Darrel E. Goll

Studies on purified α-actinin: I. Effect of temperature and tropomyosin on the α-actinin/F-actin interaction☆

α-Actinin, purified by two passages through a DEAE-cellulose column, migrates either as a single band or as a single major band with a fainter trailing band during polyacrylamide gel electrophoresis at pH 8.3. In the presence of sodium dodecyl sulfate, purified α-actinin always migrates as a single electrophoretic zone during polyacrylamide gel electrophoresis. Temperature has large effects on the interaction of α-actinin with F-actin. At 0 °C, α-actinin causes large increases in F-actin viscosity, either in the presence or absence of tropomyosin. Quantitative binding studies show that α-actinin can displace tropomyosin from F-actin at 0 °C and that F-actin will quantitatively bind 45% of its weight of α-actinin either in the presence or absence of tropomyosin. This binding ratio corresponds to one α-actinin molecule to approximately 10 to 11 G-actin subunits and suggests that one molecule of α-actinin binds to each turn of the F-actin helix at 0 °C. At 37 °C, α-actinin causes little increase in F-actin viscosity until α-actinin to F-actin ratios of 0.10 to 1.0 have been exceeded. Tropomyosin almost completely abolishes the effect of α-actinin on F-actin viscosity at 37 °C. Quantitative binding studies at 37 °C show that F-actin will completely bind added α-actinin up to an α-actinin to F-actin ratio of 0.1 to 1.0 (ww) in the absence of tropomyosin but only up to a ratio of 0.04 to 1.0 (ww) in the presence of tropomyosin. The latter binding ratio corresponds to approximately two α-actinin molecules per one F-actin strand 1 μm long. Temperature may be an important factor restricting the binding of α-actinin to the Z-line end of the F-actin filament in vivo.

Studies on purified α-actinin: II. Electron microscopic studies on the competitive binding of α-actinin and tropomyosin to Z-line extracted myofibrils☆

Abstract α-Actinin forms an extensive network of cross-connections between thin filaments in the I-band when incubated with Z-line-extracted fibrils at 0 °C; this result may be due to the very high affinity of α-actinin for actin at 0 °C rather than indicating any other in situ localization of α-actinin in addition to the Z-line. By incubating low-ionic-strength extracted, glycerinated fiber bundles in highly purified α-actinin at 37 °C, cross-connections are predominately limited to the Z-line terminus of thin filaments. Incubation of extracted fiber bundles in tropomyosin at 0 °C results in no visible binding, but at 37 °C circular tufts are bound in three I-band zones. Incubating Z-line-extracted fibers in equal quantities of α-actinin and tropomyosin at 0 °C shows that tropomyosin does not interfere with α-actinin binding. At 37 °C, tropomyosin reduces the number of α-actinin cross-connections, but still binds in the three characteristic I-band zones. Unextracted fibers incubated in α-actinin at 0 °C also show the heavy network of I-band cross-connections; this indicates that at 0 °C, α-actinin can displace tropomyosin from the thin filament in vitro.

Purification and properties of α-actinin from rabbit skeletal muscle☆

Abstract The 6-S α-actinin species can be purified from a P15–25 α actinin fraction by DEAE-cellulose chromatography. The resulting P15–25 (DEAE) fraction is eluted as a single peak upon rechromatography on 4% agarose or DEAE-cellulose columns, although the rechromatography on DEAE-cellulose removes a very small amount of aggregates from the P15–25 (DEAE) fraction. Sedimentation diagrams of the P15–25 (DEAE) fraction show that approx. 85% of the protein in this fraction sediments with an s°20,w = 6.23 and about 10–15% sediments with an observed s value of 9.1. The 9.1-S component may be an aggregate of the 6.2-S species. The P15–25 (DEAE) fraction exhibits 2–3-fold higher specific activity in the turbidity assay of α-actinin activity than the original P15–25 fraction. Amino acid composition of the P15–25 (DEAE) fraction is clearly different from the amino acid composition of actin, demonstrating that α-actinin is a separate protein component of the myofibril and is not simply an unusual form of denatured actin. These results also show that the 6-Sα actinin species does not exhibit marked aggregating tendencies and that the large aggregates prevalent in earlier α-actinin preparations were probably due to the presence of denatured actin in these preparations. By using purified α-actinin, it was shown that the stoichiometry of the α-actinin-F-actin interaction is 0.41 parts of P15–25 (DEAE) to 1 part of F-actin. This corresponds to a molecular ratio of one α-actinin to ten G-actin monomers. Tropomyosin and α-actinin compete for the same or closely located binding sites on actin, but at 0°, α-actinin appears able to displace tropomyosin from F-actin. The presence of α-actinin was demonstrated in low-ionic-strength extracts of glycerinated fiber bundles of rabbit psoas muscle; this extraction causes removal of both Z-lines and M-lines. Incubation of Z-line-extracted fibrils with the P15–25 α-actinin fraction caused moderate reconstitution of Z-lines in these fibrils. Since the Z-line constitutes about 6% of the dry mass of the myofibril, but α-actinin makes up only about 1% of the myofibrillar protein, the Z-line is probably composed of substances in addition to α-actinin.

