Level Of Light Chain Phosphorylation Research Articles

Protective effects of curcumin against rat intestinal inflammation‑related motility disorders.

Intestinal inflammation frequently occurs alongside dysmotility, which is characterized by altered myosin light chain phosphorylation levels. Curcumin, an active component from the ginger family, is reported to confer anti-inflammatory effects. However, the effects of curcumin on both diarrhea and constipation associated inflammation remains to be elucidated. The present study was designed to investigate the effects of curcumin on diarrhea and constipation and to determine the related mechanisms. Sprague-Dawley rats were used to establish diarrhea and constipation models via intracolonic acetic acid (4%) instillation or cold water gavage for 2 weeks, respectively. Blood samples were collected to measure the serum levels of the cytokines TNF-α and IL-1β using ELISA kits. Western blotting was performed to measure NF-κB, RhoA, Rho-related kinase 2, phosphorylated MLC20, phosphorylated myosin phosphorylated target subunit 1, 130k Da-MLC kinase (MLCK), c-kit tyrosine kinase protein expression, and reverse transcription-quantitative PCR was conducted to measure MLCK expression levels. The results indicated that curcumin reversed the elevations in the pro-inflammatory cytokines IL-1β and TNF-α by inhibiting the NF-κB pathway in rats with diarrhea and constipation. The results also indicated that myosin light chain (MLC) phosphorylation in intestinal smooth muscle was positively and negatively associated with the motility of inflammation-related diarrhea and constipation in rats, respectively. Curcumin significantly reversed the increased MLC phosphorylation in the jejunum of the rats with diarrhea, significantly enhanced the reductions in inflammatory mediators, including TNF-α and IL-1β, of rats with constipation and significantly ameliorated the related hyper-motility and hypo-motility in rats with both diarrhea and constipation. In conclusion, the potential roles of the MLC kinase, c-kit tyrosine and Rho A/Rho-associated kinase 2 pathways, which are involved in curcumin-induced amelioration of inflammation-related diarrhea and constipation, were explored in the present study. Results from the present study suggested that curcumin has potential therapeutic value for treating intestinal inflammation and inflammation-related motility disorders.

Maternal high-salt diet altered PKC/MLC20 pathway and increased ANG II receptor-mediated vasoconstriction in adult male rat offspring.

High-salt diet (HSD) is associated with cardiovascular diseases. This study aims at ascertaining the influence of maternal HSD on offspring's angiotensin II (ANG II)-mediated vasoconstriction and the underlying mechanisms. In comparison to a normal-salt diet, HSD used in pregnancy in rats changed the ultrastructures of the coronary artery (CA) in 5-month-old male offspring, and increased ANG II-mediated CA contractility. Measurement of [Ca(2+) ]i in CA using fluorescent fura-2, a Ca(2+) indicator, showed that ANG II-mediated increases in [Ca(2+) ]i were the same between HSD and normal-salt diet groups, but the ratio of diameter change/[Ca(2+) ]i induced by ANG II were significantly higher in HSD groups. Angiotensin II receptor type 1, not angiotensin II receptor type 2, caused ANG II-mediated vasoconstriction. Protein kinase C (PKC) inhibitor GF109203X attenuated the ANG II-mediated vasoconstriction, PKC agonist phorbol12,13-dibutyrate produced a greater contraction. There was an increase in PKCβ mRNA and the corresponding protein abundance in the offspring, whereas other PKC subunits PKCα, PKCδ, and PKCε did not change. Moreover, 20 kDa myosin light chain phosphorylation levels were increased in HSD group. Maternal HSD affected the developmental programing for the offspring CA, with increased ANG II-mediated vasoconstrictions. The angiotensin II receptor type 1-PKC-20 kDa myosin light chain phosphorylation pathway was the possible mediated cellular mechanism.

Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator-stimulated phosphoprotein

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with alpha-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with alpha-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.

