Alan G. Weeds

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.

Actin-binding proteins--regulators of cell architecture and motility.

Numerous actin-binding proteins from a variety of cell types have been described. Here I attempt to correlate the properties and functions of some of these. Three major classes have been identified: (1) cross-linking proteins which form filament bundles or isotropic gels; (2) proteins which cap filament ends and nucleate the polymerization of G-actin (many of these also sever actin filaments); (3) proteins which bind to G-actin and stabilize the monomer pool. Some of the proteins described here combine the properties of more than one class and the activities of many of them are regulated by changes in Ca2+ ion concentration.

Studies on the role of myosin alkali light chains: Recombination and hybridization of light chains and heavy chains in subfragment-1 preparations

The influence of the light chains of myosin on its ATPase activity was examined using a new method for dissociation and recombination. Initial experiments involved the interchange of the two Alkali † light chains in homogeneous subfragment-1 preparations isolated from chymotryptic digests of rabbit skeletal muscle myosin. Thus subfragment-1 containing Alkali 1 light chains was dissociated with 4·7 m -NH 4 Cl in the presence of excess Alkali 2 light chains and recombination occurred when the ammonium chloride was removed by dialysis. The recombined subfragment-1 containing Alkali 2 light chains was purified by ion exchange chromatography and its ATPase activity examined. Subfragment-1 species formed by recombination in this way had actin-activated ATPase activities similar to control preparations containing the same light chain. Thus differences observed in the actin-activated ATPase activities of isolated subfragment-1 isoenzymes ( Weeds & Taylor, 1975 ) appear to be due to the particular Alkali light chain present. Hybridization experiments were performed using light and heavy chains from different types of myosin to explore the role of these different light chains. Subfragment-1 containing a fast-twitch muscle myosin heavy chain and either cardiac or slow-twitch myosin light chains was formed by dissociating fast-twitch muscle subfragment-1 in the presence of an excess of the appropriate light chain. These hybrid subfragment-1 species had ATPase activities measured in the absence of actin almost identical to control fast-twitch muscle subfragment-1. However, in the presence of actin the ATPase activity reflected not only the source of the heavy chain but also the light chain. Similar experiments were performed to produce hybrid subfragment-1 containing Alkali 2 light chains and cardiac myosin heavy chains. The results, although less clearcut, were consistent with the observations reported above. Thus, although these light chains do not appear to influence the ATP turnover by subfragment-1 alone, they are implicated in interactions involving actin.

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.

Partial purification and characterization of an actin depolymerizing factor from brain

The finding that much of the actin present in brain [I] or platelets [2,3] exists in a non-fnamentous state under conditions where only a few % should remain unassembled has led to the search for proteins which prevent actin assembly or promote disassembly. This report describes the isolation from chick embryo brain of an actin depolymerizing protein with a polypeptide MI 19 000. This protein is distinct from profilin [4,5] in its isoelectric point and in its ability to disassemble actin filaments [6].

F-actin capping proteins

Recent research on F-actin capping proteins has concentrated on three main areas. The discovery that controlled actin polymerization is the driving force for intracellular movement suggests an important role for capping proteins in regulating filament number and length. A capping protein from Dictyostelium (related to heat-shock protein HSP70) has been characterized that is activated by external stimuli. This provides a pivotal connection between extracellular signalling, cytoskeletal reorganization and locomotory behaviour. The roles of individual actin-binding sites in the gelsolin family of severing/capping proteins and binding sites for phosphatidylinositol 4,5-bisphosphate have been identified.

Nucleotide sequence of pig plasma gelsolin: comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats

Pig plasma gelsolin (Mr = 81595; 739 residues) contains 704 identical residues out of a maximum 730 when compared to the cytoplasmic form of human gelsolin. The cDNA sequence also codes for a peptide of 33 residues N-terminal to the nine-residue plasma extension sequence previously reported: these 33 residues are highly homologous to the human signal peptide and plasma extension. Comparison of the gelsolin sequences with chicken brush border villin, severin from Dictyostelium discoideum and fragmin from Physarum polycephalum shows a strong evolutionary relationship between all these proteins. There are six large repeating segments in gelsolin and villin, and three similar segments in severin and fragmin. Although these multiple repeats cannot be related to any known function of these actin-severing proteins, this superfamily of proteins appears to have evolved from an ancestral sequence of 120 to 130 amino acid residues.

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.

Characterization of myosin light chains from histochemically identified fibres of rabbit psoas muscle

It is now clearly established that rabbit myosin, isolated from skeletal muscles defined physiologically as of the fast-twitch class, contains two chemically related but phenotypically distinct light chains (termed LCl and LC3). These ‘Alkali light chains’-so called because of their release at high pH-differ in mol. wt. by 4000, but have an identical ammo acid sequence over their C-terminal 141 residues [l] . Together with the unrelated ‘DTNB light chain’ (LC2), the alkali light chains give a characteristic band pattern on polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS), which is different from the light chain pattern of myosin from slow-twitch muscles [2,3]. Chemically related alkali light chains are present in fast-twitch muscle myosin from chicken [3], sheep and cat [4]. Their function remains unclear, though they appear to be essential for the enzymatic activity of the myosin [5]. In support of this, it is notable that cross-reinnervation of fast-twich and slow-twitch muscles results in a reciprocal transformation of the myosin light chains [6,7] which closely parallel the changes occurring in the twitch characteristics: for review of these latter, see [8]. The relative amounts of the two light chains have been determined by densitometry of the bands on SDS polyacrylamide gels, and results indicated that rabbit myosin contains 1.35 moles of the larger LCl light chain and 0.65 moles of LC3 per mole of myosin [9]. These unequal and non-integral yields of the two

Primary, secondary and tertiary myotubes in developing skeletal muscle: A new approach to the analysis of human myogenesis

Monoclonal antibodies to myosins have been used to describe and define the appearance and maturation of 3 different classes of myotube in developing human quadriceps muscle. Five monoclonal antibodies were used: (i) MAb A against human slow myosin heavy chain; (ii) MAb B against a myosin heavy chain present in most adult Type 2 fibres; (iii) MAb C against myosin heavy chain present in all mature and immature Type 2 fibres; (iv) MAb D, with similar reactivity to MAb C; (v) MAb E against human embryonic myosin. The combined use of two of these antibodies (A and B) enables the confident early identification of each of 3 classes (primary, secondary, tertiary) of myotubes, which appear sequentially during myogenesis. Our results show that induction of slow myosin heavy chain synthesis is a biphasic phenomenon in developing human skeletal muscle. Slow myosin heavy chain was present in all the earliest (9 weeks gestation) primary myotubes, but was not detected in secondary or tertiary myotubes until about 29 weeks gestation. Each stage of fetal muscle development has a characteristic immunocytochemical pattern which reveals cellular heterogeneity not evident on myosin ATPase histochemistry. Myosin immunocytochemistry may usefully be applied to assess the gestational age of fetuses. A new interpretation of human skeletal muscle development is proposed, based on the separate programming of 3 different kinds of myotube. This may be important in the analysis of diseased muscle in which developmental abnormalities or regeneration are present.