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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

One of the reactive thiols in the myosin head, SH1, was covalently labeled with a biotin derivative, N-iodoacetyl-N'-biotinylhexylenediamine. When 50% of the SH1 thiol was modified with the biotin reagent as judged from measurements of ATPase activities, the biotinylated myosin bound one mole of avidin per mole of myosin at the saturating level. The avidin-myosin complex was readily formed in the presence of MgADP or MgATP. Peptide maps of the biotinylated myosin revealed that SH1 is actually the site of biotinylation with N-iodoacetyl-N'-biotinylhexylenediamine. Electron microscopic examination of the avidin-myosin complex showed that the attachment site of avidin on the myosin head is 130 A from the head-rod junction, indicating that the SH1 thiol is located there.
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PMID:Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system. 654 25

The experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30 degrees C were studied. Substantially all RLC was released from myosin by incubation for 5 min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+, while the effect of Mg2+ was about 10,000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP. The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30 degrees C for 5 min produced a hybrid myosin. In the presence of 5 mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.
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PMID:Inhibitory effect of MgATP on the release of regulatory light chain from scallop myosin and light chain composition of scallop myosin hybridized with abalone light chain 2 at 30 degrees C. 660 40

The time course of magnesium adenosine triphosphate (Mg ATP) cleavage in chemically skinned muscle fibres of the rabbit was measured by a method in which Mg ATP cleavage was initiated by photolytic release of ATP from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and terminated by rapid freezing 50 ms to 8 s later. Up to 5 mM-ATP was released following a single 50 ns laser pulse at 347 nm. Mg ATP cleavage was measured at 19 degrees C in the presence and absence of calcium ions, for fibres near rest length and stretched beyond overlap of the myofilaments. At full overlap and in the absence of calcium (less than 10(-8) M) and nucleotide, the fibres developed rigor tension. Following the laser pulse the tension decreased to that of a relaxed fibre in two distinct phases. The first phase lasted about 40 ms and was followed by a second phase during which tension decreased to zero with an approximately exponential time course with a rate constant of 11 s-1. In the presence of 2 X 10(-5) M-free calcium ions, the initial phase following the laser flash lasted approximately 13 ms, and was followed by an exponential rise of tension with a rate constant of 28 s-1. The active tension reached by the muscle fibres was 54 kN/m2. For fibres stretched beyond overlap, no change in tension was observed following the release of Mg ATP. Under all conditions the time course of Mg ATP cleavage was biphasic, and consisted of a rapid initial burst of ADP formation, complete within 50 ms, followed by a slower steady-state rate of Mg ATP cleavage. The number of molecules of Mg ATP cleaved during the burst was approximately equal to the number of myosin subfragment 1 heads for fibres at full myofilament overlap, and equal to 0.7 molecules per myosin subfragment 1 head for fibres stretched beyond overlap. At full overlap in the presence of calcium ions, the steady-state rate equalled 1.8 mol Mg ATP cleaved per mole myosin subfragment 1 head per second. In all other cases the steady-state rate of Mg ATP cleavage was at least 10-fold less. When fibres at full overlap were pre-incubated with 2 mM-ADP, the initial phase of the tension response was somewhat prolonged, but the burst of ADP formation was also complete within 50 ms.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit. 661 12

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.
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PMID:Phosphorylation of myosin light chain and phosphorylase in tracheal smooth muscle in response to KCl and carbachol. 670 May 72

Conventional methods of assessing adequate nutrition in children may be inaccurate or change too slowly to be helpful during acute illness. Techniques to measure protein synthesis or breakdown provide more accurate information. Three-methylhistidine (3MH), an unusual amino acid found in actin and myosin, is not further metabolized and is quantitatively excreted following muscle degradation. Urinary excretion of 3MH has, therefore, been proposed as a marker of protein catabolism. The ratio of 3MH to creatinine in 24-hr urine collectons was studied in 14 healthy or stressed premature infants, and compared to nitrogen balance (N bal), caloric intake and clinical course. There is a significant inverse correlation between 3MH/Cr ratio and N Bal (R = -.507). The mean 3MH/Cr ratio was 0.140 +/- 0.037 mu mole/mg (n = 37) in healthy growing premature infants and 0.296 +/- 0.160 (n = 26) in infants who were stressed and/or had inadequate nutrient intake. Healthy growing infants almost invariably had a ratio below 0.200. Serial determinations in three infants consistently showed a rise in the ratio to above 0.200 during periods of stress or decreased intake. The 3MH/Cr ratio may be a more sensitive indicator of metabolic status and may be useful clinically, especially in infants receiving total parenteral nutrition.
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PMID:Urinary 3-methylhistidine excretion and nitrogen balance in healthy and stressed premature infants. 677 77

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg X ADP and uncomplexed ADP to the active site of myosin subfragment-1. Additionally, the binding parameters for Mg2+ in the ternary complex protein X Mg X ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg X ADP just one mole of ligand binds/mole subfragment-1. The affinity of Mg X ADP at low ionic strength is 2.1 X 10(6) M-1 and only five-times lower in the absence of Mg2+. In the ternary complex Mg2+ has a low affinity of 4.1 X 10(4) M-1. At high ionic strength the uncomplexed ADP binds with a 43-times-lower affinity than Mg X ADP, whose affinity is 6.9 X 10(5) M-1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 X 10(5) M-1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.
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PMID:Interaction of ADP and magnesium with the active site of myosin subfragment-1 observed by reactivity changes of the critical thiols and by direct binding methods at low and high ionic strength. 683 46