Partial Bladder Outlet Obstruction Selectively Abolishes Protein Kinase C Induced Contraction of Rabbit Detrusor Smooth Muscle

Despite the acute onset, partial bladder outlet obstruction in the rabbit induces detrusor remodeling similar to that in men with benign prostatic hyperplasia in terms of its impact on structural and functional alterations in smooth muscle. We determined if partial bladder outlet obstruction induced remodeling alters the protein kinase C signaling pathway that leads to contraction. Smooth muscle from control animals and those subjected to 2 weeks of partial bladder outlet obstruction were mounted for isometric force recording, measurement of myosin light chain phosphorylation and levels of adducin phosphorylation. Bladder muscle strips were stimulated by phorbol dibutyrate or carbachol in the presence and absence of bisindolylmaleimide-1. Smooth muscle strips from animals subjected to partial bladder outlet obstruction showed little to no increase in stress in response to phorbol dibutyrate and no increase in myosin light chain phosphorylation levels. Muscle strips from control animals produced a robust contraction with concomitant increases in myosin light chain phosphorylation. Inhibition of protein kinase C by bisindolylmaleimide-1 significantly depressed carbachol induced contractions of muscle strips from control animals but it had no effect on carbachol induced contractions of muscle strips from outlet obstructed animals. Phorbol dibutyrate increased phospho-adducin levels in muscle strips from the 2 animal sources, suggesting that protein kinase C could be activated. We propose that partial bladder outlet obstruction does not alter protein kinase C activation, but rather abolishes or uncouples the pathway(s) downstream of protein kinase C, leading to contraction. Loss of this pathway may contribute to the loss of normal voiding behavior and the resultant decompensated state.

Partial bladder outlet obstruction abolishes the receptor- and G protein-dependent increase in calcium sensitivity in rabbit bladder smooth muscle

Partial bladder outlet obstruction (PBOO) alters the function of the whole bladder and produces specific alterations in the contractility of the bladder smooth muscle cell. The goal of this study was to test the hypothesis that PBOO affects smooth muscle contraction at the level of the receptor- and G protein-dependent increase in myofilament Ca2+ sensitivity. To address this question, we used alpha-toxin-permeabilized strips of bladder smooth muscle from control animals and animals subjected to 2 wk of PBOO. Increasing free [Ca2+] increased force in permeabilized strips from control animals; the addition of 10 microM carbachol and 10 microM GTP increased both the Ca2+ sensitivity of the contractions and the maximal levels of force attained. In contrast, although increases in [Ca2+] increased force in permeabilized strips from PBOO animals, the addition of carbachol and GTP had no additional effects. Myosin light chain phosphorylation levels increased with [Ca2+], and although they tended to be higher in strips from PBOO animals, they did not reach statistical significance. Assessment of G protein activity from both animal models suggests this is not a site responsible for the loss of carbachol and GTP enhancement of myofilament Ca2+ sensitivity. The addition of phorbol dibutyrate increased the Ca2+ sensitivity of force development in strips from both animal models, suggesting that an alteration in PKC signaling is not involved. Our results are consistent with the hypothesis that PBOO decreases receptor-mediated myofilament calcium sensitization and that the site of action is downstream from either the G proteins or PKC.

Quinine inhibits vascular contraction independent of effects on calcium or myosin phosphorylation.

This report contains results of studies designed to determine whether quinine has direct effects on myofilament Ca2+ sensitization in addition to effects on Ca2+. Quinine decreased the EC50 value and maximal contraction of intact arterial strips to histamine. Incubation of arterial strips with indomethacin or 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one did not alter quinine inhibition, suggesting that the effect is not mediated via cyclooxygenase or cGMP. Pretreatment of strips with quinine had no effect on the histamine-dependent increases in myosin light chain phosphorylation levels. Quinine inhibited Ca2+-induced contraction in alpha-toxin permeabilized strips, but not the Ca2+-induced contraction in Triton X-100 permeabilized strips. Pretreatment of the alpha-toxin permeabilized strips with quinine before stimulation with guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) did not have any effect on the response. In conclusion, quinine inhibited Ca2+-dependent contractions of the alpha-toxin permeabilized strips, which retain modulatory pathways both upstream and downstream from the contractile proteins but did not inhibit GTPgammaS-dependent contraction of the alpha-toxin permeabilized preparation important in upstream modulation of the contraction. Moreover, quinine did not inhibit the Ca2+-dependent contractions of the Triton X-100 permeabilized strips, which are devoid of all modulatory pathways. This suggests that quinine does not act upstream from or directly on the contractile proteins. A more likely site of action may be downstream of the contractile proteins and specifically at the coupling of the contractile proteins with the physiological endpoint of force development.

