Search PubMed⌕ Search

Biomedical subjects

J Kendrick-Jones

Publications and source records attributed to J Kendrick-Jones.

At least 91 records · Page 5Linked to original sources
PubMed1988

Molecular cloning and sequencing of the chicken smooth muscle myosin regulatory light chain.

A cDNA probe was constructed from a chicken skeletal muscle regulatory light chain cDNA and was used to screen a chicken gizzard cDNA library. A clone containing the entire coding region of the chicken gizzard regulatory light chain was isolated and sequenced. The deduced protein sequence is identical to the most recently reported chemical sequence of the chicken smooth muscle regulatory light chain, and has homologies with other troponin C-like calcium-binding proteins.

Amino Acid Sequence↗
PubMed1988

Cingulin, a new peripheral component of tight junctions.

The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.

Amino Acids↗
PubMed1988

Brush border myosin filament assembly and interaction with actin investigated with monoclonal antibodies.

Monoclonal antibodies binding to epitopes in the rod portion of brush border myosin were used to study the mechanism of filament assembly and its role in myosin interaction with actin. The antibodies and their Fab fragments had specific effects on the size of the filaments assembled in vitro. Two antibodies (BM3 and BM4), directed against the tip of the myosin tail, completely inhibited myosin filament assembly. The other antibodies (BM1, BM2 and BM5), binding to other sites along the myosin rod, only partially blocked filament growth, and short filaments could be assembled. Thiophosphorylated brush border myosin filaments appeared slightly more stable to the effects of the antibodies than those composed of dephosphorylated myosin. Only one (BM3) of the antibodies which completely inhibited the assembly of new filaments was capable of disassembling preformed myosin filaments. The other antibody, BM4, partially disassembled filaments, leaving approximately 0.2-microns long 'cores', suggesting that polymerization in this myosin occurs by a biphasic mechanism, i.e. the formation of a stable nucleus of antiparallely packed molecules, followed by elongation. The antibodies BM1 and BM2 bound to myosin filaments generating a regular transverse pattern with a approximately 14-nm periodicity, and had little effect on the stability of these preformed filaments. Inhibition of filament formation and solubilization of the myosin by the antibodies appeared to be associated with inhibition of myosin interaction with actin, as measured by the actin-activated MgATPase activity. In the presence of the antibodies which completely inhibit filament assembly, we observed a decrease to approximately 20% (BM4-Fab) and to approximately 50% (BM3) of the control actin-activated myosin MgATPase activity, and this activity was kinetically different from that of the soluble myosin S1 fragment, suggesting that the rod has a profound effect on the kinetics of actomyosin interaction.

Actins↗
PubMed1988

Modulation of cellular morphology and locomotory activity by antibodies against myosin.

Three monoclonal antibodies directed against chicken brush border myosin were used to study the possible function of myosin in microfilament organization and locomotion of chicken fibroblasts. These antibodies bind to distinct and separate epitopes on the heavy chain of chicken nonmuscle myosin and display differential effects of myosin filament formation and actin-myosin interaction (Citi, S., and J. Kendrick-Jones. 1988. J. Musc. Res. Cell Motil. 9: 306-319). When injected into chicken fibroblasts, all antibodies induced breakdown of stress fibers. Concomitantly, a large proportion of the cells developed extensive lamellae which altered their morphology drastically. These cells showed also increased locomotory activity. All effects were concentration dependent and reversible. The most drastic alterations were observed with cells injected with antibody quantities exceeding the quantity of cellular myosin (molar ratios of antibody to myosin greater than 3:1). The finding that antibodies with different effects on myosin filament formation in vitro all induce similar intracellular processes suggests that it is the antibody-induced decrease in functional myosin that triggers an increase in plasma membrane dynamics and locomotory activity, rather than differences in myosin filament length or conformation.

Actin Cytoskeleton↗
PubMed1988

Spatial pattern of myosin phosphorylation in contracting smooth muscle cells: evidence for contractile zones.

We have purified a polyclonal antibody by affinity chromatography which binds specifically to the phosphorylated form of the regulatory light chain (Mr = 20,000) of smooth muscle myosin. This antibody does not stain relaxed, permeabilized smooth muscle cells isolated from guinea pig taenia coli. However, when these cells were stimulated to contract with CaCl2 (100 microM) and ATP (1 mM), the immunofluorescence staining was localized in a series of transverse bands. This distribution of activated myosin appears to reflect an underlying structural organization of the smooth muscle cell cytoskeleton into mechanically coupled contractile zones.

Animals↗
PubMed1987

Polymerization of vertebrate non-muscle and smooth muscle myosins.

