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J T Stull

Publications and source records attributed to J T Stull.

At least 109 records · Page 6Linked to original sources
PubMed1988

Calcium dependence of myosin light chain phosphorylation in smooth muscle cells.

Smooth muscle cells grown in culture may provide a model system for studying the Ca2+ dependence of myosin light chain phosphorylation. Tracheal smooth muscle cells in culture had 60% of the myosin content of tracheal tissue. Western analysis with appropriate antibodies demonstrated one 20-kDa light chain and the presence of a 150-kDa myosin light chain kinase in both tracheal smooth muscle tissue and cells. Moreover, tracheal cells contained 74% of the myosin light chain kinase activity measured in tissue. Similar types of analyses of nonmuscle cells showed a much lower myosin and myosin light chain kinase content. Carbachol (10 microM) or ionomycin (10 microM) stimulation of fura-2-containing cells resulted in a rapid increase in cytosolic free Ca2+ concentration and in the extent of myosin light chain phosphorylation. Maximal increases in Ca2+ concentrations were greater with ionomycin than with carbachol (4400 versus 492 nM). Light chain phosphorylation increased after the Ca2+ concentration exceeded 200 nM from control values of 165 nM. Half-maximal phosphorylation (33%) occurred at 260 nM Ca2+. There was a similar relationship between free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in carbachol- and ionomycin-stimulated cells. This relationship had a Hill coefficient of 2.7. These observations indicate that small changes in Ca2+ concentrations stimulate myosin light chain phosphorylation and thus presumably contraction in smooth muscle cells.

Animals↗
PubMed1988

Atrial natriuretic peptide inhibits the agonist-induced increase in extent of myosin light chain phosphorylation in aortic smooth muscle.

The effect of atrial natriuretic peptide (ANP) on angiotensin II- and histamine-induced contraction and muscle light chain phosphorylation was examined in strips of rabbit aorta smooth muscle. Preincubation of strips with 10(-7) M ANP prior to addition of either agonist inhibits both the increase in extent of myosin light chain phosphorylation and the contractile response to either 5 x 10(-8) M angiotensin II or 10(-5) M histamine without inhibiting the agonist-induced increase in the intracellular free Ca2+ concentration. Furthermore, in muscle strips precontracted with either angiotensin II or histamine, addition of ANP leads to a prompt relaxation and a prompt decrease in the extent of myosin light chain phosphorylation. These data argue that ANP uncouples the initial agonist-induced Ca2+ transient from the increase in extent of myosin light chain phosphorylation either by inhibiting the Ca2+-dependent activation of myosin light chain kinase or stimulating the activity of a phosphoprotein phosphatase capable of bringing about the rapid dephosphorylation of phosphorylated myosin light chains.

Aequorin↗
PubMed1988

Calcium control of smooth muscle contractility.

Ca2+ is a primary second messenger that binds to an intracellular receptor protein, calmodulin. Increases in cytosolic Ca2+ concentration mediated by activation of cell surface receptors result in the formation of a Ca2+ calmodulin complex that regulates many Ca2+-dependent cellular processes. In smooth muscle, Ca2+/calmodulin activates myosin light chain kinase, which phosphorylates the regulatory light chain of myosin. This phosphorylation reaction increases the actin-activated MgATPase activity of myosin and is associated with increases in contractile properties, including force, stiffness, and maximal shortening velocity. These biochemical and biomechanical responses occur rapidly (seconds) in response to physiological stimulation involving neurotransmitter activation of smooth muscle cells. Thus, the Ca2+-dependent phosphorylation of the myosin light chain is a primary event in activation of smooth muscle contraction.

Actins↗
PubMed1987

Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase.

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.

Amino Acid Sequence↗
PubMed1987

Structural studies of rabbit skeletal muscle myosin light chain kinase with monoclonal antibodies.

Monoclonal antibodies directed against rabbit skeletal muscle myosin light chain kinase have been used to study the domains of this kinase. Specificity of nine monoclonal antibodies against rabbit skeletal muscle myosin light chain kinase was demonstrated by immunoblot analysis and immunoadsorption of kinase activity. None of the antibodies reacted by immunoblot analysis with either chicken skeletal or rabbit smooth muscle myosin light chain kinases. Epitope mapping of trypsin-digested rabbit skeletal muscle myosin light chain kinase showed that antibodies 2a, 9a, 9b, 12a, 12b, 16a, and 16b are directed against the 40-kDa catalytic domain. In addition, these seven antibodies reacted with sites that are clustered within a 14-kDa fragment of the kinase generated by Staphylococcus aureus V8 protease digestion. Two monoclonal antibodies, 14a and 19a, reacted with two distinct epitopes located within the inactive, asymmetric trypsin fragment. Six of nine monoclonal antibodies (2a, 9a, 9b, 12a, 12b, and 14a) inhibited kinase activity. Kinetic analyses demonstrated that antibodies 2a, 12a, and 14a inhibited kinase activity competitively with respect to myosin phosphorylatable light chain; 2a, 12a, and 14a exhibit noncompetitive inhibition with respect to calmodulin. These data suggest that monoclonal antibodies 2a, 12a, and 14a bind at or adjacent to the active site of the kinase.