An improved method for the preparation of α-actinin from rabbit striated muscle☆

Abstract A new method has been developed for extracting α-actinin from muscle. Crude α-actinin solution is obtained from a 2 mM Tris-HCl buffer extract of myofibrils at 2° and pH 8.5. This crude extract is then fractionated between 15 and 25% (NH4)2SO4 saturation. The resulting P15–25 fraction differs from previously described conventional α-actinin preparations, which are obtained by room temperature extraction of “myosin-extracted” muscle residue, in three important ways: (1) the 6-S α-actinin species makes up 25–30% of the protein in the P15–25 fraction but only about 4–8% of the protein in the conventional α-actinin extracts; (2) the P15–25 fraction exhibits 4–6 times more specific activity in the ATPase and turbidity assays of α-actinin activity than do conventional α-actinin extracts; and (3) in contrast to the existing descriptions of α-actinin activity, the P15–25 fraction causes the largest percent increase in ATPase activity and turbidity response at 100–125 mM KCl, which is very close to the KCl concentration existing in vivo.

α-Actinin from red and white porcine muscle☆

Properties of purified α-actinin prepared from red and white sections of porcine semitendinosus muscle were compared. Although Z-lines were 2.25 times wider in red semitendinosus than in white semitendinosus, identical yields of α-actinin were obtained from these two sections of the semitendinosus. Since α-actinin is located in the Z-line, this result indicates that the Z-line contains proteins in addition to α-actinin. Reconstituted actomyosin prepared from red semitendinosus had a lower Mg2+-modified ATPase activity and a slower rate of turbidity increase than reconstituted actomyosin made from white semitendinosus; this result and the fact that red semitendinosus had wider Z-lines than the white semitendinosus confirmed that red semitendinosus contained a predominance of red fibers, whereas the white semitendinosus contained a predominance of white fibers. Red α-actinin contained more aspartic acid than white α-actinin; this difference was mainly responsible for red α-actinin having 17 more negatively charged amino acids per 1000 total amino acids than white α-actinin. Because of this difference in negatively charged, amino acid side chains, red α-actinin could be separated from white α-actinin by DEAE-cellulose chromatography or polyacrylamide gel electrophoresis. Red α-actinin did not differ from white α-actinin in sedimentation pattern (both 6.1 S), in circular dichroic spectra (both about 59% α-helical), in rate of digestion by trypsin, or in ability to increase the Mg2+-modified ATPase activity or rate of turbidity increase in suspensions of either red or white reconstituted actomyosin. The properties of porcine α-actinin are very similar to those of rabbit α-actinin, but the physiological role of α-actinin remains undetermined.

Effect of α-actinin on actin viscosity☆

Abstract As determined by analytical ultracentrifugation, purified α-actinin does not form stable complexes with G-actin, myosin, tropomyosin, or the tropomyosintroponin complex. However, α-actinin forms a stable complex with F-actin polymerized either in 100 mM KC1 or in 2mM MgCl 2 without KCl. Viscosity studies confirm that α-actinin interacts as strongly with Mg 2+ -polymerized actin as it does with KCl-polymerized actin. When measured at o°, addition of 0.02–0.05 parts of purified α-actinin to 1 part of actin, by weight, causes a 4–5-fold increase in specific viscosity of F-actin. At 37″, 0.10–0.20 parts of purified α-actinin to 1 part of actin are required to cause a 2–3-fold increase in specific viscosity of F-actin. Addition of α-actinin above this level at 37″, results in precipitation of F-actin. Tropomyosin (0.25 parts to 1 part actin, by weight) has no effect on the α-actinin-induced increase in F-actin viscosity at o° but almost completely abolishes the effect of α-actinin on F-actin viscosity at 37°. The effects of temperature on the α-actinin-induced increase in F-actin viscosity are completely reversible. Trypsin treatment of α-actinin-F-actin mixtures for 5 min at trypsin to actin ratios of 1:50, by weight, completely destroys the α-actinin-induced increase in F-actin viscosity at 37°. Subsequent incubation of the trypsin-treated mixture at o°, however, shows the ability of α-actinin to increase F-actin viscosity at o° is only partially destroyed by 15 min of trypsin digestion. Similar results are obtained by treating purified α-actinin with trypsin and then mixing the treated α-actinin with untreated actin. Neither temperature nor trypsin has any effect on viscosity of F-actin in the absence of α-actinin; hence, trypsin and temperature affect α-actinin and the α-actinin-F-actin interaction. The results are consistent with the hypothesis that at o° α-actinin cross-links F-actin by binding strongly both along the length and at one end of the F-actin strand. At 37°, however, strong binding of α-actinin is restricted primarily to one end of the F-actin strand, and any weak binding that may occur along the length of the F-actin strand is abolished by tropomyosin.