Myosin II Light Chain Phosphorylation Regulates Membrane Localization and Apoptotic Signaling of Tumor Necrosis Factor Receptor-1

Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-alpha (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.

CGMP mediates corpus cavernosum smooth muscle relaxation with altered cross-bridge function

We tested the prevailing paradigm that relaxation of corpus cavernosum smooth muscle (CCSM) and penile erection depends upon nitric oxide-induced elevation of myoplasmic cGMP and reduced Ca 2+-dependent myosin regulatory light chain phosphorylation levels. This hypothesis invokes a reversal of normal activation pathways. Upon stimulation with 250 μM phenylephrine, phosphorylation of the 20 kD myosin regulatory light chains of rabbit or human CCSM increased approximately 4-fold coincident with contraction. Removal of the agonist was followed by a slow reduction in cross-bridge phosphorylation and force to basal levels. The NO donor, sodium nitroprusside elicited a dose-dependent increase in tissue [cGMP] associated with a rapid relaxation in the continued presence of phenylephrine, although cross-bridge phosphorylation remained significantly elevated. Thus the NO-cGMP inhibitory pathway in CCSM is not simply a reversal of excitatory signal transduction mechanisms. An unidentified mechanism contributes to relaxation by decreasing the rate of cross-bridge recruitment through phosphorylation.

Mechanism of galanin-induced contraction of longitudinal smooth muscle of the rat jejunum.

Intact and alpha-toxin-permeabilized longitudinal smooth muscle were mounted for measurement of force and myosin light chain phosphorylation. Galanin contracted intact jejunum with a half-maximum effective concentration of 9.2 +/- 0.1 nM. Neither atropine, hexamethonium, guanethidine, nor tetrodotoxin affected the contraction. The contraction was also unaffected by depletion of intracellular Ca2+ or by addition of thapsigargin; removal of extracellular Ca2+ or addition of nifedipine abolished the contraction. Galanin increased myosin light chain phosphorylation levels concomitantly with force. During continued tissue stimulation, force fell to suprabasal values, whereas myosin light chain phosphorylation levels remained elevated. Galanin increased Ca2+ sensitivity of contraction in alpha-toxin-permeabilized tissues, and this was reversed by either guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin. These results suggest that galanin-induced contraction of longitudinal jejunal smooth muscle is dependent on a pertussis toxin-sensitive G protein that is apparently not coupled to the release of intracellular Ca2+ but to the influx of extracellular Ca2+ and involves an initial myofilament Ca2+ sensitization followed by Ca2+ desensitization.

Calponin and Mitogen-activated Protein Kinase Signaling in Differentiated Vascular Smooth Muscle

Contraction of smooth muscle cells is generally assumed to require Ca2+/calmodulin-dependent phosphorylation of the 20-kDa myosin light chains. However, we report here that in the absence of extracellular calcium, phenylephrine induces a contraction of freshly isolated ferret aorta cells in the absence of increases in intracellular ionized calcium or light chain phosphorylation levels but in the presence of activation of mitogen-activated protein kinase. A protein at 36 kDa co-immunoprecipitated with the mitogen-activated protein kinase and was identified as the actin-binding protein, calponin, by immunoblot. An overlay assay further confirmed an interaction between the kinase and calponin, even though the kinase did not phosphorylate calponin in vitro. Calponin also co-immunoprecipitated from smooth muscle cells with protein kinase C-epsilon. High resolution digital confocal studies indicated that calponin redistributes to the cell membrane during phenylephrine stimulation at a time when mitogen-activated protein kinase and protein kinase C-epsilon are targeted to the plasmalemma. These results suggest a role for calponin as a signaling molecule, possibly an adapter protein, linking the targeting of mitogen-activated protein kinase and protein kinase C-epsilon to the surface membrane.

The effects of caldesmon extraction on mechanical properties of skinned smooth muscle fibre preparations.