Animals↗
PubMed1987

Biochemical markers of contraction in human myometrial smooth muscle cells in culture.

Phosphorylation of a light chain subunit of myosin by Ca2+ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05 +/- 0.18 and 1.60 +/- 0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca2+ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca2+ concentration measured by quin2 fluorescence was 0.12 microM in resting cells and increased in a concentration-dependent manner with KCl to a maximal value of 0.47 microM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture.

Calcium↗
PubMed1986

Genetically engineered calmodulins differentially activate target enzymes.

Three mutant calmodulin (CaM) genes together with the normal chicken CaM cDNA have been expressed in bacteria for the purpose of determining structure/function relationships in CaM. The mutant CaM genes were generated by in vitro recombination between a chicken CaM cDNA and a processed pseudogene that encodes a full-length CaM but with 19 amino acid substitutions as compared to authentic vertebrate CaM. The calmodulin-like (CaML) proteins derived from the pseudogene are called CaML19, CaML16, and CaML3 and contain 19, 16, and 3 amino acid substitutions, respectively. CaML3 is functionally identical to CaM by all criteria tested. The functional characteristics of CaML16 and CaML19 are also indistinguishable yet quite different from normal CaM. CaML19 and CaML16 will maximally activate myosin light chain kinase but will only half-maximally activate calcineurin and CaM-dependent multiprotein kinase. In addition, CaML16 and CaML19 do not activate phosphorylase kinase. The differential activation of these enzymes does not result from the loss of Ca2+-binding sites, since CaML16 binds four Ca2+ with affinity similar to CaM or CaM23. It is more likely that the functional characteristics of the mutant proteins result from an altered tertiary structure, since the Ca2+-dependent enhancement of tyrosine fluorescence and limited proteolysis pattern of CaML16 are different from that of CaM. The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.

Amino Acid Sequence↗
PubMed1986

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases.

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.

Alanine↗
PubMed1986

Different phosphorylated forms of myosin in contracting tracheal smooth muscle.

Calmodulin-dependent myosin light chain kinase phosphorylates two light chain subunits on each myosin molecule. We have developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin in smooth muscle. Four protein bands were separated in tissue extracts by nondenaturing polyacrylamide gel electrophoresis in the presence of pyrophosphate. Immunoblots demonstrated that three forms (designated M, MP, and MP2) reacted with rabbit antisera prepared against the purified phosphorylatable light chain (P-light chain) from bovine tracheal smooth muscle. Evidence was obtained that M, MP, and MP2 represented nonphosphorylated, monophosphorylated, and diphosphorylated myosin, respectively, and that the other protein band was probably filamin. The formation of different phosphorylated forms of myosin was measured in bovine trachealis strips neurally stimulated from 1.0 to 3.5 s and quick-frozen. There was no detectable MP or MP2 in unstimulated muscles; the extent of P-light chain phosphorylation measured directly was 0.02 +/- 0.01 mol of phosphate/mol of P-light chain. After 2.5-s stimulation, maximal values of 0.63 +/- 0.06 mol of phosphate/mol of P-light chain and 0.40 +/- 0.06 MP2/myosintotal were obtained. During continuous neural stimulation from 1.0 to 3.5 s, the relationship between the extent of P-light chain phosphorylation (measured directly or calculated) and the relative amount of MP2 is consistent with a random phosphorylation process.

Animals↗
PubMed1986

Decrease in myosin light chain kinase activity of rabbit fast muscle by chronic stimulation.

Analysis of myosin light chain kinase (MLCK) activity in tibialis anterior muscles of the rabbit revealed that chronic stimulation at a frequency of 10 Hz for 24 h per day reduced the enzyme activity in a time-dependent manner. Since fast twitch muscle contains significantly more myosin light chain kinase than slow twitch muscle, the observed reductions are consistent with the type of fast-to-slow transformation observed for other type-specific muscle characteristics. The present data also indicate that the stimulation-induced decrease in MLCK activity precedes the fast-to-slow conversion of the myosin molecule as judged by pyrophosphate-polyacrylamide gel electrophoresis.

Animals↗
PubMed1986

Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness.

