Vectorial phosphorylation of filamentous smooth muscle myosin by calmodulin and myosin light chain kinase complex.

Vertebrate smooth muscle myosin extracted from myofibrils and isolated via filament assembly was co-purified with calmodulin (CaM) and myosin light chain kinase (MLCK) which are tightly associated with the filament architecture and, therefore, it may be considered as a native-like preparation. These endogenous contaminates also co-precipitated with a native-like actomyosin, for both cases, at levels sufficient to fully phosphorylate myosin within 10-20 s after addition of ATP and calcium, although their molar ratio to myosin was only about 1 to 100. Phosphorylation progress curves obtained from mixtures of the native-like, and CaM- and MLCK-free filaments indicated that the CaM/MLCK complex preferentially phosphorylated its parent filaments and, as result, the whole myosin present was not maximally phosphorylated. Solubilization of the filaments' mixtures at high ionic strength resulted in slower phosphorylation rates but with maximal phosphorylation levels being attainable. Similar observations were made on the filamentous myosin system reconstituted from the kinase- and CaM-free myosin with added purified MLCK and CaM as well as on the native-like myosin from which only one of these endogenous contaminates was removed by affinity chromatography. These data indicated that not only the MLCK but also CaM was necessary for the observed preferential phosphorylation kinetics. Thus, the native-like filamentous myosin appeared to be phosphorylated by some kind of vectorial mechanism. Similar experiments were carried out on the native-like actomyosin where these vectorial effects were even more pronounced.

Posttraumatic epilepsy risk factors: one-year prospective study after head injury.

PURPOSE: Prospective evaluation of risk factors for posttraumatic epilepsy (PTE) by using clinical, EEG, and brain computed tomography (CT) data in four assessments from the head injury (HI) acute phase to 1 year later; and evaluation of the possible epileptogenic role of hemosiderin as shown by brain magnetic resonance imaging (MRI). METHODS: Risk factors for PTE were evaluated by using Kaplan-Meier curves, log-rank test, and the Cox model in 137 consecutively enrolled adult inpatients. Percentage differences of patients with brain hyperintense and/or hemosiderin areas shown by MRI 1 year after HI were statistically evaluated by univariate tests considering two subgroups [e.g., patients with (PTE) and without (WLS) late seizures]. RESULTS: The PTE subgroup included 18 patients with at least two seizures between the second and twelfth months. Kaplan-Meier curves demonstrated that Glasgow Coma Scale low score, early seizures, and single brain CT lesions are PTE risk factors, as is the development of an EEG focus 1 month after HI. No significant percentage difference was found between PTE and WLS patients with hemosiderin spots shown by MRI 1 year after HI. CONCLUSIONS: the Cox model indicates that, for HI patients with early seizures and brain CT single temporal or frontal lesions in the acute phase, the PTE risk is 8.58 and 3.43 times higher, respectively, than for those without. An EEG focus 1 month after HI is a risk factor 3.49 times higher than for patients without such EEG changes. One year after HI, a higher percentage of PTE than WLS patients had cortical MRI hyper-intense areas including hemosiderin.

Enzyme kinetic characterization of the smooth muscle myosin phosphorylating system: activation by calcium and calmodulin and possible inhibitory mechanisms of antagonists.

A native-like smooth muscle filamentous myosin system was characterized from an enzyme kinetic point of view. The system contains endogenous myosin light chain kinase (MLCKase) and calmodulin (CM) (A. Sobieszek, J. Muscle Res. Cell Motil. 11 (1990) 114-124) and is, therefore, well suited for testing the action of CM-antagonists or other inhibitory compounds. However, this has not been done due to its complexity. The characterization of the system includes: (1) derivation of a relationship for rate of myosin phosphorylation in terms of total CM, free Ca2+ and total MLCKase concentrations, which includes only three binding constants; and (2) derivation of relationships between fractional inhibition rate (vi/vo) and total inhibitor concentration (It) which cover most of the inhibitory mechanisms applicable to the myosin system or to other CM-dependent enzymes. The three binding constants were subsequently evaluated from experimental data for filamentous myosin and for its isolated regulatory light chain (ReLC) using a non-linear regression software. They indicated differences in the interaction of myosin filament with the active CM-MLCKase complex in comparison to that of the isolated ReLC. The derived vi/vo versus It relationships, together with the software, make it possible to evaluate the inhibition constants and binding stoichiometries of CM-antagonists and other compounds inhibiting myosin phosphorylation. This approach was successfully applied to experimental data on inhibition of MLCKase by amiloride, cadmium, or CM-binding peptide (M-12) for simple mechanisms. For more complex mechanisms, inhibition by calmidozolium, trifluoperazine or melittin, the analysis showed that only calmidozolium acted specifically at the CM level in a multiple-site activator-depletion mechanism. Melittin and trifluoperazine inhibited the phosphorylation rate by a novel substrate-and-activator depletion mechanism, in which additional inhibition of the substrate resulted in the removal of the inhibition at lower range of the antagonists' concentration.

