Apolinary Sobieszek

The contractile apparatus of smooth muscle.

Publisher Summary This chapter describes the architecture of the contractile apparatus. Smooth muscle cells, when isolated, are generally spindle-like in shape with tapered ends and possess a centrally placed, elongated nucleus. In vivo, where the cells occur in bundles and overlapping layers, they adapt themselves to the contours of the tissue; in some instances, they may be essentially straight, while in others, for example in the vascular wall, they may be markedly curved along their length. This adaptation extends further to their shape in cross section, which may vary within the same muscle layer from circular to polygonal to a flattened form. Thus, the contractile machinery must have a design that not only conforms to these requirements for flexibility in cell shape, but also tolerates the presence of the cell nucleus in a central position. The organization of the contractile material, such as fibrils and contractile units is discussed in the chapter.

A Ca2+-dependent actin modulator from vertebrate smooth muscle

A protein of M r ≈ 85 000 has been isolated and purified from pig stomach smooth muscle that modulates the polymer state of actin in a Ca2+‐dependent manner. When added either to preformed F‐actin filaments or to G‐actin, prior to polymerisation, the modulator induces the formation of shorter filaments. The average filament length in the presence of the modulator is directly dependent on its molar ratio to actin indicating a stoichiometric rather than a catalytic type of interaction. When mixed with G‐actin the modulator forms a stable complex with two actin monomers; this complex is presumed to act as a potent nucleus for actin polymerisation. The dynamics of the interaction with F‐actin suggests a direct severing of actin filaments by the modulator via a binding to intrafilamentous actins.

Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments. Effects of skeletal, smooth and non-muscle tropomyosins.

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin. Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (Kapp) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (Vmax). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the Vmax. The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the Kapp as skeletal muscle tropomyosin but yields a value of Vmax that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin. On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.

Regulation of smooth muscle myosin light chain kinase: Allosteric effects and co-operative activation by calmodulin

The activation of smooth muscle myosin light chain kinase (MLCKase) by calcium and calmodulin (CM) was investigated over a wide range of concentrations of the enzyme using myosin (MY) or its isolated phosphorylatable light chain (L20) as substrates. The enzyme showed allosteric behavior. The specific phosphorylation activity was dependent on the concentration of MLCKase as well as on the concentrations of both substrates. However, at the lower (nanomolar) range of kinase the corresponding substrate rate relationships were hyperbolic. A high positive level of co-operativity of kinase was also observed for activation by CM in the presence of Ca2+. There was a pronounced CM/Ca-dependent inhibition of MLCKase activity when its molar ratio to CM was four to one or more. These kinetic data suggested that MLCKase could exist in several oligomeric forms, with an inactive high molecular size form and an active low molecular size form (protomers and/or dimers). This conclusion was confirmed by gel filtration studies. CM was not directly involved in the oligomerization process but instead, the oligomeric kinase shared an increased affinity for CM.

Diverse actions of cadmium on the smooth muscle myosin phosphorylation system

The effects of cadmium (Cd) on smooth muscle myosin phosphorylation have been investigated using an in vitro system comprising myosin filaments containing endogenous calmodulin (CM) and myosin light chain kinase (MLCKase). In the absence of calcium (Ca), Cd as well as some other divalent cations caused no activation of phosphorylation. However, when at least one (or possibly two) Ca2+ were bound per CM, the addition of 10 μM to 40 μM Cd2+ resulted in a 2 to 3 fold acceleration of the phosphorylation rate. Higher Cd concentrations caused inhibition of the system independent of Ca2+ concentration through the formation of Cd‐ATP complexes. These results explain some previously controversial data on the complex effects of Cd in intact smooth muscles.

Bulk isolation of the 20,000-Da light chain of smooth muscle myosin: Separation of the unphosphorylated and phosphorylated species

The procedure of W. T. Perrie and S. V. Perry (1970, Biochem. J. 119, 31-38) has been improved and extended to allow a convenient large-scale isolation of the 20,000-Da light chain of vertebrate smooth muscle myosin. The method utilizes as source material tropomyosin-free actomyosin or myosin. The relatively pure light chain isolated from this material could be obtained in pure form by a single gel-filtration step. Separation of the unphosphorylated and phosphorylated light chain species was achieved by subsequent chromatography on a DEAE column. The solubility properties of this light chain, relevant to its use in myosin light chain kinase assays, were also established.

