Pseudophosphorylation of the smooth muscle 20 000 dalton myosin light chain

Haeberle, Joe R.; Hott, Jeffrey W.; Hathaway, David R.

doi:10.1016/0167-4838(84)90334-0

Cited by 38 publications

(5 citation statements)

References 29 publications

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“…3A, up to the point of adding caged ATP) was <11%; this is similar to the 12% baseline value measured in paired muscles incubated in relaxing solution, and is probably due to charge modification of unphosphorylated myosin (Driska et al, 1981;Haeberle et al, 1984). Force development at low ATP concentrations in the absence of Ca ~+ in skeletal muscle is thought to be caused by cooperative switching on of the FIGURE 4.…”

Section: Mechanism Of Tension Plateausupporting

confidence: 56%

Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. A laser-flash photolysis study.

Somlyo

Goldman

Fujimori

et al. 1988

The Journal of General Physiology

138

106

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A B STR ACT The effects of laser-flash photolytic release of ATP from caged ATP [P3-1(2-nitrophenyl)ethyladenosine-5'-triphosphate] on stiffness and tension transients were studied in permeabilized guinea pig portal vein smooth muscle. During rigor, induced by removing ATP from the relaxed or contracting muscles, stiffness was greater than in relaxed muscle, and electron microscopy showed cross-bridges attached to actin filaments at an -45 ~ angle. In the absence of Ca 2+, liberation of ATP (0.1-1 mM) into muscles in rigor caused relaxation, with kinetics indicating cooperative reattachment of some crossbridges. Inorganic phosphate (Pi; 20 mM) accelerated relaxation. A rapid phase of force development, accompanied by a decline in stiffness and unaffected by 20 mM P~, was observed upon liberation of ATP in muscles that were released by 0.5-1.0% just before the laser pulse. This force increment observed upon detachment suggests that the cross-bridges can bear a negative tension. The second-order rate constant for detachment of rigor cross-bridges by ATP, in the absence of Ca 2+, was estimated to be 0.1-2.5 x 105 M-~s -~, which indicates that this reaction is too fast to limit the rate of ATP hydrolysis during physiological contractions. In the presence of Ca "+, force development occurred at a rate (0.4 s -~) similar to that of intact, electrically stimulated tissue. The rate of force development was an order of magnitude faster in muscles that had been thiophosphorylated with ATP~,S before the photochemical liberation of ATP, which indicates that under physiological conditions, in non-thiophosphorylated muscles, light-chain phosphorylation, rather than intrinsic properties of the actomyosin cross-bridges, limits the rate of force development. The release of micromolar ATP or CTP from caged ATP or caged CTP caused force development of up to 40% of maximal active tension in the absence of Ca 2+, consistent Address reprint requests to Dr. Avril V. Somlyo,

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Section: Mechanism Of Tension Plateausupporting

confidence: 56%

Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. A laser-flash photolysis study.

Somlyo

Goldman

Fujimori

et al. 1988

The Journal of General Physiology

138

106

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“…Multiple phosphorylation of myosin regulatory light chains has been reported previously (14,27,34). However, the validity of using only the migration of the light chain on nondenaturing gels as an indication of its state of phosphorylation has recently been called into question (18), because of the potential for similar shifts in migration on these gel systems in the absence of phosphate incorporation. Nevertheless, it is clear from our results that the initial shift in migration of most, if not all, of the light chain corresponds to incorporation of phosphate (Fig.…”

Section: Discussionmentioning

confidence: 99%

Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders.

Keller¹,

Conzelman²,

Chasan³

et al. 1985

The Journal of Cell Biology

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We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37°C under conditions that stimulate terminal web contraction (1 #M Ca ++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0°C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCI and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only -20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.Myosin is a major component of the cytoskeletal apparatus associated with the brush border of intestinal epithelial cells (for recent reviews of brush border structure see references 2 and 30). In brush borders, myosin is localized in the terminal web (3,9,19,22,33). Although its function in vivo remains unknown, this myosin has been implicated as the force producer (5, 26) in ATP-dependent contraction of the terminal web first observed in isolated brush borders by Rodewald et al. (41). Further support for this possibility has come from the demonstration that contraction of the terminal web correlates directly with Ca++-calmodulin-dependent phosphorylation of the regulatory light chain of brush border myosin (26). A similar type of contraction has also been stimulated in glycerinated cells from intestinal epithelia (5, 2 l) and retinal epithelia (35).Based on the morphologic changes that occur during contraction, it appears that at least some of the force for this contraction is produced within a ring of filaments that circumscribes the apical ends of both intestinal (24) and retinal epithelial cells (35) at the level of their zonula adherens junctions. This circumferential ring has been isolated from retina and stimulated to contract in vitro (36). In intestinal brush borders, this ring of filaments contains closely apposed, antiparallel actin filaments (21, 22) and myosin (21,22). Presumably, the terminal web contraction that is observed could result from an interaction between actin and myosin within this ring that constricts it in a "purse-string" fashion. However, in our original studies of contraction in isolated brush borders (26) and in glycerinated intestinal epithelial cells (21), we observed that most of the myosin was dissociated from the cytoskeleton during contraction. This observation made us question the role of myosin as a producer of force in terminal web contraction. In this and a companion study (31), we have investigated the nat...

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“…Since the description of this procedure in 1970, modifications include pre-electrophoresis to minimize pseudophosphorylation artifacts [84], and use of antibodies specific toward different isoforms of RLC to avoid nonspecific staining contributions from other proteins that co-migrate with RLC and to increase the sensitivity of the measurements [31, 38] . An alternative method includes Phos-tag SDS-PAGE where denatured proteins are solubilized in SDS sample buffer [85, 86].…”

Section: Myosin Regulatory Light Chain Phosphorylation: Understandmentioning

confidence: 99%

Role of myosin light chain phosphatase in cardiac physiology and pathophysiology

Chang

Kamm

Stull

2016

Journal of Molecular and Cellular Cardiology

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Maintenance of contractile performance of the heart is achieved in part by the constitutive 40% phosphorylation of myosin regulatory light chain (RLC) in sarcomeres. The importance of this extent of RLC phosphorylation for optimal cardiac performance becomes apparent when various mouse models and resultant phenotypes are compared. The absence or attenuation of RLC phosphorylation results in poor performance leading to heart failure, whereas increased RLC phosphorylation is associated with cardiac protection from stresses. Although information is limited, RLC phosphorylation appears compromised in human heart failure which is consistent with data from mouse studies. The extent of cardiac RLC phosphorylation is determined by the balanced activities of cardiac myosin light chain kinases and phosphatases, the regulatory mechanisms of which are now emerging. This review thusly focuses on kinases that may participate in phosphorylating RLC to make the substrate for cardiac myosin light chain phosphatases, in addition to providing perspectives on the family of myosin light chain phosphatases and involved signaling mechanisms. Because biochemical and physiological information about cardiac myosin light chain phosphatase is sparse, such studies represent an emerging area of investigation in health and disease.

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Pseudophosphorylation of the smooth muscle 20 000 dalton myosin light chain

Cited by 38 publications

References 29 publications

Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. A laser-flash photolysis study.

Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. A laser-flash photolysis study.

Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders.

Role of myosin light chain phosphatase in cardiac physiology and pathophysiology

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