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J T Stull

Publications and source records attributed to J T Stull.

At least 127 records · Page 7Linked to original sources
PubMed1985

Characterization of chicken skeletal muscle myosin light chain kinase. Evidence for muscle-specific isozymes.

Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.

Animals↗
PubMed1985

Phosphorylation of rabbit skeletal muscle myosin in situ.

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.

Animals↗
PubMed1985

Myosin light chain phosphorylation and isometric twitch potentiation in intact human muscle.

The effects of a single voluntary contraction of the quadriceps muscle group on phosphate incorporation into the phosphorylatable light chains (P-light chains) of fast and slow myosin isolated from the vastus lateralis muscle and potentiation of the electrically stimulated twitch tension was studied in intact human muscle. Twitch potentiation was maximal 20 s after the voluntary contraction. Thereafter, twitch potentiation declined, but was still significantly higher than pre-contraction values 2 min after the voluntary contraction. Phosphate incorporation into the P-light chain of fast myosin followed a similar time course to twitch potentiation, but no phosphate was incorporated into slow myosin P-light chains. These observations suggest that myosin light chain kinase activity is mainly associated with fast-twitch muscle fibers and, in agreement with previous studies, suggests that twitch potentiation associated with P-light chain phosphorylation is confined to the fast-twitch fibers of human muscle.

Adult↗
PubMed1985

Myosin light chain kinases and myosin phosphorylation in skeletal muscle.

Myosin light chain kinases appear to exist as a family of tissue- and species-specific isozymes. The skeletal muscle kinases, although differing widely in molecular weight among vertebrate species, are catalytically similar and antigenically related. The smooth muscle kinases are catalytically and antigenically distinct from the skeletal muscle kinases. The functional basis for the existence of myosin light chain kinase isozymes has not been determined. Phosphorylation of fast-twitch skeletal muscle myosin P-light chain occurs at physiologically relevant contraction frequencies and durations, and the extent of P-light chain phosphorylation correlates with enhancement of isometric twitch tension in fast-twitch muscle under a variety of experimental conditions. Phosphorylation of myosin P-light chain in vertebrate fast-twitch skeletal muscle may play a modulatory role in calcium regulation of muscle contractility.

Animals↗
PubMed1985

Myosin phosphorylation, force, and maximal shortening velocity in neurally stimulated tracheal smooth muscle.

Field stimulation of intrinsic nerves in bovine tracheal smooth muscle strips elicited atropine-sensitive contractions that were more rapid than those obtained by addition of carbamylcholine to the bathing medium. These stimulus conditions were used to improve estimates of maximal rates of activation as indicated by myosin light chain phosphorylation, maximal shortening velocity (Vo), and isometric force. Maximal values of Vo [0.25 X optimal muscle length X s-1] and light chain phosphorylation (0.65 mol phosphate/mol light chain) were attained after 5 s of stimulation and preceded maximal force (60 s). Force gradually fell to 0.85 times maximal values during 30 min of stimulation, while both light chain phosphorylation and Vo declined to 0.3 times the maximal value. The temporal correlation between light chain phosphorylation and Vo supports the hypothesis that myosin phosphorylation in smooth muscle functions in regulating cross-bridge cycling rates. Myosin was dephosphorylated during relaxation with a half time of 2.7 s. Calculated maximal cellular rates of light chain phosphorylation were similar to measured values, indicating that most of the kinase was activated on stimulation.

Animals↗
PubMed1984

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity.

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1984

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity.

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.

Animals↗
PubMed1984

Myosin phosphorylation decreases the ATPase activity of cardiac myofibrils.

Our previous work showed that myosin phosphorylation decreased the ATPase activity of skeletal muscle myofibrils that were lightly fixed with glutaraldehyde. The fixation process prevented sarcomere shortening and destruction of the ordered filament array upon the addition of ATP. We have now extended these results to myofibrils prepared from hearts of rabbits, dogs and rats. Myofibrils were phosphorylated by incubation with myosin light chain kinase, calmodulin and either ATP-gamma s or ATP, for 15 minutes at 25 degrees C. The extent of myosin light chain phosphorylation was 50% to 80%. The ATPase activity of unphosphorylated myofibrils was not altered by reaction with 0.01% glutaraldehyde for 5 minutes at 0 degrees C, and the ATPase activity of unfixed myofibrils was not changed by phosphorylation. However, phosphorylation decreased the ATPase activity of fixed myofibrils by 50%. The effect on myocardial myofibrillar ATPase activity of phosphorylation was similar in the three animal species. These results suggest that in both skeletal and cardiac muscle, myosin phosphorylation decreases the rate of cross-bridge cycling resulting in decreased energy expenditure. It also appears that the effect of myosin light chain phosphorylation on ATPase activity requires an ordered myofilament structure.