Purification and Characterization of a Smooth Muscle Myosin Light Chain Kinase-Phosphatase Complex

We show that a myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) forms a multienzyme complex with myosin light chain kinase (MLCKase). The stability of the complex was indicated by the copurification of MLCKase and MLCPase activities through multiple steps that included myofibril preparation, gel filtration chromatography, cation (SP-Sepharose BB) and anion (Q-Sepharose FF) exchange chromatography, and affinity purification on calmodulin and on thiophosphorylated regulatory light chain columns. In addition, the purified complex eluted as a single peak from a final gel filtration column in the presence of calmodulin (CaM). Because a similar MLCPase is present in varying amounts in standard preparations of both MLCKase and myosin filaments, we have named it a kinase- and myosin-associated protein phosphatase (KAMPPase). The KAMPPase multienzyme complex was composed of a 37-kDa catalytic (PC) subunit, a 67-kDa targeting (PT) subunit, and MLCKase with or without CaM. The approximate molar ratio of the PC and PT subunits was 1:2 with a variable and usually higher molar content of MLCKase. The targeting role of the PT subunit was directly demonstrated in binding experiments in which the PT subunit bound to both the kinase and to CaM. Its binding to CaM was, however, Ca2+-independent. MLCKase and the PT subunit potentiated activity of the PC subunit when intact myosin was used as the substrate. These data indicated that there is a Ca2+-independent interaction among the MLCPase, MLCKase, and CaM that are involved in the regulation of phosphatase activity.

Purification and Characterization of a Kinase-associated, Myofibrillar Smooth Muscle Myosin Light Chain Phosphatase Possessing a Calmodulin-targeting Subunit

A myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) was purified from turkey gizzard myofibrils, and it was found to be closely associated with the myosin light chain kinase (MLCKase). For this reason we have named this phosphatase the kinase- and myosin-associated protein phosphatase (KAMPPase). Subunits of the KAMPPase could be identified during the first ion exchange chromatography step. After further purification on calmodulin (CaM) and on thiophosphorylated regulatory myosin light chain affinity columns we obtained either a homogenous preparation of a 37-kDa catalytic (PC) subunit or a mixture of the PC subunit and variable amounts of a 67-kDa targeting (PT) subunit. The PT subunit bound the PC subunit to CaM affinity columns in a Ca2+-independent manner; thus, elution of the subunits required only high salt concentration. Specificity of interaction between these subunits was shown by the following observations: 1) activity of isolated PC subunit, but not of the PTC holoenzyme, was stimulated 10-20-fold after preincubation with 5-50 μM of CoCl2; 2) the pH activity profile of the PC subunit was modified by the PT subunit (the specific activity of the PTC holoenzyme was higher at neutral pH and lower at alkaline pH); and 3) affinity of the holoenzyme for unphosphorylated myosin was 3-fold higher, and for phosphorylated myosin it was 2-fold lower, in comparison with that of the purified PC subunit. KAMPPase was inhibited by okadaic acid (Ki = 250 nM), microcystin-LR (50 nM) and calyculin A (1.5 μM) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.

Smooth muscle myosin as a calmodulin binding protein. Affinity increase on filament assembly

SummarySmooth muscle myosin is normally copurified with myosin light chain kinase (MLCKase) and calmodulin (CM). We have now established the binding affinities and stoichiometries of these two components with respect to monomeric and filamentous myosin. The relative amounts of CM and MLCKase in fresh synthetic myosin filaments were approximately stoichiometrical but for both in a molar ratio to myosin of about 1 to 30 or less. A 107 dilution of filaments did not result in any significant decrease in the amount of endogenous MLCKase and CM except in the absence of Ca2+ when the CM content was reduced around five-fold.Binding assays were performed with myosin depleted of CM and MLCKase by passage over melittin- and CM-affinity columns, arranged in tandem. For binding to myosin preassembled into filaments three classes of CM binding sites could be demonstrated. (1) A high affinity binding characterized by a dissociation constant of 20–30nm and a rather low binding stoichiometry of below 1 to 500. (2) An intermediate affinity, characterized by a dissociation constant of 1.2μm and 1 to 100 binding stoichiometry. (3) A low affinity with a Kd > 10μm and with an approximate 1 to 1 binding ratio relative to myosin. If CM was made available during filament assembly the high affinity binding predominated, with a stoichiometry in the presence of Ca2+ of about 1 to 50. The binding affinity but not the stoichiometry was reduced several fold by the removal of Ca, excluding a non-specific trapping of CM within the filament architecture. Collectively, these data demonstrate an independent and specific association of MLCKase and CM with myosin, that is strengthened by filament assembly.

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation.

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation‐related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded moysin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.