Contraction and myosin light-chain phosphorylation were measured in electrically stimulated tracheal smooth muscle. Latencies for the onset of force, stiffness, and light-chain phosphorylation were 500 milliseconds. Myosin light chain was phosphorylated from 0.04 to 0.80 mole of phosphate per mole of light chain with a pseudo-first-order rate of 1.1 per second with no evidence of an ordered or negatively cooperative process. Following the period of latency, stiffness increased with phosphorylation and both increased more rapidly than isometric force. The linear relation between stiffness and phosphorylation during activation suggests independent attachment of each myosin head upon phosphorylation.

Animals↗
PubMed1986

Frequency-dependent myosin light chain phosphorylation in isolated myocardium.

A specific light chain subunit (P-light chain) of myosin from striated and smooth muscles is phosphorylated by Ca2+ calmodulin-dependent myosin light chain kinase. Phosphorylation of cardiac P-light chain was examined in isolated perfused rabbit ventricular septae to determine the effect of contraction frequency on this Ca2+-dependent reaction. Muscles stimulated at 42 beats/min had 0.23 mol phosphate/mol P-light chain which decreased to 0.12 mol phosphate/mol P-light chain when the muscles were made quiescent (0 beats/min in the presence of 22 mM K+ for 30 min). Rephosphorylation of P-light chain to 0.24 mol phosphate/mol P-light chain occurred in muscles stimulated at 84 beats/min for 90 min but not in muscles stimulated at 42 beats/min for 30 min (0.15 mol phosphate/mol P-light chain). Stimulation at frequencies ranging from 0 to 126 beats/min for 30 min produced a frequency-dependent increase in P-light chain phosphorylation from 0.1 to 0.4 mol phosphate/mol P-light chain. Increased inotropy for 30 s with isoproterenol was not associated with significant increases in P-light chain phosphorylation in muscles stimulated at 42 beats/min. The rates of myosin P-light chain phosphorylation and dephosphorylation in ventricular muscle are much slower than the reported rates of phosphorylation in either fast-twitch skeletal or smooth muscles. The extent of cardiac P-light chain phosphorylation appears dependent upon the steady-state frequency of contraction.

Animals↗
PubMed1986

Phosphorylation of myosin in permeabilized mammalian cardiac and skeletal muscle cells.

The effect of myosin phosphorylation on tension production at less than 50% maximal activation by Ca2+ was examined in rabbit psoas and ventricular muscle. For psoas fibers, tension was determined at pCa 6.0, 5.8, 5.6, 5.5, and 5.4. Myosin light chain kinase (0.15 microM) and calmodulin (2 microM) were added, and the fibers were incubated at pCa 5.4, which resulted in an increase in light chain phosphorylation (P-light chain) from 5-10 to 60-75%. After 5 min, the sequence of pCa activations was repeated. An identical protocol was followed for cardiac muscle, except the activation solutions were pCa 6.2, 6.0, 5.9, 5.8, and 5.6. Phosphorylation of P-light chain increased tension in both permeabilized cardiac and skeletal muscle fibers. The effect manifested itself as a leftward shift in the pCa-tension relationship at levels below 50% maximal activation, with a decrease in the slope of the pCa-tension relationship. These results indicate that P-light chain phosphorylation affects actin-myosin interactions in cardiac and skeletal muscles at submaximal levels of Ca2+ activation.

Animals↗
PubMed1985

The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers.

We have studied the effect of myosin P-light chain phosphorylation on the isometric tension generated by skinned fibers from rabbit psoas muscle at 0.6 and 10 microM Ca2+. At the lower Ca2+ concentration, which produced 10-20% of the maximal isometric tension obtained at 10 microM Ca2+, addition of purified myosin light chain resulted in a 50% increase in isometric tension which correlated with an increase in P-light chain phosphorylation from 0.10 to 0.80 mol of phosphate/mol of P-light chain. Addition of a phosphoprotein phosphatase reversed the isometric tension response and dephosphorylated P-light chain. At the higher Ca2+ concentration, P-light chain phosphorylation was found to have little effect on isometric tension. Fibers prepared and stored at -20 degrees C in a buffer containing MgATP, KF, and potassium phosphate incorporated 0.80 mol of phosphate/mol of P-light chain. Addition of phosphoprotein phosphatase to these fibers incubated at 0.6 microM Ca2+ caused a reduction in isometric tension and dephosphorylation of the P-light chain. There was no difference before and after phosphorylation of P-light chain in the normalized force-velocity relationship for fibers at the lower Ca2+ concentration, and the extrapolated maximum shortening velocity was 2.2 fiber lengths/s. Our results suggest that in vertebrate skeletal muscle, P-light chain phosphorylation increases the force level at submaximal Ca2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament.

Animals↗