The role of caldesmon in the regulation of smooth muscle contraction was investigated in chemically skinned smooth muscle fibres from the guinea-pig taenia coli. A 19-kDa C-terminal fragment of caldesmon gave a minor (<5%) reduction of force in fully thiophosphorylated fibres, but reduced force by about 50% at intermediate activation levels without affecting the level of light chain phosphorylation. An extraction procedure was developed using incubation in solutions containing high Mg2+ concentrations. Protein analysis revealed a selective decrease in the amount of caldesmon in the fibres. Maximal active force per cross-sectional area was unaffected. The Ca2+ dependence of active force was shifted towards lower Ca2+ concentrations and became less steep. The effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. The results are consistent with the hypothesis that caldesmon in smooth muscle thin filaments inhibits force generation and plays a role in regulating cooperative attachment of cross-bridges at sub-maximal levels of activation in smooth muscle.

Rate of active tension development from rigor in skinned atrial and ventricular cardiac fibres from swine following photolytic release of ATP from caged ATP.

We investigated the rate of tension development (kappa td) after photolytical release of ATP from P3-1-(2-nitrophenyl)-ethyladenosine-5'-triphosphate ('caged ATP') of atrial and ventricular fibre bundles from pig. Contraction was initiated from high-tension (HT) and low-tension (LT) rigor at maximal Ca2+ activation (pCa 4.5). The kappa td of atrial fibre bundles was 6.8 s-1 from LT and 6.9 s-1 from HT rigor. Rate of tension development of ventricular fibre bundles was significantly lower (P < 0.001) being 1.06 s-1 and 0.94 s-1 from LT and HT rigor, respectively. The kappa td of skinned ventricular fibre bundles incubated in a high [K+], low [Ca2+] (cardioplegic) solution prior to the skinning procedure decreased significantly (P < 0.05) to 0.73 s-1 and 0.63 s-1 from LT and HT rigor, respectively, whereas that of skinned atrial fibre bundles remained at 7.1 s-1 and 6.9 s-1 from LT and HT rigor, respectively. Phosphorylation levels of the myosin light chain 2 isoform in the atrial fibre bundles (ALC-2) was 15.6 +/- 2.7%. The corresponding values for the two ventricular isoforms, VLC-2 and VLC-2*, were 31.2 +/- 0.4% and 25.1 +/- 2.1%, respectively. Phosphorylation levels of fibre bundles incubated in cardioplegic solution prior to skinning were 11.6%, 18.9%, and 15.4% of the ALC-2, VLC-2 and VLC-2*, respectively. The results show that the rate of tension development is more than seven-fold higher in the atrial compared with ventricular fibre bundles. These results correlate with the differences in ATPase activity of the contractile proteins in solution and, most likely, reflect differences in the myosin isoform composition. In ventricular fibre bundles the increased levels of light chain phosphorylation were associated with increased rate of contraction.

Effect of unphosphorylated smooth muscle myosin on caldesmon-mediated regulation of actin filament velocity

The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20 +/- 0.046 microns s-1 at 30 degrees C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the actin filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.

Force-velocity relation and myosin light chain phosphorylation in bovine coronary arterial smooth muscle.

We have investigated in bovine left ventricular coronary arteries the relation between the biochemical regulatory event of myosin light chain phosphorylation and the mechanical events of isometric stress and isotonic shortening, under conditions of stimulation by depolarization (65 mM KCl) or receptor occupancy (2 microM 5-hydroxytryptamine [5-HT]). At rest, levels of light chain phosphorylation were 0.07 +/- 0.01 mol phosphate/mol light chain. Maximal values were significantly different for KCl (0.42 +/- 0.02 mol phosphate/mol light chain at 1 minute) and 5-HT stimulation (0.58 +/- 0.01 mol phosphate/mol light chain at 30 seconds). Increases in light chain phosphorylation preceded isometric stress development, and values remained elevated at approximately 0.35 mol phosphate/mol light chain for up to 2 hours with both KCl and 5-HT. The sites of phosphorylation were identical for KCl and 5-HT at 2 hours. Maximal stresses for each stimulus were also maintained for 2 hours. Values of maximum velocity of shortening (Vo in muscle lengths [ML]/sec), obtained from the force-velocity relation, did not change significantly between 1 minute and 2 hours with KCl (0.070 +/- 0.008 ML/sec at 1 minute and 0.056 +/- 0.007 ML/sec at 2 hours, p greater than 0.2). However, during 5-HT stimulation, Vo declined significantly (0.053 +/- 0.006 ML/sec at 1 minute and 0.032 +/- 0.003 ML/sec at 2 hours, p less than 0.025). The relation between Vo and light chain phosphorylation was different for KCl and 5-HT, indicating that factors in addition to myosin light chain phosphorylation may modulate smooth muscle shortening velocity.

