Differences in cardiac myosin light chain LC1 among human,monkey and sheep

Summary The atrial and ventricular myosin light chains of human, monkey and sheep hearts were compared by dodecylsulfate polyacrylamide gel electrophoresis. The atrial light chain 2 and ventricular light chain 2 are similar among these mammals. However, the atrial light chain 1 of monkey has different electrophoretic mobility from those of human and sheep. The monkey ventricular light chain 1 has same mobility as that of sheep but different from that of human.Acknowledgments. This work was supported by MRC of Canada No. MA-8559. Dr G. Jackowski is a scholar of the Medical Research Council of Canada, and is the author to whom all correspondence should be addressed.     

Fragmentation of myosin A-1 light chain of fast muscle by trypsin]

Limited proteolysis of myosin by such proteolytic enzymes as trypsin, chymotrypsin or papain produces typical fragmentation of its heavy chain. Presently evidence is given that trypsin treatment cleaves the alkali light chain A-1 (20,700 dalton) to a shorter (ca 20,000 dalton) chain. The two "essential" thiols (SH-1 and 2) of moysin were alkylated with 17-C-N-ethylmaleimide and a non-negligible amount of radioactivity was also found in the two alkali light chains. Using the specific radioactivity of alkali light chain A-1 it was possible to identify it among heavy chain fragmentation products. The molecular weight of the newly formed A-1 indicates that limited tryptic cleavage of this A-1 confers on it a closer similarity with alkali light chain A-2.     

A Ca2+-sensitive myosin light chain kinase,regulating pig carotid smooth muscle actomyosin ATPase1

Summary In this paper the correlation between phosphate incorporation into the regulatory light chain of myosin by a Ca²⁺-dependent myosin light chain kinase, and the Ca²⁺-sensitive ATPase activity and superprecipitation behaviour of arterial actomyosin, is described.Acknowledgment. The financial support of the Deutsche Forschungsgemeinschaft (SFB 90) is gratefully acknowledged.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

In smooth muscle the Mr 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of Mr 17,000 that binds 4 moles of Ca2+; and a larger protein of Mr circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca2+. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of Mr 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of Mr 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca2+-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

Summary In smooth muscle the M_r 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of M_r 17,000 that binds 4 moles of Ca²⁺; and a larger protein of M_r circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca²⁺. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of M_r 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of M_r 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca²⁺-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

A Ca2+-sensitive myosin light chain kinase, regulating pig carotid smooth muscle actomyosin ATPase

In this paper the correlation between phosphate incorporation into the regulatory light chain of myosin by a Ca2+-dependent myosin light chain kinase, and the Ca2+-sensitive ATPase activity and superprecipitation behaviour of arterial actomyosin, is described.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

The contraction induced by a Ca2+-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (Fo), immediate elastic recoil (SE), unloaded shortening velocity (Vus), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca2+ -induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. Fo developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. Vus in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3 +/- 8.9% less than Vus induced by Ca2+ (1.6 X 10(-6) M) in the control fibers. Addition of Ca2+ (1.6 X 10(-6) M) to a contraction induced by MLCK-resulted in small increases in both Fo and Vus. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca2+. We conclude that with respect to actin-myosin interaction, MLCK-and Ca2+ -induced contractions are similar.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

Summary The contraction induced by a Ca²⁺-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (F_o), immediate elastic recoil (SE), unloaded shortening velocity (V_us), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca²⁺-induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. F_o developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. V_us in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3±8.9% less than V_us induced by Ca²⁺ (1.6x10^–6M) in the control fibers. Addition of Ca²⁺ (1.6x10^–6M) to a contraction induced by MLCK-resulted in small increases in both F_o and V_us. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca²⁺. We conclude that with respect to actin-myosin interaction, MLCK- and Ca²⁺-induced contractions are similar.     

Heavy and light roles: myosin in the morphogenesis of the heart

Myosin is an essential component of cardiac muscle, from the onset of cardiogenesis through to the adult heart. Although traditionally known for its role in energy transduction and force development, recent studies suggest that both myosin heavy-chain and myosin light-chain proteins are required for a correctly formed heart. Myosins are structural proteins that are not only expressed from early stages of heart development, but when mutated in humans they may give rise to congenital heart defects. This review will discuss the roles of myosin, specifically with regards to the developing heart. The expression of each myosin protein will be described, and the effects that altering expression has on the heart in embryogenesis in different animal models will be discussed. The human molecular genetics of the myosins will also be reviewed.     

