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J T Stull

Publications and source records attributed to J T Stull.

At least 145 records · Page 8Linked to original sources
PubMed1982

Quantitation of myosin light chain phosphorylation in small tissue samples.

A method to quantitate the extent of phosphorylation of the 20,000-dalton phosphorylatable myosin light chain (P-light chain) in cardiac, smooth, or skeletal muscle samples which have limited amounts of tissue and myosin is presented. Native myosin is isolated from other cellular proteins in crude homogenates prepared from a few milligrams of muscle by pyrophosphate-polyacrylamide gel electrophoresis. The extent of P-light chain phosphorylation is maintained throughout this procedure by the inclusion of myosin light chain kinase and phosphatase inhibitors. Myosin obtained by pyrophosphate-gel electrophoresis is subjected to isoelectric focusing on polyacrylamide gels to separate the phosphorylated from the nonphosphorylated forms of the P-light chain. Following staining with ammonial-silver phosphorylation is quantitated on a mole of phosphate incorporated per mol of P-light chain basis by densitometric scanning of the isoelectric focusing gels. Direct comparison of this methodology with another method used for quantitating phosphorylation revealed no difference between the techniques in measuring the extent of P-light chain phosphorylation in muscle biopsy samples. This methodology provides the means for examination of the possible regulatory roles of P-light chain phosphorylation in contraction of cardiac, smooth, skeletal muscle.

Animals↗
PubMed1982

Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase.

The reversible association of Ca42+-calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochemistry 19, 5608--5614]. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (greater than 90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (less than 20% inhibition at mu = 0.22), and displayed a marked temperature dependence (Q10 congruent to 2; Ea = 10.4 kcal mol-1). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase activation by calmodulin had a narrower pH optimum (pH 6.0--7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (greater than 70% inhibition at mu = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 degrees C, activation was primarily entropically driven (delta S degrees congruent to 40 cal mol-1 deg-1; delta H degrees = -900 cal mol-1), but between 20 and 30 degrees C, enthalpic factors predominated in driving the activation process (delta S degrees congruent to 10 cal mol-1 deg-1; delta H degrees = -9980 cal mol-1). The apparent change in heat capacity (delta Cp) accompanying activation was estimated to be -910 cal mol-1 deg-1. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interactions also make significant and probably obligatory contributions to the activation process.

Animals↗
PubMed1982

The effect of low frequency stimulation on myosin light chain phosphorylation in skeletal muscle.

Phosphorylation of the P-light chain of myosin from skeletal muscle by myosin light chain kinase is dependent upon calmodulin and Ca2+. Investigations were performed to determine if the rapid Ca2+ transients that occur during low frequency repetitive stimulation are sufficient to activate myosin light chain kinase with significant P-light chain phosphorylation. In addition, P-light chain phosphorylation was correlated with potentiation of isomeric twitch tension (staircase phenomenon). Stimulation of rat gastrocnemius muscle at 5 Hz in situ results in a time-dependent phosphorylation of the P-light chain of myosin. Initially, there was a rapid rate of phosphorylation within the first 10 muscle twitches (0.19 to 0.40 mol of phosphate/mol of P-light chain) followed by a slower rate of phosphorylation. These data indicate that myosin light chain kinase can be activated during repetitive stimulation at a low frequency in the range that occurs in vivo, despite the fact that the muscle is in the relaxed state during most of the period between each stimulation. Potentiation of isometric twitch tension was found to be temporally correlated to light chain phosphorylation at 5 Hz. It is postulated that the transient changes in intracellular Ca2+ concentration associated with low frequency stimulation are sufficient to activate myosin light chain kinase, and, furthermore, the magnitude of the potentiation of isometric twitch tension may be related to the extent of phosphorylation of myosin during a stimulus train.

Animals↗
PubMed1982

Phosphorylation of myosin light chain by a protease-activated kinase from rabbit skeletal muscle.