We investigated how light chain phosphorylation controls the stability of filaments of vertebrate non-muscle myosins (from bovine thymocytes and chicken intestine epithelial brush border cells) and smooth muscle myosin (from chicken gizzard) in vitro. Using a sedimentation assay, the solubilities of the myosins were determined by measuring the amounts of myosin monomers (Cm) and filaments (Cp) present under a given set of conditions as a function of the total myosin concentration (Ct). Below 200 mM-NaCl, each myosin displayed distinct "critical monomer concentrations" (Cc) for polymerization, which were dependent on the salt concentration, the state of light chain phosphorylation and the presence of MgATP. At 150 mM-NaCl, MgATP increased the Cc of non-phosphorylated brush border myosin approximately five to tenfold, thymus myosin approximately 10 to 15-fold, and gizzard myosin approximately 25 to 50-fold. When these myosins were phosphorylated, MgATP had little effect on their solubilities, and their Cc values remained low. Analytical ultracentrifugation and electron microscopy demonstrated that the myosins were present in three different conformational states under the conditions used in the sedimentation assays, i.e. filaments, extended monomer (6 S) and folded monomer (10 S). Since at equilibrium only filaments and monomers were observed, we suggest that the polymerization pathway for these myosins can be analysed in terms of a dynamic monomer-polymer equilibrium (polymer in equilibrium 6 S monomer in equilibrium 10 S monomer). At roughly physiological ionic strength, light chain dephosphorylation (in the presence of MgATP) promotes the folded state (10 S), whereas phosphorylation promotes the extended state (6 S), and thereby favours filament assembly. The relevance of the monomer-polymer equilibrium to the state of organization of the myosin in vivo is discussed.

Animals↗
PubMed1987

Effects of light chain phosphorylation and skeletal myosin on the stability of non-muscle myosin filaments.

The effect of light chain phosphorylation and the presence of skeletal muscle myosin on the stability of non-phosphorylated non-muscle myosin filaments was investigated. Purified skeletal, brush border and thymus myosins were assembled in vitro into hybrid filaments consisting of varying proportions of (1) non-muscle and skeletal myosins, or (2) phosphorylated and non-phosphorylated non-muscle myosins. The stability of these hetero- and homopolymers in the presence of MgATP was determined using sedimentation, gel electrophoresis and immunochemical techniques. In addition, the effect of a monoclonal antibody, binding to the tip of brush border myosin tail, on the assembly of the homo- and heteropolymers, was tested. Filamentous non-phosphorylated non-muscle myosin was disassembled by MgATP to the same extent whether in homo- or heteropolymers, indicating that skeletal myosin has no stabilising effect on the hybrid filaments. The presence of small amounts of phosphorylated non-muscle myosin was, however, found to prevent the complete disassembly by MgATP of non-phosphorylated non-muscle myosin filaments, indicating that light chain phosphorylation stabilizes co-operatively non-muscle myosin filaments. The monoclonal antibody prevented the assembly of brush border myosin into both homo- and heteropolymers, and its effect on the filaments was compared with that of MgATP.

Adenosine Triphosphate↗
PubMed1987

Studies on the structure and conformation of brush border myosin using monoclonal antibodies.

We have produced and characterised five monoclonal antibodies against myosin isolated from chicken intestinal epithelial brush border cells. The binding sites of the antibodies on the rod portion of brush border myosin were localised using rotary shadowing/electron microscopy of myosin-antibody complexes. Two antibodies were shown to bind to the tip of the myosin tail, two antibodies to sites about two thirds down the length of the rod, and one antibody about one third down the length of the rod. Brush border myosin was digested with papain, trypsin and alpha-chymotrypsin, and they myosin fragments obtained were analysed by western blots with the monoclonal antibodies and polyclonal antiserum, and by gel overlay with 125I-labelled light chains. Using this approach we were able to identify and map the protease cleavage sites and thus characterise the proteolytic substructure of brush border myosin. Solid-phase assays, western blots and immunofluorescence were used to study the cross-reactivity of these monoclonal antibodies against a variety of myosins from different species and cell types, to assess the immunological relatedness between brush border myosin and homologous molecules present in different tissues and species. Finally, we used a competitive solid-phase assay to measure the 'relative affinities' of the antibodies towards the three possible conformational states of brush border myosin, i.e. filament, extended monomer and folded monomer.

Animals↗
PubMed1987

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments.

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.

Actin Cytoskeleton↗
PubMed1987

Effect of N-ethylmaleimide on Ca-inhibition of Physarum myosin.

