Brian Pope

Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility.

Experiments have been carried out to explore the specificity of proteolytic cleavage of rabbit skeletal muscle myosin by chymotrypsin. Whilst heavy meromyosin is obtained most readily from monomeric myosin in 0·6 m -NaCl, polymeric myosin in 0·12 m -NaCl also yields heavy meromyosin when digested in the presence of divalent cations. Digestion of polymeric myosin in the absence of divalent cations produces subfragment-1. Thus the two regions of myosin susceptible to proteolytic cleavage may be discriminated by the presence or absence of divalent cations when polymeric myosin is exposed to brief chymotrytic digestion. This metal dependence suggests that the flexibility of the two sites is modulated by divalent cations. Calcium ions are more effective than magnesium ions in protecting the subfragment-1 site. This protective effect appears to be due to the presence of the DTNB light chains, which are known to bind calcium ions. Chymotryptic digestion of isolated DTNB light chains or myosin shows that these light chains are readily susceptible to proteolysis, and the specific site of proteolytic attack varies when divalent cations are present. The two principal sites of cleavage within the DTNB light chain have been identified by N-terminal analysis, and these sites are located on either side of the putative calcium binding site, identified from the amino acid sequence ( Collins, 1976 ). Brief digestion of polymeric myosin in the absence of divalent cations shows that the DTNB light chains are cleaved more readily, than the subfragment-1 site in the heavy chains. Thus these light chains become degraded in myosin molecules with uncleaved heavy chains. Further digestion of this material in the presence of divalent cations shows no protection of the subfragment-1 site. DTNB light chains cleaved in myosin that has been digested with chymotrypsin in the presence of divalent cations still protect the subfragment-1 site during furthur exposure to chymotrypsin. These results suggest that protection of the subfragment-1 site requires the presence of DTNB light chains with intact calcium binding sites. The different proteolytic fragments obtained from myosin by chymotryptic cleavage have been identified by polyacrylamide gel electrophoresis and their apparent molecular weights are discussed in relation to myosin fragmentation products obtained with other proteinases.

The ATPase activities of rat cardiac myosin isoenzymes

Rat ventricular myosin contains three isoenzymes which can be separated by polyacrylamide gel electrophoresis in the presence of pyrophosphate buffers [ 11. Two of these isoenzymes, Vr and Vs (the fastest and slowest migrating components, respectively), contain homodimers of two chemically distinct heavy chains, while the intermediate component, V2, contains 1 mol of each of these two heavy chains [2]. The light chains in these isoenzymes have identical electrophoretic mobilities suggesting that the main structural differences reside in the heavy chains [ 11. The distribution of these isoenzymes varies with the age and thyroid status of the rat. Va predominates in rats made hypothyroid by hypophysectomy or thyroidectomy [ 1,2], while replacement therapy with physiological doses of the hormone leads to a shift in the distribution of the isoenzymes towards Vr, which appears to be due to stimulation of specific mRNA synthesis [3]. In 3-4-week-old rats, only Vr is present [I]. Thus it is possible to prepare myosin isoenzymes of both Vr and V3 forms from suitable animals without the need to separate the mixture. Staining the gels for enzymatic activity suggests that the Ca2+activated ATPase of Vr is substantially higher than’ that of Vs. However, this activity is not physiologically meaningful, nor is it possible to measure other ATPase activities on gels. Here we report studies on the ATPase activities of these two isoenzymes, measured under a number of different conditions to show that these phenotypes differ in their kinetic properties. Vr has a higher ATPase activity than V3 except for the activity measured in the absence of divalent cations. Marked differences are observed in the physiologically important actin-activated ATPase.

Expression of the N‐terminal domain of dystrophin in E. coli and demonstration of binding to F‐actin

The N‐terminal head domain of human dystrophin has been expressed in soluble form and high yield in E. coli, allowing us to test the previously unconfirmed assumption that dystrophin binds actin. DMD246, the first 246 amino acid residues of dystrophin, binds F‐actin in a strongly co‐operative manner with a Hill constant of 3.5, but does not bind G‐actin. Dystrophin heads are thus functionally competent actin‐binding proteins. This result opens the way to identifying critical residues in the actin‐binding site and encourages us that the other domains of dystrophin might also be treated as functionally autonomous modules, accessible to a similar approach.

