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

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.     

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.     

Effect of phosphorylation of myosin light chains on interaction of heavy meromyosin with regulated F-actin in ghost fibers

Summary The binding of phosphorylated heavy meromyosin to regulated F-actin in ghost fibers at high Ca²⁺ concentration increases, and at low Ca²⁺ concentration decreases, the anisotropy of intrinsic tryptophan fluorescence of F-actin. The effect is opposite to the effect of the binding of dephosphorylated heavy meromyosin.     

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.     

Complex formation betweenγ-immunoglobulin and calmodulin in calcium-free conditions

We show that -immunoglobulin (IgG) binds calmodulin (CaM) in a Ca²⁺-independent manner, with Kd value of (1.7±0.5)×10^–7M. A single IgG molecule maximally bound 10 CaM molecules. The binding is to the heavy chain or Fab portion, but not the Fc portion, of the IgG molecules. Ca²⁺ greatly diminished the interaction between IgG and CaM, with IC₅₀=8–9M. These data give a novel insight into protein-protein interactions.     

Expression of alpha-cardiac myosin heavy chain in mammalian skeletal muscle

We have investigated the reactivity of different human, rat and cat muscles to a monoclonal antibody directed against human -cardiac myosin heavy chain. We have found that special fiber subpopulations of human massetr and extraocular muscles, as well as the bag fibers of human, rat and cat muscle spindles, were reactive to this antibody, indicating that these fibers expressed -cardiac myosin heavy chain or a closely related isoform. This isomyosin was present in the spindle bag fibers at early fetal stages, whereas its expression in masseter and extraocular muscle fibers was not detected during the first 22 weeks of gestation. Our results add to the list of muscle proteins which are expressed in locations or at developmental stages other than those initially described, suggesting that a revision of the present nomenclature of the subgroups of myosin heavy chains might be considered in the future.     

Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1

Summary Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K⁺- and Ca²⁺-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.The support of this work by an EMBO short time fellowship (to R. L.) and by the Deutsche Forschungsgemeinschaft (No. SFB 90, B 7) and the excellent technical assistance byM. Troschka andC. Köhler are gratefully acknowledged.     

Dynamic laser light scattering studies of the effects of pyrophosphate on cyclic motions of cross-bridges in isolated thick myofilaments fromLimulus striated muscle

Pyrophosphate (PP_i) is a non-hydrolyzable ATP analogue known to affect the binding between myosin heads and actin. By using a dynamic laser light scattering method, we have shown that 1–2 mM PP_i enhances the increase in values induced by Ca²⁺ in isolated thick myofilaments fromLimulus striated muscle. However, similar treatment has no effect on the values of filaments suspended in either relaxing solution or ATP-free solution. is the average linewidth of the photoelectron count autocorrelation function of the light scattered. PP_i had no effect on the increase of values by Sr²⁺ but it substantially increased the values of the thick myofilaments suspended in Ba²⁺-substituted Ca²⁺ activating solution. The results show that PP_i also affects the energy-requiring cyclic cross-bridge motions.     

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction

Summary Zn²⁺ (10–100 M) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca²⁺. Botulinum type A toxin (BTX_A, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn²⁺ (100 M) restored MEPPs and increased their frequency after they had been abolished by BTX_A in Ca²⁺-free solutions. The antagonistic effect of Zn²⁺ in the Ca²⁺-free solution was reduced by exposing the diaphragm to the toxin in the Ca²⁺-free solutions containing high K⁺. Thus, the action of BTX_A is probably enhanced by depolarization of the motor nerve terminals.     

Functional characterization of the human myosin-7a motor domain

Myosin-7a participates in auditory and visual processes. Defects in MYO7A, the gene encoding the myosin-7a heavy chain, are causative for Usher syndrome 1B, the most frequent cause of deaf-blindness in humans. In the present study, we performed a detailed kinetic and functional characterization of the isolated human myosin-7a motor domain to elucidate the details of chemomechanical coupling and the regulation of motor function. A rate-limiting, slow ADP release step causes long lifetimes of strong actin-binding intermediates and results in a high duty ratio. Moreover, our results reveal a Mg²⁺-sensitive regulatory mechanism tuning the kinetic and mechanical properties of the myosin-7a motor domain. We obtained direct evidence that changes in the concentration of free Mg²⁺ ions affect the motor properties of human myosin-7a using an in vitro motility assay system. Our results suggest that in a cellular environment, compartment-specific fluctuations in free Mg²⁺ ions can mediate the conditional switching of myosin-7a between cargo moving and tension bearing modes.     