[Effect of preincubation on smooth muscle myosin light chain kinase activity]. / Zalezhnist' aktyvnosti kinazy lehkykh lantsiuhiv miozynu hladen'kykh m'iaziv vid umov peredinkubatsiï.

Phosphorylation of myosin regulatory light chain (RLC) catalysed by myosin light chain kinase (MLCK) is a key reaction in the regulation of actin-myosin interaction in smooth muscle. The activation of MLCK by calmodulin (CaM) and Ca2+ was investigated over a wide range of the enzyme concentrations using myosin or its RLC with Mw = 20 kDa as substrates. Kinase activation by CaM (at saturating Ca2+ concentrations) was characterized by positive cooperativity even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between MLCK and CaM. The activation of the kinase by Ca2+ was also cooperative but only at relatively low CaM levels. This cooperativity was shown to result from time dependent changes in MLCK that take place during its incubation with Ca2+ and CaM before substrate addition in phosphorylation assays. As a result the kinase activity as a function of its concentration at constant CaM level was biphasic: there was the activity optimum at 1:1 ratio of CaM to MLCK and almost complete inhibition at 3 to 7 molar excess of kinase over CaM. Such changes that take place during 10 to 15 min preincubation with Ca2+ and CaM may involve the kinase supramolecular structure formation or/and its conformational rearrangements.

[Conformational modifications of smooth muscle light chain kinase]. / Konformatsiina modyfikatsiia kinazy lehkykh lantsiuhiv miozynu hladenkykh m'iaziv.

Our recent investigations have shown that smooth muscle myosin light chain kinase (MLCK) exists in solution as a mixture of oligomeric, dimeric and monomeric species; besides during preincubation (maintaining of the activated enzyme without substrate) with substoichiometric amounts of calmodulin (CaM) it undergoes definite changes leading to several fold lowering of its activity. Fluorescent data obtained in this work suggest that such kinase inhibition must not be connected with quantitative redistribution of different kinase species but rather it is the result of conformational modifications of this enzyme activated molecules leading to the reduction of their affinity to CaM. Such conformational rearrangements took place also at equimolar kinase to CaM ratio (or CaM excess) but in this case they were characterized by lower depth and insignificant MLCK activity fall. The nature of these conformational changes is discussed.

[Formation and regulation of myosin light chain kinase and phosphatase complex in smooth muscle: the outlook]. / Formirovanie i regulatsiia kompleksa kinazy i fosfatazy legkikh tsepei miozina v gladkoi myshtse: perspektivy issledovanii.

Myosin kinase and phosphatase, phosphorylating and dephosphorylating regulatory light chain of myosin, are the key regulatory enzymes of smooth muscle. They are tightly associated not only with myosin filament but also with each other and, therefore, appear to form a functional complex which is responsible for regulation of contraction and relaxation of smooth muscle. In our recent studies we have shown that the complex includes the kinase with its activator (calmodulin; CM) and the phosphatase in a form of catalytic and targeting subunits. The targeting subunit, being a CM binding protein, links the catalytic subunit to the kinase and to CM in a Ca-independent manner. In solution, the kinase is not exclusively monomeric but also dimerise and forms small amounts of oligomers, and all these forms are in equilibrium with each other. The dimers are responsible for a cooperative activation of the kinase by CM as well as for its intramolecular autophosphorylation, while the oligomeric form is involved in kinase localization on the myosin filament and also participates in formation of the complex with phosphatase. A kinase related protein (telokin) acts as a very effective modulator of the oligomeric state of the kinase by transferring the oligomers into the dimers and/or monomers. Telokin was effectively releasing the kinase from myosin filaments and the phosphatase from the complex with resulting inhibition of myosin phosphorylation and acceleration of myosin dephosphorylation. These modulating effects were reversed by a very slow phosphorylation of telokin by the kinase.

Calorimetric studies of the thermal unfolding of smooth muscle myosin fragments and their complexes with ADP and phosphate analogs.