Adenosine Triphosphatases↗
PubMed1984

Myosin light chain phosphorylation in fast and slow skeletal muscles in situ.

The physiological properties of contraction-induced phosphate incorporation into the phosphorylatable light chain (P-light chain) of myosin were examined in fast-twitch white, fast-twitch red, and slow-twitch skeletal muscles in situ. Neural stimulation of rat gastrocnemius muscles between 0.5 and 100 Hz produced an increase in the phosphate content of the P-light chain from the white portion of the muscle, and the rate of P-light chain phosphorylation was frequency dependent. The extent of phosphorylation of P-light chain from the fast-twitch red portion of the gastrocnemius muscle was less. In contrast to fast-twitch skeletal muscle, only high-frequency stimulation (30-100 Hz) produced a small increase in the phosphate content of P-light chain from the slow-twitch soleus muscle. Fast white muscle contained 2.2 and 3.5 times more myosin light chain kinase activity than did the fast red and slow muscle, respectively. The rate of P-light chain dephosphorylation was four times faster in slow muscle than in fast white muscle. Thus the greater extent of phosphorylation of P-light chain in fast-twitch white skeletal muscle fibers may be due in part to the presence of more kinase and less phosphatase activities. Isometric twitch tension potentiation was correlated to the extent of phosphorylation of P-light chain from fast white muscle. The physiological consequences of P-light chain phosphorylation are likely to be of greatest importance in fast-twitch white muscle.

Animals↗
PubMed1984

Phosphorylation of myosin light chain and phosphorylase in tracheal smooth muscle in response to KCl and carbachol.

It has been proposed that Ca2+-dependent myosin light chain (P-light chain) phosphorylation in smooth muscle permits cycling of myosin cross-bridges within myofibrillar elements for muscle shortening, but a second Ca2+-dependent regulatory mechanism is responsible for force generation. Accordingly, we examined P-light chain phosphorylation and another Ca2+-dependent protein phosphorylation reaction, phosphorylase a formation, in bovine tracheal smooth muscle during isometric force generation elicited by the cholinergic agonist carbachol or KCl depolarization, two stimuli thought to increase the concentration of sarcoplasmic free Ca2+ by mobilizing different pools of Ca2+. Increases in P-light chain phosphorylation reached maximal values of 0.79 and 0.59 mole of phosphate per mole of P-light chain at 1 min and then declined during maintained isometric force developed in response to 1 microM carbachol and 60 mM KCl, respectively. Carbachol elicited approximately twice the amount of force as found in the presence of KCl, and yet a more rapid rate of decline in the phosphate content of P-light chain was apparent. Decreases in maximal levels of phosphorylase a also occurred during carbachol-mediated isometric force maintenance, yet did not occur with KCl stimulation. Concentration-dependent responses with carbachol and KCl showed a positive relationship between the extent of P-light chain phosphorylation and extent of developed isometric force after 1 min of contraction with both stimuli. Under no conditions was force generated without P-light chain phosphorylation. The concentration dependence of phosphorylase a formation with KCl was similar to isometric force and P-light chain phosphorylation. However, concentrations of carbachol necessary to stimulate phosphorylase a formation were much higher than those required for stimulation of isometric force and P-light chain phosphorylation. Furthermore, carbachol attenuated the stimulation of phosphorylase a formation by isoproterenol. Thus, carbachol appears to have both an inhibitory and stimulatory effect on phosphorylase a formation in bovine tracheal smooth muscle. These results also indicate that maintained isometric force in smooth muscle may be dependent upon the maximal extent of P-light chain phosphorylation obtained during an early temporal transient in phosphorylation.

Animals↗
PubMed1983

The role of myosin light chain kinase phosphorylation in beta-adrenergic relaxation of tracheal smooth muscle.