Antibodies were elicited against turkey gizzard myosin light chain kinase (MLCK), purified by affinity chromatography on the enzyme bound to Sepharose, and used to localize myosin kinase--in rabbit fast skeletal, slow skeletal, cardiac, and smooth muscles--by indirect immunofluorescence. When studied on nitrocellulose replicas of NaDodSO4/polyacrylamide gel electrophoretograms, antibodies were specific for the Mr 140,000 MLCK of gizzard smooth muscle. By using the same technique, they were shown to recognize the Mr 140,000 MLCK and a Mr 75,000 polypeptide--presumably derived from the former by proteolysis--in rat arterial and stomach smooth muscle as well as in rat thyroid cells. The same antibodies reacted only with a Mr approximately equal to 75,000 protein from rat cardiac and skeletal muscle. Antibodies inhibited the activity of smooth and skeletal myosin kinases in an in vitro assay with approximately equal to 11 mole of antibody needed for 50% inhibition of 1 mole of gizzard enzyme. The antibodies stain vascular and gizzard smooth muscle cells with no apparent segregation of the enzyme in a specific part of the cell. In contrast, sarcomeric muscles exhibit a striated staining pattern, superimposable to the staining by antiactin antibodies. This shows that (i) antibodies are not species- or tissue-specific, (ii) they recognize kinases that differ in their molecular weight and ability to be phosphorylated, probably at the level of their common catalytic and calmodulin-binding domains, and (iii) sarcomeric muscle kinases are at least in part bound to the contractile apparatus and their distribution is restricted to a specific part of the sarcomere. This raises the possibility that myosin phosphorylation may be controlled not only by the Ca2+ concentration but also by actin-myosin interaction.
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PMID:Subcellular localization of myosin light chain kinase in skeletal, cardiac, and smooth muscles. 695 92

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.
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PMID:Quantitation of myosin light chain phosphorylation in small tissue samples. 707 67

Treatment of relaxed skinned rabbit psoas muscle fibers with 0.1 mM N-phenylmaleimide (NPM) for 1 h locks all of the crossbridges in a weakly-binding state resembling that of the myosin.ATP crossbridge. Under these conditions, NPM reacts mainly with myosin heavy chain (Barnett et al. (1992) Biophys. J. 61, 358-367). Here the specific sites for that reaction are explored. Small bundles of rabbit psoas muscle fibers were treated with Triton X-100 to make the fiber sarcolemmas permeable. The bundles were treated with 0.1 mM [14C]NPM for 1 h, and homogenized for SDS-PAGE. 43 +/- 2.2% of the muscle fiber protein ran in the myosin heavy chain band, the same as for untreated fibers. An alkylating stoichiometry of 2.2 +/- 0.33 moles NPM per mole myosin heavy chain was determined. Exhaustive trypsin digestion followed by two-dimensional thin-layer chromatography and reverse-phase HPLC revealed two major sites on myosin heavy chain for NPM binding. The sites contained about the same amount of linked NPM, suggesting that the reaction stoichiometry of each site under the conditions studied is approx. 1 mol NPM/mol myosin heavy chain. Comparison of the labeled tryptic peptides with NPM-reacted synthetic SH1 and SH2 tryptic peptides and analysis of the treated fiber bundles' ATPase activity suggested that the sites for NPM reaction on myosin heavy chain when it locks crossbridges in a weakly-binding state are Cys-697 (SH2) and Cys-707 (SH1).
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PMID:The site and stoichiometry of the N-phenylmaleimide reaction with myosin when weakly-binding crossbridges are formed in skinned rabbit psoas fibers. 749 34

Affinity-purified polyclonal antibodies prepared against a synthetic peptide corresponding to sequence 18-29 from the N-terminus of rabbit alpha-skeletal actin reacted with G- and F-actin. Epitope mapping experiments with thrombin and hydroxylamine cleaved actin, and immunochemical assays verified the specificity of antibodies for the 18-29 sequence on actin. The binding of up to 0.5 mol of IgG per mole of actin did not affect the rigor binding of myosin subfragment 1 (S-1) to actin. Similarly, the binding of IgG to actin was not changed by a complete saturation of actin by S-1. In contrast to this, the weak acto-S-1 interactions in the presence of ATP were strongly inhibited by the 18-29 antibodies. At 25 degrees C, the acto-S-1 ATPase activity was inhibited by IgG stronger than the binding of S-1.ATP gamma S to actin. Thus, at this temperature, a catalytic inhibition of the acto-S-1 system appears to account at least in part for the antibody effect. Acto-S-1 ATPase activities at 25 degrees C were inhibited also by F(ab)(18-29). At 5 degrees C, the acto-S-1 ATPase activity and the binding of S-1.ATP to actin were inhibited approximately to the same extent by IgG(18-29). These results are discussed in terms of S-1 binding sites on actin and the possible role of sequence 18-29 in actomyosin interactions.
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PMID:Role of sequence 18-29 on actin in actomyosin interactions. 768 58


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