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

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.     

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.     

东亚三角涡虫肌球蛋白轻链（MLC）融合蛋白原核表达载体的构建及其在大肠杆菌中的表达

构建了涡虫肌球蛋白轻链融合蛋白原核表达载体,并进行表达,根据涡虫基因文库中肌球蛋白轻链（Mlc）基因完整的ORF序列设计合成特异性51物,通过PCR扩增涡虫Mlc基因,并插入到融合蛋白原核表达载体PET-28a中,转化宿主菌B121（DE3）细胞．0．4mMol／L的IPTG诱导表达MLc蛋白．重组质粒测序和酶切结果显示Mlc基因已正确插入PET-28a中,重组蛋白经SDS—PAGE在18．2KD处有一条明显的蛋白表达条带。western blot检测得到同样大小的条带。结果表明,涡虫His-MLC融合蛋白已成功表达。     

Minoxidil-induced cardiac hypertrophy in guinea pigs

To investigate whether during cardiac hypertrophy changes occur in contractile protein composition and in mechanical and energetic properties of the myocardium, contractile protein composition, isometric force and adenosine triphosphate (ATP) consumption were studied in control and hypertrophied guinea-pig hearts. Cardiac hypertrophy was induced by adding minoxidil (120 or 200 mg/l) to the drinking water. Protein analysis was performed by one-dimensional gel electrophoresis. The myosin heavy-chain (MHC) composition was determined in an enzyme-linked immunosorbent assay (ELISA). ATP consumption and force development were simultaneously measured during isometric contraction in chemically skinned trabeculae. Histochemical analysis of cross-sectional area of cardiomyocytes and interstitial space was performed on the left ventricular tissue of 200 mg/l minoxidil-treated and control guinea pigs. Minoxidil treatment (120 and 200 mg/l) significantly increased left ventricular dry weight normalized for body weight by 19 ± 4 and 24 ± 4%, respectively. No significant differences were found in the cellular cross-sectional area, while interstitial space was slightly decreased in minoxidil-treated hearts. In left ventricular trabeculae of 200 mg/l minoxidil-treated guinea pigs, ATPase activity was slightly less than in those of control guinea pigs, whereas force did not differ significantly. Calcium sensitivity of force and ATPase activity were not affected by minoxidil treatment. Gel electrophoresis revealed no difference in contractile protein composition, but a tendency towards a lower amount of α-MHC in the minoxidil-treated hearts was found in ELISA. Received 1 February 1999; accepted 15 March 1999     

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.     

Myosin regulation in the migration of tumor cells and leukocytes within a three-dimensional collagen matrix

The migration of cells is a complex regulatory process which results in the generation of motor forces through the reorganization of the cytoskeleton. Here we present a comparative study of the expression and involvement of myosin in the regulation of the physiological migration of leukocytes and the pathological migration of tumor cells. We show that the involvement of myosin in the migration is distinct in these two cell types. In leukocytes, the activity of non-muscle myosin II is essential for both the spontaneous (matrix-induced) migration and the migration induced by ligands to G protein-coupled receptors, i.e. chemokines and neurotransmitters. In contrast, spontaneous tumor cell migration is largely independent of non-muscle myosin II activity, whereas the norepinephrine-induced migration is completely inhibited by either direct inhibition of non-muscle myosin II or of the kinases phosphorylating the myosin light chain, namely ROCK or the calcium/calmodulin-dependent myosin light-chain kinase.Received 31 August 2004; accepted 26 October 2004     

Selenoprotein W: a review

Purification of selenoprotein W (Se-W) from rat and monkey muscles was shown to exist in multiple forms: with or without reduced glutathione and/or a 41-Da moiety (identity still unknown). TGA is located at coding position 13 in Se-W complementary DNA (cDNA) from all five species studied (rats, mice, sheep, human and monkey). TGA is also the stop codon in the rodents and sheep cDNA, but TAA is the stop codon in primates. There is an 80% homology of the nucleotide sequence in the coding region among the five species of animals, and the predicted amino acid sequences are 83% identical (rodents identical and primates identical). Se-W levels are highest in muscle, heart and brain from sheep and primates, but very low in rodent hearts. Studies with tissue cultures of muscle and brain cells indicated that selenium influenced Se-W levels. Although the metabolic function of Se-W is unknown, preliminary data suggest that it has an antioxidant function.     