A protease-activated protein kinase that phosphorylates the P light chain of myosin in the absence of Ca2+ and calmodulin has been isolated from rabbit skeletal muscle. The enzyme has properties similar to protease-activated kinase I from rabbit reticulocytes [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11588-11564], which has been shown to phosphorylate the P light chain of myosin [P. T. Tuazon, J. T. Stull, and J. A. Traugh (1982) Biochem. Biophys. Res. Commun. 108, 910-917]. The protease-activated kinase from skeletal muscle has been partially purified by chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The enzyme phosphorylates histone as well as the P light chain of myosin following activation by proteolysis. Stoichiometric phosphorylation of myosin light chain was observed with the protease-activated kinase and myosin light chain kinase. The sites phosphorylated by the protease-activated kinase and myosin light chain kinase were examined by two-dimensional peptide mapping following chymotryptic digestion. The phosphopeptides observed with the protease-activated kinase were different from those obtained with the Ca2+-dependent myosin light chain kinase, indicating that the two enzymes phosphorylated different sites on the P light chain of skeletal muscle myosin. When actomyosin from skeletal muscle was examined as substrate, the P light chain was phosphorylated following activation of the protease-activated kinase by limited proteolysis.

Animals↗
PubMed1982

Myosin light chain phosphorylation-dephosphorylation in mammalian skeletal muscle.

Phosphorylation of the myosin light chain 2 (LC2) subunit was examined in rat fast-twitch and slow-twitch skeletal muscles in response to repetitive stimulation at 23 and 35 degrees C and on incubation of fast-twitch skeletal muscle with isoproterenol. After a 1-s tetany at 35 degrees C, LC2 phosphate content in extensor digitorum longus muscle increased rapidly and transiently from 0.21 to 0.51 mol phosphate/mol LC2. This pattern of phosphorylation was similar to that observed at 23 degrees C. Increases in LC2 phosphate content were dependent on the frequency and duration of stimulation. In soleus muscle LC2 phosphate content was minimal following a 1-s tetany but increased markedly following more prolonged tetanies. On incubation of extensor digitorum longus muscle with isoproterenol (20 microM), LC2 phosphate content did not change, whereas phosphorylase a levels increased. A positive correlation existed between LC2 phosphate content and potentiation of peak twitch tension in both types of muscles, suggesting a physiological function for LC2 phosphorylation.

Action Potentials↗
PubMed1980

Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin.

Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin for form the complex, Ca42+-calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simply hyperbolic kinetics, indicating 1:1 stoichiometry of Ca42+-calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa2+ and KCM were found to have values of 10 microM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.

Animals↗
PubMed1980

Myosin phosphorylation during contraction and relaxation of tracheal smooth muscle.

The role of myosin phosphorylation in regulating smooth muscle contraction has been investigated by quantitating the myosin phosphate content of tracheal smooth muscle frozen during contraction or relaxation. Myosin was purified from quick-frozen muscle samples with the aid of antibodies prepared against tracheal smooth muscle myosin, and the phosphate content was determined after separation of nonphosphorylated and phosphorylated myosin subunits by isoelectric focusing. The myosin phosphate content increased from an initial value of 0.50 to 1.1 mol of phosphate/mol of myosin within 3 min after the addition of 100 microM methacholine to resting tracheal smooth muscle. Myosin phosphorylation coincided temporally with the increase in isometric tension. Tracheal smooth muscles relaxed and the phosphate content decreased from 1.2 to 0.50 mol of phosphate/mol of myosin upon the addition of 10 microM atropine to muscles which had been previously contracted with 100 microM methacholine. Incubating methacholine-contracted muscles in a calcium-free Krebs buffer relaxed tracheal smooth muscles and reduced the phosphate content to 0.2 mol of phosphate/mol of myosin. Addition of 3 mM CaCl2 to these muscles elicited an increase in isometric tension concomitant with phosphorylation of myosin. These results support the hypothesis that myosin phosphorylation is important in regulating smooth muscle contraction.

Animals↗
PubMed1980

Protein phosphorylation: quantitative analysis in vivo and in intact cell systems.

