Extensible and Less-Extensible Domains of Connectin Filaments in Stretched Vertebrate Skeletal Muscle Sarcomeres as Detected by Immunofluorescence and Immunoelectron Microscopy Using Monoclonal Antibodies1

Abstract

Two kinds of monoclonal antibodies (3B9 and SM1) against connectin, muscle elastic protein, reacted with both alpha- and beta-connectins. Immunofluorescence studies revealed that 3B9 stained both edges of the A band of chicken breast muscle myofibrils and remained as such upon stretching to a sarcomere length of 3.5 microns. On the other hand, SM1 stained the I band very close to the edges of the A band and the SM1-stained stripes moved considerably upon stretching to a sarcomere length of 3.5 microns. Immunoelectron microscopic observations with frog semitendinosus muscle revealed that three distinct stripes bound with 3B9 in the edges of the A band did not move on stretching up to 3.5 microns. On the other hand, the two stripes stained with SM1 in the I band clearly moved to the same extent as the stretching. However, when a sarcomere was stretched to 4.0 microns, all the stripes with 3B9 or SM1 disappeared and diffused deposits of the antibodies were observed. Thus it is concluded that connectin filaments in the I band region are more extensible than those at both edges of the A band.

Proteolysis of specific muscle structural proteins by mu-calpain at low pH and temperature is similar to degradation in postmortem bovine muscle.

“…This antibody (4C7), which labels at the A-I junction as determined by immunofluorescence microscopy (results not shown), may recognize a single epitope of titin at the A-I junction, but we cannot rule out other possibilities (e.g., it may recognize closely spaced, repeated epitopes that are common to both the 1,200 kDa and the T2 portion of the molecule). Reports of monoclonal titin antibodies that recognize multiple epitopes along titin are not uncommon (Itoh et al, 1988;Furst et al, 1989). The possibility also exists that the degraded titin polypeptide bands seen in these PM MF also may represent a heterogeneous population of polypeptides, resulting from cleavage on either side of a single epitope, giving rise to fragments migrating at approximately 1,200 kDa and approximately 2,400 kDa (T2), at least some of which contain the epitope recognized by the antibody used in this study.…”

Dynamics of actin and assembly of connectin (titin) during myofibrillogenesis in embryonic chick cardiac muscle cells in vitro

“…13-15). Since it is known that the epitope portions of P1200 are extensible while those of 4C9 are less extensible (Itoh et al, 1988;Matsuura et al, 1991)) it is conceivable that during myofibrillar growth and elongation, connectin filaments are stretched a t their extensible domains into which Z band and other sarcomeric materials are in turn inserted to increase sarcomere number. However, the possibility also exists that this uneven distribution of P1200 epitopes in these regions may be artificially produced during specimen preparation.…”

Identification of High Molecular Weight Proteins in Squid Muscle by Western Blotting Analysis and Postmortem Rheological Changes

“…It also reacted with some other antibodies, but not with all the connectin antibodies. 3B9 is an antibody which has three epitopes in the connectin filament at the edge of the A band, 15) so it is presumed that there was also a connectin filament in the squid mantle muscle having a similar amino acid sequence to that of the A band of vertebrate connectin. Moreover, similarly to the rabbit sample, connectin was detected as doublet bands in the squid sample.…”

“…The primary antibodies used were the following: 3B9, mouse anti-chicken skeletal muscle connectin monoclonal antibody; SM-1, mouse antihuman skeletal muscle connectin monoclonal antibody; and Pc1200, goat anti-rabbit skeletal muscle connectin polyclonal antibody. 15,27) In addition, NB2, mouse antichicken pectoral nebulin monoclonal antibody supplied by Sigma Co.; PcNeb, rabbit anti-chicken pectoral nebulin polyclonal antibody; 28) and C20, goat antihuman skeletal muscle nebulin polyclonal antibody supplied by Santa Cruz Biotechnology were used.…”

“…[11][12][13] Immunohistochemical studies of -and -connectin using antibodies have suggested the location and function by which connectin affects the elasticity in muscle sarcomeres. [14][15][16][17] As for connectin-like protein in invertebrates, Hu et al reported that there was a band of high molecular protein working by SDS PAGE.…”