Myosin light chain kinase from smooth muscle has been shown to be phosphorylated by cyclic AMP-dependent protein kinase, which leads to a decrease in the affinity of the kinase for Ca2+ . calmodulin and, hence, a decrease in enzymatic activity. This event has been proposed as a mechanism for the relaxation of smooth muscle in response to increased intracellular concentrations of cyclic AMP. The ratio of myosin light chain kinase activities measured in the presence of 4 microM or 100 microM Ca2+, at 1 microM calmodulin, permits evaluation of such a change in the calmodulin activation properties of myosin light chain kinase. This activity ratio was decreased by phosphorylation of either purified bovine tracheal smooth muscle myosin light chain kinase, or the endogenous myosin light chain kinase in a homogenate of tracheal smooth muscle, with the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. The ratio was unchanged, however, by activation of the endogenous cyclic AMP-dependent protein kinase in homogenates of tracheal smooth muscle by the addition of cyclic AMP. Incubation of tracheal smooth muscle with isoproterenol, at a concentration sufficient to relax the muscle and to increase phosphorylase a formation, had no effect upon the activity ratio. Incubation of tracheal smooth muscle for 2 hr in the presence of carbachol resulted in a transient increase and then a decrease in myosin light chain phosphate content to control values with no decrease in isometric force. The addition of isoproterenol at 2 hr still resulted in relaxation. These findings are inconsistent with a role of myosin light chain kinase phosphorylation in mediating relaxation of tracheal smooth muscle by beta-adrenergic agonists. Cyclic AMP-dependent effects on cytoplasmic calcium concentrations may be more important in mediating relaxation.

Adrenergic beta-Agonists↗
PubMed1983

Phosphorylation of myosin light chain in skeletal and smooth muscles.

Significant differences in the properties of myosin light chain phosphorylation in skeletal and smooth muscles may be important in considering the role of myosin phosphorylation in contraction. Repetitive, low-frequency stimulation of fast-twitch skeletal muscle resulted in phosphorylation of myosin light chain. Some of the factors leading to phosphorylation under these conditions include 1) partial activation of myosin light chain kinase with each stimulus, 2) a slow rate (t1/2 = 1 s) of inactivation of the kinase activity, and 3) a very slow rate of dephosphorylation by myosin light chain phosphatase. Myosin light chain phosphorylation was correlated with potentiation of isometric twitch tension in posttetanic potentiation and staircase responses. Stimulation of contraction in bovine tracheal smooth muscle by the cholinergic agonist carbachol was correlated with phosphorylation of the myosin light chain. The initial rate and maximum extent of phosphorylation during the first minute of stimulation was dependent on the concentration of carbachol. Phosphate incorporation into light chain declined after 1 min whereas isometric tension was maintained. beta-Adrenergic inhibition of tension development was accompanied by a decrease in the rate and extent of phosphorylatable myosin light chain phosphorylation but was not associated with reduced affinity of myosin light chain kinase for calcium-calmodulin.

Animals↗
PubMed1983

Effects of the calmodulin antagonist, fluphenazine, on phosphorylation of myosin and phosphorylase in intact smooth muscle.

The activities of phosphorylase kinase and myosin light-chain kinase are regulated by Ca2+ binding to calmodulin. However, differences in the activation properties of the purified enzymes are apparent, since calmodulin binds to phosphorylase kinase in the absence of Ca2+ whereas prior formation of a Ca2+ . calmodulin complex is necessary for calmodulin to bind to and activate myosin light chain kinase. Since the phenothiazines have been implicated as anticalmodulin drugs and inhibit contractile activity in smooth muscle, we examined the effects of the phenothiazine, fluphenazine, on isometric tension development and phosphorylation of phosphorylase and the phosphorylatable light chain (P-light chain) of myosin in intact bovine tracheal smooth muscle. Preincubation with 50 microM fluphenazine for 5 min inhibited the maximal rate and extent of isometric tension development and P-light chain phosphorylation in the presence of 60 mM KCl. Application of fluphenazine after tension and the phosphate content of the P-light chain had reached steady-state levels in response to 60 mM KCl produced little relaxation or dephosphorylation of the P-light chain. KCl-mediated phosphorylase a formation was not inhibited by preincubation with fluphenazine for 5 min. However, long periods of preincubation (30-60 min) produced significant inhibition of phosphorylase a formation and proportionally greater inhibition of tension and P-light chain phosphorylation. Since phosphorylase a formation was not inhibited during short-term preincubation with fluphenazine, KCl-dependent increases in the concentration of free intracellular Ca2+ may not have been affected. Moreover, since both isometric tension development and P-light chain phosphorylation were attenuated in a parallel manner, inhibition of contractile activity in intact smooth muscle by anticalmodulin agents may be directly related to inhibition of P-light chain phosphorylation.

Animals↗