MSAP enhances migration of C6 glioma cells through phosphorylation of the myosin regulatory light chain

A key regulatory mechanism in cell motility is the control of myosin activity, which in non-muscle cells is determined by phosphorylation of the myosin regulatory light chain (MRLC). Here we show that MRLC-interacting protein (MIR)-interacting saposin-like protein (MSAP) enhances cell spreading in fibroblasts and migration of rat C6 glioma cells through increases in MRLC phosphorylation. Overexpression of MSAP enhanced the motility of glioma cells measured in matrigel invasion chambers and using a scratch assay. Downregulation of MSAP by RNA interference significantly decreased glioma cell migration and phosphorylation of MRLC. Inhibition of the corresponding MRLC kinase by ML-7 did not affect migration of MSAP-overexpressing cells. The present results show that MSAP controls glioma cell migration via enhancement of MRLC phosphorylation. This effect is independent of the activity of MRLC kinase. Thus, MSAP is a novel modulator of cell motility that influences migration of glioma cells and possibly other tumors.Received 9 February 2005; received after revision 2 March 2005; accepted 21 March 2005     

The role of myosin phosphorylation in the contraction-relaxation cycle of smooth muscle

Summary Considerable evidence from a variety of experimental procedures indicates that the phosphorylation of myosin is involved in the regulation of contractile activity in smooth muscle. Phosphorylation of the 20,000-dalton myosin light chains is required to initiate crossbridge cycling and this is consistent with the observation that the actin-activated Mg²⁺-ATPase activity of myosin is phosphorylation-dependent. In the simplest interpretation of this process it may be proposed that phosphorylation acts as an on-off switch. Clearly this cannot explain the observed complexity of smooth muscle contractile behavior and such may imply either that additional mechanisms are involved or that the role of myosin phosphorylation is not fully appreciated. Recently it has been shown that monomeric smooth muscle myosin can exist in a folded and an extended conformation and that each form is characterized by distinct enzymatic properties. Under appropriate solvent conditions phosphorylation of myosin favors the extended conformation. It is tentatively suggest that this, or an analogous, transition might be involved in the regulation of the smooth muscle contractile apparatus, and this possibility is discussed.The authors are supported by grants HL 23615 and HL 20984 from the National Institutes of Health.     

Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion

Summary Transients in myoplasmic [Ca²⁺] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K⁺-stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca²⁺ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an imporoved estimate of activation rates.Supported by a grant from the National Institutes of Health 5-PO1-HL19242. K. E. Kamm was supported by a National Heart, Lung, and Blood Institute Research Service Award HL-05957.     

Acid-base equilibrium in the blood of sheep.

The acid-base equilibrium in the blood of sheep is different from that of human blood mainly because of a lower concentration of 2,3-DPG. A nomogram relating pH, pCO2, total CO2 content and base excess has been developed.     

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.     

Energetics and regulation of crossbridge states in mammalian smooth muscle

Conclusion On the basis of measurements of the high energy phosphate usage associated with different mechanical states, as well as the degree of myosin light chain phosphorylation and mechanical properties, information has been gained concerning the existence and regulation of different crossbridge states in smooth muscle. Although incomplete, a general operational scheme is shown in figure 5. At very low intracellular calcium concentrations, actin and myosin are dissociated, as shown by a loss of resistance to stretch in resting muscles. At somewhat higher intracellular calcium concentrations in atonic, resting muscles, crossbridges can attach and be manifest mechanically as an increased resistance to stretch without ATP-driven crossbridge cycling and active force production. When the muscle is activated, intracellular calcium increases further, the light chains of myosin are phosphorylated through the calcium-calmodulin activation of myosin light chain kinase, actin-activated myosin ATPase activity increases and crossbridges cycle. Calcium also appears to modulate the ATPase activity and the rate of cycling of the phosphorylated crossbridge. The crossbridge cycling rate is highest during force development and slows with time as maximum isometric force is maintained reflecting a change in the rate at which phosphorylated crossbridges cycle. This may result from a decrease in the intracellular free calcium concentration with continued stimulation. During relaxation, the intracellular calcium concentration decreases, there is net dephosphorylation of the myosin light chains, the rate at which phosphorylated crossbridges cycle slows further with a gradual return to the attached, but non-cycling state or the detached state.     

Acid-base equilibrium in the blood of sheep

Summary The acid-base equilibrium in the blood of sheep is different from that of human blood mainly because of a lower concentration of 2,3-DPG. A nomogram relating pH, pCO₂, total CO₂ content and base excess has been developed.     

Relationship of specific granules to the natriuretic and diuretic activity of rat atria

Summary The isolation of several fractions from rat atrial homogenates, by the use of differential and sucrose gradient centrifugation, indicates that the diuretic and natriuretic activity is restricted to the fractions rich in specific granules. Our preliminary results suggest that the active substance is a small peptide which is probably different from the natriuretic substance(s) already known.Supported by a group grant given by the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension of the Clinical Research Institute of Montreal.Acknowledgments. The authors thank Suzanne Diebold for technical assistance.     

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.     

Different intrafusal fiber composition of spindles in sheep and pig extraocular muscles

Summary Histochemical profiles of intrafusal fibers have been examined in muscle spindles of extraocular muscles of sheep and pig. Results show that in the sheep the intrafusal content presents, in addition to chain fibers, at least one bag₁ and one bag₂ fiber, whereas in the pig almost all the spindles are bag₁-fiber spindles.Acknowledgment. This study was supported by grants from C.N.R. and M.P.I.     

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.