Localization of human A,B and H isoantigens in Cynomolgus monkey tissues

Summary The presence and distribution of human A, B and H isoantigens were demonstrated in Cynomolgus monkey (Macaca fascicularis) by means of red cell adherence test. Although no human antigens were found on primate erythrocytes, various epithelial tissues revealed the presence of A, B or H antigenic substance. The distribution and localization was similar to that found in human tissues. Majority of specimens from each individual animal possessed only 1 human type isoantigen with the exception of the salivary and sweat glands, where all animals showed the presence of H antigen in addition to other specificity, and of Brunner's gland, where all sections reacted positively also for A antigen.     

Intramuscular comparison of myosin isozymes and light chains in rat extensor digitorum longus muscle

Summary Complete muscle cross sections were obtained from the proximal and distal third regions of ten rat extensor digitorum longus muscles. Electrophoretic methods were then used to quantify the various myosin isozymes and light chains in each muscle specimen. The results demonstrated that the relative distribution of the various myosin isozyme and light chain variables do not vary significantly between the two sampling regions.     

Intramuscular comparison of myosin isozymes and light chains in rat extensor digitorum longus muscle

Complete muscle cross sections were obtained from the proximal and distal third regions of ten rat extensor digitorum longus muscles. Electrophoretic methods were then used to quantify the various myosin isozymes and light chains in each muscle specimen. The results demonstrated that the relative distribution of the various myosin isozyme and light chain variables do not vary significantly between the two sampling regions.     

Difference in the sensitivity to Ca ion among glycerinated skeletal, cardiac and smooth muscles

Glycerinated smooth muscle was contracted almost maximally with 15 mM Mg and 5 mM ATP, while extracted skeletal and cardiac muscles needed Ca ion with 15 mM Mg and 5 mM ATP for producing contraction.     

Difference in the sensitivity to Ca ion among glycerinated skeletal,cardiac and smooth muscles

Summary Glycerinated smooth muscle was contracted almost maximally with 15 mM Mg and 5 mM ATP, while extracted skeletal and cardiac muscles needed Ca ion with 15 mM Mg and 5 mM ATP for producing contraction.Acknowledgments. I thank Prof. T. Suzuki and Dr H. Nakanishi for helpful suggestions, and Mr G. Ito for technical assistance.     

Localization of ceruloplasmin biosynthesis in human and monkey liver cells and its copper regulation

Zusammenfassung Aus Affenserum (Macacus rhesus) ist das Zeruloplasmin in Form einer homogenen Eiweisssubstanz isoliert und in ihren physikalisch-chemischen Konstanten näher bestimmt worden. Der Vergleich der Eigenschaften des Zeruloplasmins des Menschen und des Affen ergibt eine Ähnlichkeit dieser beiden Substanzen. Bei der Untersuchung mittels spezifischer Präzipitation im Agar haben sich beim Antiserum des Kaninchens Beweisgründe für eine antigene Identität beider Zeruloplasmine ergeben. An Gewebeschnitten der Affenleber inkubiert bei aktiv oxydativer Phosphorylierung ergab sich die Bindung des Zeruloplasmins in vitro. Resynthetisierte Eiweißsubstanz bildet einen spezifischen Niederschlag mit dem Antiserum des Kaninchens, welches gegen das Zeruloplasmin des Menschen immunisiert ist. Mit Hilfe lumineszierender Antikörper ist die spezifische Lumineszenz des Zeruloplasmins nur in parenchimatösen Leberzellen gefunden worden. Die Biosynthese des Zeruloplasmins ist in der Leber lokalisiert. Aus In-vitro-Versuchen an Gewebeschnitten der Leber beim Affen und entsprechenden In-vivo-Versuchen ergibt sich, dass Kupfersalze in geringen Konzentrationen die Biosynthese verstärken, in grossen Konzentrationen hingegen die Biosynthese des Zeruloplasmins hemmen.     

Molecular cloning of the fibroin light chain complementary DNA and its use in the study of the expression of the light chain gene in the posterior silk gland of Bombyx mori

Fibroin light chain (L-chain) mRNA (mol. wt 4.0 X 10(5) daltons) was purified from the posterior silk gland of the silkworm, Bombyx mori (J-131 strain). Double-stranded complementary DNA was synthesized and inserted into the PstI site of pBR322 employing the oligo(dC)-oligo(dG) tailing method. Several recombinant plasmids containing the inserts of about 800 base pairs were isolated. Hybridization-translation assay demonstrated that these clones hybridized specifically with the fibroin L-chain mRNA. One of these clones (pLA23) was used as a probe to investigate relative concentrations of the fibroin L-chain gene and mRNA in the posterior silk glands at different stages of late larval development.