Protein phosphorylation-dephosphorylation appears to be an essential component in the regulation of many cellular processes by hormones and drugs. This concept has developed primarily from in vitro biochemical studies in which various purified proteins have been phosphorylated and dephosphorylated by distinct protein kinases and phosphoprotein phosphatases. However, the more difficult, but essential, task of demonstrating the physiological occurrence of these reactions in intact tissue or cell preparations in many cases has not been undertaken in a quantitative manner. There are 4 basic approaches for assessing the extent of protein phosphorylation in vivo and in intact cell systems, each having particular advantages and disadvantages. These are summarized in Table 2. The applicability of any one procedure will be highly dependent upon the protein under investigation. For instance, chemical measurements of total protein-bound phosphate may provide only limited information for proteins which are phosphorylated at multiple sites but could be highly useful for those proteins such as glycogen phosphorylase which are phosphorylated at single sites. The relative ease and the high sensitivity of measuring 32P incorporation into proteins will tempt many investigators to rely heavily on this approach. It is a very powerful procedure, particularly for the initial identification of phosphoproteins, but ultimately quantitative conclusions regarding 32P incorporation must be corroborated by one or more of the other procedures. There is no simple, single experimental approach that may be used under all circumstances, but by integrating these procedures firm conclusions may be drawn regarding the physiological importance of phorphorylation of specific proteins.

Adenosine Diphosphate↗
PubMed1980

Phosphorylation of myosin in perfused rabbit and rat hearts.

Working perfused rabbit and rat hearts were used to determine whether changes in myosin P-light chain phosphate content could affect cardiac contractility. Control perfused rabbit and rat hearts contained 0.48 +/- 0.02 and 0.61 +/- 0.02 mol phosphate/mol P-light chain, respectively. Perfusion of 1) rabbit hearts with 0.1 microM isoproterenol for 30 s, 2) rabbit or rat hearts with a fivefold increase in perfusate [Ca2+] for 30 s, or 3) rabbit hearts with a threefold increase in [Ca2+] for 5 and 15 min caused significant increases in LV dP/dtmax, but had no effect on P-light chain phosphate content. Perfusion of rabbit hearts with Ca2+-free buffer or buffer containing 22 mM K+ caused complete cessation of contractile activity within 30 s and reduced P-light chain phosphate content to 50 and 70%, respectively, of control values after 30 min. Reperfusion of hearts exposed to 22 mM K+ with control buffer restored LV dP/dtmax to control values within 10 min, whereas P-light chain phosphate content remained at 70% of control value. Thus, changes in cardiac contractile state were not accompanied by changes in the extent of phosphorylation of P-light chain.

Animals↗
PubMed1980

Phosphorylation of contractile proteins in heart and skeletal muscle.

Contractile performance of cardiac and skeletal muscles may be regulated by cyclic AMP or Ca2+, two second messengers that stimulate the phosphorylation of specific myofibrillar proteins. Cyclic AMP-dependent protein kinase catalyzed the rapid phosphorylation of a single site in the inhibitory subunit of cardiac troponin in vitro and in perfused hearts. Skeletal muscle troponin was not phosphorylated by this enzyme in vivo. Although there was a correlation between cardiac troponin phosphorylation and the positive inotropic response to catecholamines, a biochemical mechanism that could account for a functional relationship between the two processes has not been discovered. Phosphorylation of skeletal muscle myosin was catalyzed by myosin light chain kinase in the presence of Ca2+ and the ubiguitous, multifunctional Ca2+-dependent regulator protein (CDR). The activation of kinase activity appeared to proceed via a trimolecular reaction process in which Ca2+ bound to CDR and the Ca2+.CDR complex then interacted with the enzyme. In rat extensor digitorum longus muscle, a 1 sec tetanic contraction resulted in phosphorylation of myosin light chain with the maximal phosphate incorporated 20 sec after the contraction. The light chain phosphate content declined slowly and correlated to post-tetanic potentiation of isometric twitch tension. Phosphorylation of skeletal muscle myosin may be important in modulating contraction.

Animals↗