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion☆☆☆

Abstract Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [3H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.

Nemaline myopathy, an integrated study: Selective extraction

Abstract Low ionic strength extraction of glycerinated skeletal muscle containing nemaline rods removes Z lines before rods or M lines. Low ionic strength extraction also removes Z lines in nemaline muscle more rapidly than in control muscle. Partial extraction of nemaline rods uncovers a highly ordered filament lattice consisting of a set of longitudinal filaments and another set of filaments that pass obliquely to the longitudinal filaments. Spacing and intercepts of the filaments in this lattice are responsible for the longitudinal and transverse periodicities seen in the intact rod. The structure in the rod lattice between adjacent minor transverse periods appears similar to the structure of native Z lines and permits the interpretation that the nemaline rod is a lateral polymer of Z-line subunits. Polyacrylamide gel electrophoretic analysis of the low ionic strength extracts of nemaline muscle, done both in denaturing (SDS) and nondenaturing solvents, reveals that the extracts contain α-actinin and tropomyosin, but do not contain large quantities of new or unknown proteins. Nemaline rods may owe their resistance to low ionic strength extraction to their highly ordered, crystalline lattice, to their sequence of very stable longitudinal filaments, or to both these features. Large deposits of spherical, 200-A particles in nemaline muscle were identified as glycogen.

N- and C-terminal amino acids of purified α-actinin

Abstract Highly purified bovine cardiac α-actinin is obtained by successive chromatography on DEAE-cellulose and hydroxyapatite of a crude fraction obtained by salting out low ionic strength extracts of bovine cardiac muscle between 0 and 30% ammonium sulfate saturation. Hydroxyapatite chromatography removes a 43 000-dalton polypeptide chain that is difficult to remove by successive DEAE-cellulose columns. Removal of all 43 000-dalton material by hydroxyapatite chromatography is accompanied by disappearance of a very small 9 to 10 S boundary in analytical ultracentrifuge diagrams of DEAE-cellulose-purified 6.2S α-actinin. Approximately 95% of the protein in DEAE-cellulose and hydroxyapatite-purified α-actinin is the 100 000-dalton α-actinin polypeptide as estimated by SDS-polyacrylamide gel electrophoresis. Purified bovine cardiac, porcine skeletal, chicken gizzard, and chicken breast α-actinins all contain leucine as the C-terminal amino acid of both polypeptide chains in the α-actinin molecule. Bovine cardiac and porcine skeletal α-actinins contain arginine as the amino acid penultimate to C-terminal leucine. None of the four different α-actinins studied had a N-terminal amino group available for reaction with dansyl chloride, but all four α-actinins contained 1.6 to 1.8 acetate residues per molecule (200 000 daltons) of α-actinin. It seems likely that the N-terminal amino groups of both polypeptide chains in these four α-actinins are acetylated. A peptide having the composition N-Ac-Asp 2 -Glu 4 was isolated from a proteolytic digest of bovine cardiac α-actinin. α-Actinin seems to be a conserved protein molecule found in many different motile systems.

Accumulation of myosin, actin, tropomyosin, and α-actinin in cultured muscle cells☆

Abstract In an effort to understand the conditions that promote the assembly of myofibrillar proteins in muscle cells, the temporal sequence of accumulation of four myofibrillar proteins, actin, myosin, tropomyosin, and α-actinin, was monitored during the period of de novo assembly of myofibrils in differentiating muscle cells. Isotope dilution experiments indicated that all four proteins were accumulated simultaneously. Therefore, assembly of myofibrils may be occurring in the presence of a full complement of myofibrillar proteins.