, no difference could be demonstrated in the light-chain pattern of atrial and ventricular myosin. Complete identity was also found between anterior latissimus dorsi and cardiac light chains. 4. Electrophoretic analysis of soluble peptides released by tryptic digestion of myosin and electron microscopic study of light meromyosin paracrystals showed significant differences between the heavy chains of atrial and ventricular myosins, as well as between the heavy chains of cardiac and skeletal myosins. 5. The results confirm previous immunochemical findings and provide direct biochemical evidence for the existence of a new, unique type of myosin in the chicken atrial tissue.
Abbreviation: SDS, sodium dodecyl sulphate.
284 Introduction
Cardiac muscle has been generally considered as a homogeneous tissue in biochemical studies concerned with contractile proteins. However, in the last years it has been shown that different types of myosin are present in cardiac muscle cells of mammals and birds [1--3]. In a immunohistochemical study of the chicken heart we found that muscle cells from atrial, ventricular and conduction tissue contain antigenically distinct myosins [2]. In particular atrial myosin was antigenically different from ventricular myosin and showed immunological cross-reactivity with myosin from fast skeletal muscle. We have therefore undertaken a biochemical study to compare the structural and functional properties of chicken atrial and ventricular myosin with fast and slow skeletal myosin. The results show that atrial myosin can be clearly distinguished from all other types of cardiac and skeletal myosins investigated and must therefore represent a new type of myosin. Materials and Methods
Myosin preparation. Freshly excised cardiac and skeletal muscles of adult chicken were used for myosin preparation. Atrial and ventricular myosins were obtained from pooled right and left atria and ventricles, respectively. The skeletal muscles used were the fast pectoralis and the slow anterior latissimus dorsi. Myosin was purified according to previously reported procedures [4,5] as specifically detailed in Dalla Libera et al. [6]. Protein concentration was determined b y the method of Lowry et al. [7]. Myosin ATPase activity. (K÷/EDTA) - or Ca2÷-activated ATPase activity was assayed as described by Balint et al. [8]. The assay mixture for (K÷/EDTA) ATPase contained 0.6 M KC1, 10 mM EDTA, 2.5 mM ATP, 50 mM Tris-HC1, pH 7.6. The assay mixture for Ca2÷-ATPase contained 25 mM KC1, 10 mM CaC12, 2.5 mM ATP, 50 mM Tris-HC1, pH 7.6. Protein concentration was 0.1 mg/ml in a final volume of 1.0 ml. The reaction was carried out for 5 min at 25 ° C. The reaction was started by adding ATP, and the Pi liberated was measured as described by Lindberg and Ernster [ 9]. Alkali lability of the myosin ATPase activity was assayed by preincubating myosin at pH 9.2 for 10 min before the reaction [10]. The actin-stimulated ATPase activity of myosin was determined as described by Barany and Close [4]. Protein concentration of myosin was 0.2 mg/ml. Gel electrophoresis. One