The relationship between ATPase activity, isometric force, and myosin light-chain phosphorylation and thiophosphorylation in skinned smooth muscle fiber bundles from chicken gizzard.

Isometric force developed by skinned gizzard muscle fiber bundles and levels of phosphorylation and thiophosphorylation of the 20,000-dalton myosin light chain were determined. These data showed a highly non-linear relationship between isometric force and myosin light-chain phosphorylation. Maximum force was developed at approximately 0.2 mol of phosphate/mol of light chain as reported previously (Hoar, P. E., Kerrick, W. G. L., and Cassidy, P. S. (1979) Science 204, 503-506). In contrast, the relationship between isometric force and myosin light-chain thiophosphorylation was linear, with maximum force occurring at 1.0 mol of thiophosphate/mol of myosin light chain. These observations are consistent with the latch-bridge hypothesis for conditions of varying myosin light-chain phosphatase/myosin light-chain kinase activity ratios as discussed by Hai and Murphy [1988) Am. J. Physiol. 254, C99-C106). To further test the latch-bridge hypothesis, ATPase activity was also measured during isometric force development in these fiber bundles. The relationship between isometric force and ATPase activity was linear whether the myosin light chains were phosphorylated or thiophosphorylated. Thus the number of cycling myosin cross-bridges, as measured by ATPase activity, was directly proportional to the force the muscle developed, not to the level of myosin light-chain phosphorylation. This finding that high levels of tension generated at low levels of light-chain phosphorylation are associated with high levels of ATPase activity is inconsistent with the latch-bridge model (Hai and Murphy, 1988).

Phosphatase inhibition with okadaic acid does not alter the relationship between force and myosin light chain phosphorylation in permeabilized smooth muscle

The phosphatase inhibitor, okadaic acid, has been used to test the hypothesis that myosin light chain phosphatase activity plays a central role in latchbridge formation in smooth muscle. In the permeabilized rabbit portal vein there is a non-linear relationship between myosin light chain phosphorylation and force production such that maximum force output occurs with about 50% phosphorylation. Treatment of the muscle with okadaic acid does not change this relationship even though there is a profound inhibition of phosphatase activity. The data suggest that dephosphorylation of the myosin light chain while the myosin is in the force producing state does not account for the high force output with low levels of light chain phosphorylation in smooth muscle.

The turnover of phosphate bound to myosin light chain-2 in perfused rat heart.

The rate of exchange of phosphate bound to ventricular myosin light chain-2 (LC2-P) was measured in rat hearts perfused with [32P]Pi at various levels of perfusate Ca2+. Computer simulations of the light-chain labelling suggested the presence of two isotopically distinct pools of LC2-P, one large pool comprising 90% of the total and a small pool consisting of the remaining 10%. At control levels of perfusate Ca2+ the phosphate of the large pool turned over very slowly (t 1/2 congruent to 250 min), whereas that of the small pool turned over much more rapidly (t 1/2 congruent to 1 min). At high levels of perfusate free Ca2+ (5mM) the turnover of the phosphate of the small pool decreased markedly, whereas that of the large pool remained little changed. Conversely, at low perfusate free Ca2+ (0.2 mM), the turnover of the large pool decreased, whereas that of the small pool remained unchanged. The possible identity of these two pools is discussed. The total myosin-light-chain kinase activity of rat ventricle was found to be only 2-3-fold higher than the kinase activity expressed in the heart under control conditions. This, coupled with the very low turnover of most of the LC2-bound phosphate, implies that, in heart, there is insufficient myosin-light-chain kinase activity to cause a rapid rise in the overall level of light-chain phosphorylation, even under conditions of increased cytoplasmic Ca2+.

A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle.