We have established a quick method for preparing Physarum myosins whose actin-activated ATPase activities are inhibited by microM levels of Ca2+ (from plasmodial stage: Kohama, K. & Kendrick-Jones, J. (1986) J. Biochem. 99, 1433-1446; and from amoebal stage: Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, Y., & Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). N-Ethylmaleimide alkylates sulfhydryl (SH) groups on the heavy chains in the heads of the plasmodial myosin. The actin-activated ATPase activity of the modified myosin was significantly decreased when assayed in low Ca2+ concentrations. Moreover, the activity remained low even when the Ca2+ concentrations was increased, i.e., the myosin was desensitized. For complete desensitization, about 4 mol SH per mol myosin (500,000 Mr) must be modified. These residues are probably the "reactive thiols" which have been predicted from primary structure studies to be conserved among myosins of higher and lower eukaryotes. Ultraviolet absorption spectra of the modified and intact myosins showed a peak at 277 nm. The height of this peak in intact myosin was reduced when the Ca2+ concentration was increased. This Ca-induced reduction was hardly detectable in the modified myosin although Ca-binding activity to myosin did not appear to be affected by the modification. We interprete these results that Ca2+ may change the conformation of the myosin heavy chain by binding to myosin and speculate that impairment of this process upon modification could cause the desensitization to Ca2+ in the ATPase activity.

Actins↗
PubMed1986

Amino acid sequence of myosin essential light chain from the scallop Aquipecten irradians.

The amino acid sequence of the scallop myosin essential light chain (SELC) was determined from analysis of the intact, S-carboxymethylated protein and peptides produced by cleavage at its four methionine residues by cyanogen bromide digestion and at its six arginine residues by citraconylation and tryptic digestion. SELC contains 156 amino acid residues, including three cysteines, four tyrosines, one tryptophan, two histidines, and an unblocked amino-terminal proline. The protein has a calculated Mr of 17,616. SELC is an acidic protein, with a net charge of 18- at physiological pH. Comparative analysis reveals four homologous domains (I-IV), which arose by reduplication of a gene for a small, ancestral calcium binding protein. Each domain has a helix-loop-helix structure, with all the ligands for calcium binding located within a 12-residue segment that spans the loop and the first turn of the following helix. Potential calcium binding sequences were found in the ancestral sites III (residues 94-105) and IV (residues 132-143). Mutations in critical positions in domains I and II seem to preclude the possibility of calcium binding in the amino-terminal half of SELC. An unexpected third potential calcium binding segment (at residues 119-130, predicted to be in helical conformation) was found in domain IV. A reactive thiol group (Cys-78) that is involved in binding of regulatory light chains was tentatively located in an extended "linker region", which connects the two halves of the molecule.

Amino Acid Sequence↗
PubMed1986

Localisation of light chain and actin binding sites on myosin.

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.

Actins↗
PubMed1986

Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site.

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.

Adenosine Triphosphate↗
PubMed1986

Regulation in vitro of brush border myosin by light chain phosphorylation.

Myosin was purified from chicken brush border cells to greater than 95% homogeneity and in a predominantly non-phosphorylated state. The effects of light chain phosphorylation by a Ca2+-calmodulin-dependent myosin light chain kinase on the conformational, enzymatic and filament assembly properties of this myosin were investigated. The actin-activated MgATPase activity of the non-phosphorylated myosin was low, and upon light chain phosphorylation an eight- to ninefold increase in this activity was observed, which was further potentiated by tropomyosin. Light chain phosphorylation was shown to control the assembly and disassembly of brush border myosin filaments. For example, turbidity measurements and electron microscopy demonstrated that MgATP disassembled non-phosphorylated myosin filaments; the disassembled myosin could reassemble when the light chains were phosphorylated, and could be disassembled again by dephosphorylating the light chains with phosphatase. In the electron microscope, the disassembled non-phosphorylated myosin molecules appeared in a folded conformation, and they were extended when phosphorylated. Proteolytic digestion was used to probe further the conformation of these folded and extended molecules, and their subunit organizations were characterized by a gel overlay technique. Quantitative analysis further demonstrated that light chain phosphorylation alters dramatically the monomer/polymer equilibrium of brush border myosin, shifting it towards filament formation. Comparison of analogous data for myosin from gizzard and thymus shows that each myosin has distinct solubility properties.

Animals↗
PubMed1986

The inhibitory Ca2+-regulation of the actin-activated Mg-ATPase activity of myosin from Physarum polycephalum plasmodia.

Myosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (greater than 95%), in a high yield (about 10 mg/100 g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500,000 Mr myosin). Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when Ca2+ concentration was increased to 50 microM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3 microM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+. The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2 microM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin. The involvement of this myosin-linked regulatory system in the Ca2+ -control of cytoplasmic streaming is discussed.

Actins↗
PubMed1985

Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets.

Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.

Aminoquinolines↗