Purification and some properties of hemagglutinin from the Myxomycete,Physarum polycephalum

A new hemagglutinin was isolated from the plasmodium ofPhysarum polycephalum by salting out with ammonium sulphate followed by chromatography on DE-32, DEAE-Toyopearl and hydroxyapatite. This hemagglutinin, named physarumin, was purified 1000-fold over crude extracts. The molecular weight of physarumin was determined to be 22,000 by size exclusion chromatography on Bio-Gel P-60 and 8,700 by SDS-polyacrylamide gel electrophoresis. Physarumin agglutinated rabbit, guinea pig, horse and human erythrocytes. Physarumin-induced hemagglutination was inhibited by fetuin and ₁ glycoprotein, but not by commercially available mono-and disaccharides. Hemagglutinating activity was blocked by EDTA, and was restored by adding Ca²⁺ but not by Mg²⁺.     

The Ca2+-transport ATPases in smooth muscle

Summary A calmodulin stimulated Ca²⁺-transport ATPase which has many of the characteristics of the erythrocyte type Ca²⁺-transport ATPase has been purified from smooth muscle. In particular, the effect of calmodulin on these transport enzymes is mimiced by partial proteolysis and antibodies against erythrocyte Ca²⁺-transport ATPase also bind to the smooth muscle (Ca²⁺+Mg²⁺)ATPase. A correlation between the distribution of the calmodulin stimulated (Ca²⁺+Mg²⁺)ATPase and (Na⁺+K⁺)ATPase activities in smooth muscle membranes separated by density gradient centrifugation suggests a plasmalemmal distribution of this (Ca²⁺+Mg²⁺)ATPase. A phosphoprotein intermediate in smooth muscle which strongly resembles the corresponding phosphoprotein in sarcoplasmic reticulum of skeletal muscle may indicate the presence in smooth muscle of a similar type of Ca²⁺-transport ATPase.     

Zur biologischen Bedeutung von Fe3+ und Cu2+ als Komplexbildner

Summary The possible role of the metabolism of heavy metal ions in the process of ageing is discussed. It is suggested that, during this process, Cu²⁺ and Fe³⁺ as strong complexing ions, inhibit the activity of other metal enzymes by replacing their metal ion-activator. The relative stability of Cu²⁺- and Fe³⁺-complexes with various chelating compounds related to biological systems has been determined.     

Zur Metallspezifität der Hexokinase

Summary The activity of hexokinase has been determined in the presence of different metal ions. Besides Mg²⁺, the ions Co²⁺, Ni²⁺, Mn²⁺, Zn²⁺, and Cd²⁺ show remarkable activation. The differences are explained by superposition of an activating and an inhibiting function. The specifity problem is discussed.     

Temperature acclimation of Mg2+Ca2+-myofibrillar ATPase from a cold-selective teleost,Salvelinus fontinalis: a compromise solution

Summary Brook trout (Salvelinus fontinalis, Mitchill) were acclimated over 15 weeks to either +4°C or +24°C. The effects of temperature on myofibrillar Mg²⁺Ca²⁺-ATPase activities were investigated. In contrast to goldfish, temperature acclimation does not alter the kinetic properties of the brook trout myofibrillar ATPase. Activation energy (G^#) is lower and substrate turnover number is higher than values previously reported for cold-adapted stenotherms. Properties of brook trout ATPase appear to be a compromise enabling function across a broad temperature range. The different strategies of adapting to seasonal temperature variations are briefly discussed.The authors are grateful to the Wellcome Trust for financial support.-Correspondence should be addressed to I.A.J.     

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.     

The effect of hydrocortisone and thyroxine on development of calcium homeostasis in embryonic intestinal epithelium

Cytoplasmic Ca²⁺ concentration of epithelial cells from 14-day embryonic chick duodena decreased during 72 h of organ culture to a value 54% of that found at 17 days in vivo. The ability of cells to maintain a constant Ca²⁺ concentration when challenged with high extracellular calcium was also significantly reduced. Addition of 1 M hydrocortisone during culture restored both parameters of Ca²⁺ homeostasis to that of 16-day uncultured duodena, and rise in cytoplasmic Ca²⁺ was significant within 4 h of hormone treatment. Thyroxine influenced epithelial Ca²⁺ similarly, but to a lesser degree and only after 48–72 h of culture. These data indicate that glucocorticoids, and possibly thyroid hormones, influence the development of calcium homeostasis in intestinal epithelium.     

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca²⁺ mobilization and Ca²⁺ sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca²⁺]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca²⁺]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.