Characterisation of the F-actin binding domains of villin: classification of F-actin binding proteins into two groups according to their binding sites on actin

The F‐actin binding properties of chicken villin, its headpiece and domains 2–3 (V2‐3) have been analysed to identify sites involved in bundle formation. Headpiece and V2‐3 bind actin with K d values of ~7 μM and ~0.3 μM, respectively, at low ionic strength. V2‐3 binding, like that of villin, is weakened with increasing salt concentration; headpiece binding is not. Competition experiments show that headpiece and V2‐3 bind to different sites on actin, forming the two cross‐linking sites of villin. Headpiece does not compete with the F‐actin binding domains of gelsolin or α‐actinin, but it dissociates actin depolymerizing factor. We suggest that the F‐actin binding domains of actin severing, crosslinking and capping proteins can be organized into two classes.

Wild-type p53 protein shows calcium-dependent binding to F-actin.

Nuclear localization of p53 is required for p53 to detect and respond to DNA strand abnormalities and breaks following DNA damage. This leads to activation of the tumour suppressive functions of p53 resulting in either cell cycle arrest and DNA repair; or apoptosis. Critical functional changes in DNA which require strand breaks, including gene rearrangement, may transiently mimic DNA damage: here it is important not to trigger a p53 response. The fine control of p53 in these different circumstances is unknown but may include transient sequestering of p53 in the cytoplasm. Reversible nuclear-cytoplasmic shuttling is an intrinsic property of p53 (Middeler et al., 1997) associated with cell cycle-related changes in p53s subcellular distribution. Takahashi and Suzuki (1994) described p53 inactivation by shuttling to the cytoplasm and Katsumoto et al. (1995) found wild-type p53 to be closely associated with cytoplasmic actin filaments during DNA synthesis. Here we show that, in the presence of free calcium ions, p53 binds directly to F-actin with a dissocation constant of about 10 μM. Thus, part of the regulatory machinery in normal cell cycling may involve p53-actin interactions regulated by calcium fluxes and the dynamic turnover of F-actin.

Changes in myosin light chains in the rat soleus after thyroidectomy

The occurrence of physiological and structural changes in skeletal muscle in hypothyroidism is well-documented...

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

The X‐ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin‐actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH. Mutation of acidic residues in either actin or segment 1 involved in ligation of the intermolecular calcium ion resulted in loss of one of the bound calcium ions at pH <7, but not at pH 8. Thus the calcium ion trapped in the segment 1‐actin complex is that located at the intramolecular site. The implications of this for gelsolin function are discussed.

Heterogeneity of myosin heavy chains in subfragment-1 isoenzymes from rabbit skeletal myosin

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The ADF/Cofilin Family: Accelerators of Actin Reorganization

Actin polymerization and cytoskeletal reorganization play an essential role in cell locomotion and many forms of motility, including phagocytosis and cytokinesis. The rate of assembly of actin filaments in vitro is virtually diffusion controlled (Drenckhahn and Pollard 1986), but depolymerization rates are too slow to regenerate the monomer pool required for cells to advance at rates of up to 30µm/min −1 (Zigmond 1993). Actin Depolymerizing Factors (ADF/cofilin) are ideal candidates to aid in this process. They are localized together with actin in motile regions of cells (Fig. 1A) and are essential for cell viability in yeast, C. elegans, Drosophila and Dictyostelium. Here we describe the structure and properties of these proteins and highlight recent work on their interactions with actin.

Molecular biology of actin binding proteins: evidence for a common structural domain in the F-actin binding sites of gelsolin and α-actinin

Summary We review the impact of molecular biology on actin binding proteins, in particular on sequence relationships and expression of clones to dissect properties in vitro. Significant homologies exist between proteins in each class, but we propose, in addition, that common structural features exist between the F-actin binding sites of severing and cross-linking proteins.