The thermal unfolding of turkey gizzard smooth muscle myosin subfragment 1 (S1) and heavy meromyosin (HMM) in the absence of added nucleotides, in the presence of ADP, and in S1 or HMM ternary complexes with ADP and Pi analogs, orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4-), have been studied by differential scanning calorimetry (DSC). It has been shown that the formation of these ternary complexes causes significant structural changes in S1 or in the heads of HMM which are reflected in a pronounced increase of the protein thermal stability. The effect of BeFx was less distinct than that of AlF4- or Vi. Phosphorylation of regulatory light chains (RLC) in S1 or in HMM had practically no influence on these effects. In general, the changes caused by various Pi analogs in smooth muscle S1 or HMM were similar to those observed earlier with skeletal muscle S1 devoid of RLC. It is concluded that RLC and their phosphorylation do not significantly affect the character of structural changes induced in motor domains of the HMM heads by the formation of ternary complexes HMM--ADP--Vi, HMM--ADP--AlF4-, and HMM--ADP--BeFx--stable analogs of the intermediate states of the HMM ATPase reaction, HMM.ADP.Pi and HMM. ATP.

Oligomerization of smooth muscle myosin light chain kinase and its modifications by melittin and calmodulin.

The catalytic activity of smooth muscle myosin light chain kinase (MLCKase) requires the presence of calcium and calmodulin [CM; J. T. Stull et al. (1993) Molecular and Cellular Biochemistry, Vols. 127/128, pp. 229-237] and can also be modified through its own oligomerization [E. B. Babiychuk et al. (1995) Biochemistry, Vol. 34, pp. 6366-6372]. In the present report we demonstrate that melittin, one of the most potent CM antagonists, interacted reversibly with the MLCKase apoenzyme with affinities comparable to those of CM and influenced the oligomeric state of the kinase. At low melittin to kinase ratios the kinase formed insoluble oligomers (aggregates) while at higher melittin concentrations it existed predominantly as soluble oligomers revealed by cross-linking as octamers and hexamers. The kinase alone exhibited similar biphasic solubility within a 5-30 microM range and its solubility was strongly influenced by the ionic strength of the medium. Melittin was also found to promote both the aggregation of the purified 24-kDa C-terminal fragment of the kinase and its analogue telokin, as well as of myosin light chains, but had no effect on the solubility of bovine serum albumin, caldesmon, or calmodulin. These data and our cross-linkage experiments indicate that the insoluble kinase oligomers arose via melittin-induced aggregation of the kinase dimers in which the relokin-like domain played a main role. The soluble oligomers, in turn, were formed after saturation of the kinase with melittin, which resulted in a weakening of the interaction between the protomers with an increase of the long-range order within the oligomers. This interpretation is consistent with the observed effects of melittin on MLCKase catalytic and autocatalytic activities. At concentrations of melittin required to produce soluble oligomers, the binding of the kinase to myosin filaments was considerably enhanced. A plausible mechanism for the formation of the soluble oligomers and aggregates is suggested and its relation to the possible MLCKase assemblies discussed in terms of a model.

Smooth muscle myosin light chain kinase, supramolecular organization, modulation of activity, and related conformational changes.

It has recently been suggested that activation of smooth muscle myosin light chain kinase (MLCK) can be modulated by formation of supramolecular structures (Sobieszek, A. 1991. Regulation of smooth muscle myosin light chain kinase. Allosteric effects and co-operative activation by CaM. J. Mol. Biol. 220:947-957). The present light scattering data demonstrate that the inactive (calmodulin-free) MLCK apoenzyme exists in solution as a mixture of oligomeric (2% by weight), dimeric (53%), and monomeric (45%) species at physiological ionic strength (160 mM salt). These long-living assemblies, the lifetime of which was measured by minutes, were in equilibrium with each other. The most likely form of the oligomer was a spiral-like hexamer, the dimensions of which fit very well the helical structure of self-assembled myosin filaments (Sobieszek, A. 1972. Cross-bridges on self-assembled smooth muscle myosin filaments. J. Mol. Biol. 70:741-744). After activation of the kinase by calmodulin (CaM) we could not detect any appreciable changes in the distribution of the kinase species either when the kinase was saturated with CaM or when its molar concentration exceeded that of CaM. Our fluorescent measurements suggest that the earlier observed inhibition of kinase at substoichiometric amounts of CaM (Sobieszek, A., A. Strobl, B. Ortner, and E. Babiychuk. 1993. Ca2+-calmodulin-dependent modification of smooth-muscle myosin light chain kinase leading to its co-operative activation by calmodulin. Biochem. J. 295:405-411) is associated with slow conformational change(s) of the activated (CaM-bound) kinase molecules. Such conformational rearrangements also took place with equimolar kinase to CaM; however, in this case there was no decrease in MLCK activity. The nature of these conformational changes, which are accompanied by reduction of the kinase for CaM affinity, is discussed.

Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins.

Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.

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 microM 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 microM) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.

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.

Telokin (kinase-related protein) modulates the oligomeric state of smooth-muscle myosin light-chain kinase and its interaction with myosin filaments.

Telokin, an abundant gizzard protein, inhibited phosphorylation of regulatory light chain when filamentous myosin was used as the substrate but no inhibition was observed with myosin subfragment 1. At physiological telokin-to-myosin molar ratio (1:1), the inhibition amounted to a 3.5-fold reduction in the initial phosphorylation rate whereas at high molar excess of telokin over myosin, we observed an up to 20-fold decrease in this rate. In agreement with previous observations [Shirinsky, Vorotnikow, Birukov, Nanaev, Collinge, Lukas, Sellers and Watterson (1993) J. Biol. Chem. 268, 16578-16583], telokin did not inhibit phosphorylation of the isolated regulatory light chain of myosin and only moderately (35%) inhibited that of heavy meromyosin. To gain a better understanding of the mechanism of this inhibition, we investigated the effects of telokin on the recently described [Babiychuk, Babiychuk and Sobieszek (1995) Biochemistry 34, 6366-6372] oligomeric properties of smooth-muscle myosin light-chain kinase (MLCK). We showed, on the one hand, that telokin rapidly solubilized the large kinase oligomers formed at low ionic strength. With soluble kinase, on the other hand, telokin acted to increase the relative concentration of MLCK dimers and to decrease that of the hexamers and octamers. This, in turn, resulted in a reduction in the amount of MLCK bound to myosin because filamentous myosin appeared to exhibit a higher affinity for the hexamers than for the dimers. Telokin by itself was also shown to dimerize and oligomerize in solution and this oligomerization was greatly enhanced in the presence of MLCK. We suggest that telokin affects myosin phosphorylation by modulation of the oligomeric state of MLCK and its interaction with myosin filaments.

Kinase-related protein (telokin) is phosphorylated by smooth-muscle myosin light-chain kinase and modulates the kinase activity.

Telokin is an abundant smooth-muscle protein with an amino acid sequence identical with that of the C-terminal region of smooth-muscle myosin light-chain kinase (MLCK), although it is expressed as a separate protein [Gallagher and Herring (1991) J. Biol. Chem. 266, 23945-23952]. Here we demonstrate that telokin is also similar to smooth-muscle myosin regulatory light chain (ReLC) not only in its gross physical properties but also as an MLCK substrate. Telokin was slowly phosphorylated by MLCK in the presence of Ca2+ and calmodulin and could be readily dephosphorylated by myosin light-chain phosphatase. A threonine residue was phosphorylated with up to 0.25 mol/mol stoichiometry. This low stoichiometry, together with the observed dimerization of telokin [Sobieszek and Nieznanski (1997) Biochem. J. 322, 65-71], indicates that the telokin dimer was acting as the substrate with a single protomer being phosphorylated. Our enzyme kinetic analysis of the phosphorylation reaction confirms this interpretation. Because telokin phosphorylation also required micromolar concentrations of MLCK, which also facilitates the formation of kinase oligomers, we concluded that the oligomers are interacting with telokin. Thus it seems that telokin modulates the phosphorylation rate of myosin filaments by a mechanism that includes the direct or indirect inhibition of the kinase active site by the telokin dimer, and that removal of the inhibition is controlled by slow phosphorylation of the telokin dimer, which results in MLCK dimerization.

Calmodulin-dependent autophosphorylation of smooth muscle myosin light chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation.

In the presence of Ca2+ and calmodulin (CM), purified smooth muscle myosin light chain kinase (MLCKase) was found to undergo autophosphorylation at a rate that was about 200-fold slower than its catalytic activity. Up to 1.7 mol of phosphate were incorporated per mole of kinase. Lower levels of incorporation could be correlated with the presence of an endogenous protein phosphatase which could be inhibited with okadaic acid or Microcystin-LR. The major autophosphorylation site was identified as Thr-863 or Thr-865 and was located on the 24-kDa C-terminal fragment of the kinase. In addition, there was a relatively low and variable contribution of a Ca/CM-independent autophosphorylation at Ser-814 or Ser-815. The initial autophosphorylation rates and maximal incorporation levels were highest at a molar ratio of 2 MLCKase to 1 CM and were inhibited at higher CM levels. This indicated that binding of one molecule of the kinase apoenzyme by a CM-kinase complex was necessary for the reaction to occur. Kinetic analysis of the autophosphorylation reaction was consistent with this interpretation and indicated a second-order intermolecular process that included MLCKase dimerization or oligomerization. In contrast, the low Ca/CM-independent contribution was of intramolecular type since it did not depend on the kinase concentration. The autophosphorylation appeared to be involved in a relatively slow modification of the oligomeric properties of the kinase leading to a 2-4-fold amplification of the kinase catalytic activity which followed its activation by CM. Oligomerization and dimerization of the kinase was independently demonstrated by light scattering measurements.