A method for measuring the molar stoichiometry of myosin light chain phosphorylation in intact smooth muscle has been developed. Antiserum to the 20,000-Da light chains of bovine aortic smooth muscle was harvested from rabbits and used to label light chains by a radioimmunoblotting procedure. In the initial characterization it was found that the 20,000-Da light chains could be transferred by electroblotting from polyacrylamide gels to nitrocellulose paper with an efficiency of approximately 80% over a protein range of 0.1-5.0 micrograms. At a dilution of 1:500, the unpurified light chain antiserum required approximately 10-12 h at 22 degrees C to reach equilibrium binding to the transferred light chains. Moreover, equilibrium labeling of the light chain-antibody complex with 125I-protein A required 4-6 h of incubation at 22 degrees C. By using these conditions, a radioimmunoassay for the 20,000-Da light chains was developed that was linear over a protein range of 0.1-5.0 micrograms (5-250 pmol). As little as 20 ng of light chains could be measured if a second antibody procedure (goat anti-rabbit immunoglobulin G Fab fragments) was used. Phosphorylated and unphosphorylated myosin light chains were separated by glycerol-urea polyacrylamide gel electrophoresis. This procedure, combined with radioimmunoblot, gave similar estimates of phosphorylation levels when compared with direct assay for phosphate or scanning of Coomassie blue-stained gels. Moreover, when applied to intact uterine smooth muscle, the glycerol-urea gel radioimmunoblot gave values of myosin light chain phosphorylation for relaxed and contracted muscles that were not statistically different from those obtained with a two-dimensional electrophoretic method.(ABSTRACT TRUNCATED AT 250 WORDS)

Random phosphorylation of the two heads of thymus myosin and the independent stimulation of their actin-activated ATPases.

Like other vertebrate nonmuscle myosins, thymus myosin contains two phosphorylatable light chains. Phosphorylation of these light chains regulates the actin-activated ATPase of this myosin. The time courses for the phosphorylation of both monomeric and filamentous thymus myosin by gizzard myosin light chain kinase fitted single exponentials to greater than 85% phosphorylation. This indicates that the two heads of thymus myosin are phosphorylated at the same rate and suggests that these phosphorylations are random processes. The actin-activated ATPases of thymus myosins with different levels of light chain phosphorylation were also determined. A linear relationship was obtained between the extent of light chain phosphorylation and stimulation of the actin-activated ATPase. Since thymus myosin appears to be phosphorylated randomly, this linear relationship indicates that phosphorylation of one head of thymus myosin stimulates the actin-activated ATPase of that head independently of the phosphorylation of the second head. The apparent random phosphorylation of thymus myosin light chains contrasts with the reported ordered phosphorylation of the light chains of filamentous smooth (gizzard) muscle myosin. Also, while the actin-activated ATPases of the two heads of thymus myosin are regulated independently, both heads of gizzard myosin must be phosphorylated before the ATPase of either head is activated by actin.

Myosin light chain phosphorylation during contraction of chicken fast and slow skeletal muscles

A modified automatic freezing apparatus ( K. M. Kretzschmar and D. R. Wilkie, 1962, J. Physiol. (London), 202, 66–67 ) was used for studying light chain phosphorylation during the early phase of contraction of the fast, posterior latissimus dorsi, and slow, anterior latissimus dorsi, muscles of chicken at 37 °C. The frozen muscles were worked up under conditions which avoid artifacts in quantitating the level of light chain phosphorylation in contracting and resting muscles. The posterior latissimus dorsi muscle reached 80% of its maximal isometric tension at 0.1 s of tetanic stimulation. At the same time, light chain phosphorylation increased by 60% of its maximal extent. The peak tension of the posterior muscle at 0.2 s of stimulation was accompanied by maximal light chain phosphorylation. In case of the slow anterior latissimus dorsi muscle, maximal tetanic tension was developed in 2.5 – 5 s and light chain phosphorylation also proceeded at a much slower rate than in the fast posterior muscle. When contralateral posterior latissimus dorsi muscles were stimulated for 0.2 s and one muscle was frozen at the height of tetanus while the other muscle was allowed to relax and frozen 0.4 s after terminating the stimulation, both contracted and relaxed muscles exhibited maximal light chain phosphorylation. However, when the muscle was allowed to relax for 0.8 s before freezing, half of the phosphorylated light chain became dephosphorylated. The resting level of phosphate content of the light chain was restored in both the posterior and anterior muscles during a longer time after relaxation.