Richard M. Robson

Identification of the 30 kDa polypeptide in post mortem skeletal muscle as a degradation product of troponin-T.

Although a 30 kDa polypeptide frequently is seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of post mortem (pm) skeletal muscle and in turn is used as an indicator of proteolysis, its origin has not been conclusively identified. We used antibodies to selected myofibrillar proteins, including some known to be degraded pm, to identify this polypeptide. The left side of eight beef carcasses was electrically stimulated (ES) within 1 h after slaughter, and the right side served as the non-stimulated (NS) control. The longissimus lumborum (LL) muscle was removed from the carcass at 24 h pm and was stored at 2 degrees C. Myofibrils were prepared from the LL muscle immediately after stimulation (0 day) and from the stored muscle sample at 1, 3, 7, 14 and 28 days pm for analysis of SDS-PAGE and Western blots. By SDS-PAGE, troponin-T (TN-T) decreased in amount more rapidly pm in ES samples than in NS samples. By SDS-PAGE, a 30 kDa band increased and became a prominent band by 7 days pm in both NS and ES samples. A monoclonal antibody (mAb) to TN-T labeled purified TN-T, as well as the TN-T in myofibrils, a prominent 30 kDa polypeptide and a family of lower molecular mass polypeptides in pm muscle. This mAb also labeled a 30 kDa band that had been electrophoretically purified from pm muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.

Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture.

A model of the structure of vertebrate Z-lines and Z-line analogs is introduced and supported by evidence from electron microscope studies of wide Z-lines (rat and feline soleus, and feline and canine cardiac muscles), narrow Z-lines (guppy, newt and frog skeletal muscles), and Z-rods (from a patient with nemaline myopathy and from cardiac muscles of aged dog). The model is based on a pair of Z-filaments (termed a Z-unit), which are linked near their centers at a 90 degrees angle and form bridges between neighboring antipolar thin (actin) filaments. A square lattice of four Z-filament pairs (the basic structure of the Z-line, termed a Z-line unit) defines the geometrical position of the I-square unit. In this native state of the Z-line, small square and large square net forms appear in cross-section. Other cross-sectional patterns of Z-lines, including basket-weave and diagonal-square net patterns, can be explained by detachment of the Z-filament from the Z-filament binding region within each Z-filament pair due to chemical or physical stress. Dissection of Z-lines and Z-line analogs with calcium-activated neutral protease provides evidence that the width of all wide Z-line structures is determined by the amount of overlap of antipolar thin filaments from adjacent sarcomeres. Longitudinal patterns of narrow and wide Z-lines are shown and described in relation to the model. To test the proposed model, the dynamics of the Z-line unit structure were computer-simulated. An attempt was made to correlate longitudinal (z direction) and cross-sectional (x and y directions) patterns and to determine the amount of movement of thin or Z-filaments that is required to explain the diversity observed in cross-sectional patterns of Z-lines. The computer simulations demonstrated that the structural transitions among the small square, and therefore large square net, as well as basket-weave and diagonal-square net forms seen in cross-sections could be caused by movements of thin filaments less than 10 nm in any direction (x, y or z).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Nemaline myopathy rod bodies

Ca2+-activated protease (CAF) digestion of glycerinated nemaline myopathy muscle removed the electron-dense material covering rods and Z-lines and exposed longitudinal backbone filaments, 6-7 nm wide, which span the lengths of the original rods. Decoration of the exposed filaments (which are responsible for the periodicity parallel to the long axis of intact nemaline rods) with heavy meromyosin (HMM) proved they are actin filaments. After CAF treatment, cross-striated periodical patterns in longitudinal sections and Z-filament-like proteins connecting actin filaments seen in cross-section disappeared. This suggests that alpha-actinin may be involved in formation of this pattern because of the specificity of CAF toward alpha-actinin. Gel electrophoresis of CAF-treated nemaline muscle showed that most alpha-actinin is released into the supernatant, whereas the residue is mainly actin and myosin. Electron microscope examination of longitudinal sections of intact rods shows an oblique filament pattern, thin (7 nm) lines, thick (11 nm) lines, and an amorphous-appearance previously observed in normal Z-lines, patterns observed depend on sectioning angle and section thickness. In cross-section, rods show small square net (SS) and basket-weave (BW) forms. The SS form predominates and coexistence of the 2 forms, which also occur in normal Z-lines, is observed. Results support the idea that rods are lateral polymers of Z-line units. We think that the length of rods, as well as the width of Z-lines, is determined by the amount of overlap of actin filaments of opposite polarity. Initiation of rod formation may be due to deregulation of actin filament length.

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.

Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice

Abstract. Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.

Interaction Of α-actinin, filamin and tropomyosin with F-actin

Abstract The abilities of α-actinin, filamin and tropomyosin to bind F-actin were examined by cosedimentation experiments. Results indicated that smooth muscle α-actinin and filamin can bind to actin filaments simultaneously with little evidence of competition. In contrast, tropomyosin exhibits marked competition with either filamin or α-actinin for sites on actin filaments.

Purified desmin from adult mammalian skeletal muscle: A peptide mapping comparison with desmins from adult mammalian and avian smooth muscle

Abstract Comparative one-dimensional peptide maps were prepared by the electrophoresis of digests derived from treatment of desmins with Ca 2+ -activated muscle protease, trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. Desmins from adult mammalian skeletal and smooth muscles were very similar. Avian smooth muscle desmin, although homologous with respect to many peptides, was different from the mammalian smooth and skeletal desmins. The amino acid compositions of the three desmins were quite similar.

Molecular Characteristics of the Novel Intermediate Filament Protein Paranemin SEQUENCE REVEALS EAP-300 AND IFAPa-400 ARE HIGHLY HOMOLOGOUS TO PARANEMIN

Paranemin was initially found to copurify with the intermediate filament (IF) proteins vimentin and desmin from embryonic chick skeletal muscle and was described as an IF-associated protein (IFAP). We have purified paranemin from embryonic chick skeletal muscle, prepared antibodies, and demonstrated that they label at the Z-lines of both adult avian and porcine cardiac and skeletal muscle myofibrils. We determined the cDNA sequence of paranemin by immunoscreening a λgt22A cDNA library from embryonic chick skeletal muscle. Northern blot analysis revealed a single transcript of 5.3 kilobases, which is much smaller than predicted from the size of paranemin (280 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The derived amino acid sequence of paranemin (1,606 residues; 178,161 kDa) contains the conserved IF rod domain (308 amino acids), which has highest homology to the rod domains of nestin and tanabin. Thus, paranemin is an IF protein rather than an IFAP. Sequence analysis also revealed that the partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, are almost identical to regions of the cDNA sequence of paranemin. The complete paranemin cDNA was expressed in a cell line (SW13) with, and without, detectable cytoplasmic IFs. Antibody labeling of these cells suggests that paranemin does not form IFs by itself, but rather is incorporated into heteropolymeric IFs with vimentin.