Modulation of smooth muscle myosin light chain kinase activity by Ca2+/calmodulin-dependent, oligomeric-type modifications.

Oligomerization of turkey gizzard myosin light chain kinase (MLCKase) was demonstrated by a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a standard reagent used in investigations of specific protein-protein interaction [Mornet et al. (1989) J. Muscle Res. Cell Motil. 10, 10-24]. This approach revealed that in solution the kinase was not monomeric but the monomers were in equilibrium with the kinase dimeric and oligomeric forms. Addition of Ca2+/calmodulin (CM) shifted this equilibrium in the direction of the kinase dimers, accompanied by a 2-fold decrease of the kinase catalytic activity, in addition to a 2-fold decrease of its apparent affinity for CM [Sobieszek et al. (1993) Biochem. J. 295, 405-411]. The dimer (and/or oligomer) formation was shown to result from an interaction of the kinase autoinhibitory domain with its 24 kDa tryptic fragment containing titin-like domain II-3. The possible significance of the oligomerization in regulation of MLCKase activity is discussed.

Regulation of myosin light chain kinase: kinetic mechanism, autophosphorylation, and cooperative activation by Ca2+ and calmodulin.

Phosphorylation of the regulatory light chain of myosin catalyzed by myosin light-chain kinase (MLCK) is the key reaction in the regulation of actin-myosin interaction in smooth muscle. It is shown that this reaction is of an ordered type, whereby kinase first binds ATP and then the light chain, and following phosphate transfer, the phosphorylated light chain is released before ADP. The MLCK also phosphorylates itself, and this intramolecular autophosphorylation is Ca2+ and calmodulin (CaM) dependent. It has, however, no pronounced effect on the kinase activity or on its affinity for Ca2+ and CaM. With the aim of understanding the cooperativity of MLCK activation, the activity of the kinase was systematically measured as a function of different ligands involved. In these measurements the isolated light chain and intact filamentous myosin, as well as native actomyosin, were used as substrates. The activation of the kinase by Ca2+ was positively cooperative but only at relatively low CaM levels. The activation by CaM (at saturating Ca2+ levels) was also cooperative, even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between CaM and the kinase. This cooperativity was shown to result from time-dependent changes in the MLCK that take place during incubation with Ca2+ and CaM before addition of ATP in phosphorylation assays. As a result, activity of the kinase as a function of its concentration at constant CaM was biphasic: there was optimum activity at a ratio of 1:1 CaM to kinase and almost complete inhibition of the activity at a three- to six-fold excess of the kinase over CaM.(ABSTRACT TRUNCATED AT 250 WORDS)

Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins.

Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.

Heterogeneity of smooth muscle myosin light chain kinase. Characterization of isoelectric variants.

Purified chicken gizzard myosin light chain kinase (MLCK) analyzed by anion-exchange high-performance liquid chromatography (HPLC) can be consistently resolved into three well separated peaks, termed alpha, beta, gamma. These peaks are shown to correspond to differently charged forms of MLCK with the charge difference between alpha and beta twice as large as between beta and gamma. The isoelectric point and elution position of the peaks as well as their amplitudes are modified by phosphorylation or by autophosphorylation of MLCK suggesting that the observed charge differences are related to their different phosphate content. The three forms appear to have similar apparent affinity for both the substrates, ATP and the isolated regulatory light chain, but their specific activities are different.

Carbamazepine effects on afterdischarge, memory retrieval, and conditioned avoidance response latency in hippocampally kindled cats.

Carbamazepine (CBZ) effects on (a) complex partial seizures and afterdischarges (AD), (b) memory retrieval, and (c) conditioned avoidance response (CAR) latencies were studied in a group of 9 hippocampally kindled cats. Significant AD and epileptic seizure suppression was observed in kindled cats after administration of CBZ concurrent with significant improvement in percentage scores on the memory retrieval test. The CAR latencies were longer in kindled cats with or without CBZ than in the conditioned group of cats without both kindling and CBZ. CAR latencies were not significantly different between kindled and kindled-CBZ-treated cats. Thus, the longer latencies in kindled animals cannot be due to CBZ but apparently are related to the effects of kindling. In some kindled animals, longer CAR latencies were reversed by CBZ.