Search PubMed⌕ Search

Biomedical subjects

J Kendrick-Jones

Publications and source records attributed to J Kendrick-Jones.

At least 109 records · Page 6Linked to original sources
PubMed1984

Phosphorylation site sequence of smooth muscle myosin light chain (Mr = 20 000).

The amino terminal sequence of the myosin light chain (Mr = 20 000) isolated from chicken gizzards was found to be (sequence in text). This sequence assignment differs from that reported by Maita et al. [(1981) European J. Biochem. 117, 417] in the order of the tryptic peptides. The revised amino acid sequence exhibits greater homology with the phosphorylation site sequences of the regulatory light chains from cardiac and skeletal muscle. Moreover it is now apparent why synthetic peptides corresponding to the previously reported sequence were very poor substrates for the myosin light chain kinase.

Amino Acid Sequence↗
PubMed1983

Regulation of myosin filament assembly by light-chain phosphorylation.

Myosins isolated from vertebrate smooth muscles and non-muscle cells such as lymphocytes and platelets contain regulatory light chains (Mr = 20000), which are phosphorylated by a Ca2+-calmodulin-dependent kinase and dephosphorylated by a Ca2+-insensitive phosphatase. Phosphorylation of the regulatory light chains of these myosins in vitro regulates not only their interactions with actin but also their assembly into filaments. Under approximately physiological conditions (0.15 M NaCl, pH 7.0) stoichiometric levels of Mg-ATP disassemble these non-phosphorylated myosin filaments into species with sedimentation coefficients (So20,w) of approximately 11S. Hydrodynamic and electron microscope observations have indicated that this 11S species is a monomer with a folded conformation (Trybus et al., Proc. natn. Acad. Sci. U.S.A. 79, 6151 (1982)). Rotary shadowing reveals that the tails of disassembled gizzard and thymus myosins are folded twice at two hinge points to form a folded three-segment structure. Phosphorylation of the regulatory light chains of these myosins causes these folded 11S molecules to unfold into the conventional extended monomeric form (6S), which is able to assemble into filaments. Thus in vitro these myosin filaments can be assembled or disassembled by phosphorylation or dephosphorylation of their light chains. Whether these results have any relevance to the situation within living non-muscle and smooth muscle cells remains to be established.

Actins↗
PubMed1983

Studies on the effect of phosphorylation of the 20,000 Mr light chain of vertebrate smooth muscle myosin.

Myosin was rapidly prepared from turkey gizzard muscle to a high level of purity, in high yield and in a non-phosphorylated state. It was consistently observed that the actin-activated Mg2+ ATPase activity of this myosin was dependent on the level of phosphorylation of the 20,000 Mr light chain, for example, in the non-phosphorylated state, the myosin Mg2+ ATPase activity was not activated by actin whereas, when the light chains were phosphorylated, the Mg2+ ATPase activity of the myosin was activated approximately ninefold by actin. Using the "desensitized" scallop myosin test system (Kendrick-Jones et al., 1976; Sellers et al., 1980) it was further demonstrated that phosphorylation of the 20,000 Mr gizzard light chain has a regulatory role. These results also suggest that the regulatory mechanisms mediated by smooth muscle myosin light chains and molluscan myosin regulatory light chains are similar, i.e. in the absence of Ca2+, both types of light chain inhibit myosin interaction with actin and this inhibition is relieved by either phosphorylation in smooth muscle or by direct calcium binding in molluscan myosins. The basis of regulation exerted by these light chains is therefore repression derepression. Using a variety of techniques, i.e. turbidity measurements, quantitative high speed centrifugation, electron microscopy and dark field light microscopy, it was observed that the stability of gizzard myosin filaments at approximately physiological conditions (0.15 M-NaCl, 1 mM-MgATP, pH 7.0) was dependent on the level of light chain phosphorylation. Using purified calmodulin-dependent light chain kinase and phosphatase, it was further shown that these gizzard myosin filaments can be reversibly assembled and disassembled as a result of phosphorylation-dephosphorylation of the 20,000 Mr light chain.

Actins↗
PubMed1983

Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells.

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++-independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++-dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament-intermediate filament organization.

Adenosine Triphosphate↗
PubMed1983

Antibody to thymus myosin: its immunological characterization and use for immunocytochemical localization of myosin in vertebrate nonmuscle cells.

Thymus myosin differs immunologically from smooth muscle and striated muscle myosin isoenzymes. In the enzyme linked immunosorbent assay a moderate degree of cross reaction was observed between anti-thymus myosin and myosin from chicken gizzard (about 50% of the titer of the homologous reaction). In contrast, the cross reactivity between thymus myosin and antibodies to gizzard myosin was very low (about 5%) and no significant cross reaction was observed between thymus myosin and antibodies to striated muscle myosin and vice versa (below 1%). Antibodies to thymus myosin were further distinguished from antibodies to gizzard and striated muscle myosin by their reaction with both smooth muscle and a very broad spectrum of vertebrate nonmuscle cells. Nonmuscle cells reacting with anti-thymus myosin included (1) cell types which did not display any detectable affinity for anti-gizzard myosin (e.g. lymphocytes, polymorphonuclear leucocytes, vascular endothelium, adrenal chromaffine cells) and (2) cell types which reacted with anti-gizzard myosin as well (e.g. intestinal epithelial brush border, thymic epithelial cells, liver cells and stress fibres of cultured cells). These results illustrate, that anti-thymus myosin is a potent tool for investigating the intracellular localization of myosin in most if not all vertebrate nonmuscle cells. With respect to lymphatic tissue the present findings indicate that lymphocyte maturation appears to be accompanied by an increased level of expression of myosin and filamentous actin (the latter was visualized by labelled phalloidin). On the ultrastructural level, gold labelled antibodies to thymus myosin bound preferentially to the head region of in vitro assembled thymus myosin filaments. In cultured cells (PtK1) the antibodies showed a particular affinity for stress fibre densities, and in lymphocytes the anti-myosin label (immunoperoxidase) displayed a more or less diffuse distribution which was similar to the distribution of actin filaments (identified by decoration with heavy meromyosin).

Actins↗
PubMed1980

Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase.

The presence of actin and myosin in non-muscle cells suggests that they may be involved in a wide range of cellular contractile activities. The generally accepted view is that interaction between actin and myosin in these cells and in vertebrate smooth muscle, is regulated by the level of phosphorylation of the 20,000-molecular weight (MW) light chain. In the absence of calcium, this light chain is not phosphorylated and the myosin cannot interact with actin. Calcium activates a specific calmodulin-dependent kinase which phosphorylates the light chain, initiating actin-myosin interaction. Although most studies on the role of phosphorylation have concentration on the regulation of actin-activated myosin Mg-ATPase activity, phosphorylation of the light chain also seems to control the assembly of smooth muscle myosin into filaments. Using purified smooth muscle light chain kinase, we have confirmed this observation. We report here studies of myosins isolated from the two non-muscle sources, thymus cells and platelets. We observed that these myosins are assembled into filaments at physiological ionic strength and Mg-ATP concentrations, only when the 20,000-MW light chain is phosphorylated.

Actins↗