Isolation and Characterization of 1,200 kDa Peptide of α-Connectin1

Abstract

When rabbit skeletal muscle myofibrils were kept for 12 h at 4 degrees C, alpha-connectin was partially degraded and 1,200 kDa peptide was newly formed [Takahashi, K. & Takai, H. (1988) Abst. 80th Jpn. Soc. Zootech. Sci. p-102]. The latter was isolated together with remaining alpha-connectin. Ultracentrifugation of the mixture at low ionic strength resulted in sedimentation of alpha-connectin, leaving the 1,200 kDa peptide in the supernatant. Physicochemical properties of the isolated 1,200 kDa peptide were investigated: UV absorption spectra, circular dichroism spectra, amino acid composition, and molecular size and shape. Polyclonal antibodies against the 1,200 kDa peptide [PcAb(1200)] bound the Z line and I-band. The position of the stripe in the I band near the N1 line due to the binding of PcAb(1200) moved both away from the Z lines and from the A band as sarcomeres were elongated. Therefore, it is considered that the 1,200 kDa portion of alpha-connectin is elastic.

Effects of postmortem aging time, animal age, and sex on degradation of titin and nebulin in bovine longissimus muscle

“…results herein with those in Lusby et al, 1983;Zeece et al, 1986;Fritz and Greaser, 1991;Fritz et al, 1993). This was necessary to help reduce the confusion that can occur when inadequate separation causes high-molecularweight titin degradation products (e.g., the 1,200 kDa band of Matsuura et al, 1991) to migrate very closely to T2 or nebulin. The system used in our study ( 5 % separating gel, acrylamide:N,N-bis-methylene acrylamide = 100: 1) also permitted easier identification of the TI and T2 bands due to their greater separation (Huff-Lonergan et al, 1994).…”

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

“…A major degradation product that migrates only slightly faster under SDS-PAGE conditions than T1 is termed T2 (approximately 2,400 kDa) (Kurzban and Wang, 1988). Another titin degradation product that has been observed migrates at approximately 1,200 kDa by SDS-PAGE analysis (Matsuura et al, 1991;Takahashi et al, 1992). This latter polypeptide has been shown to contain the portion of titin that extends from the Z-line to near the putative N 2 line in the I-band (Kimura et al, 1992;Kawamura et al, 1995), although the exact position that the 1,200-kDa polypeptide reaches in the sarco-mere is still not certain.…”

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

“…The following polyclonal (pAbs) and monoclonal antibodies (mAbs) were used: anti-connectin pAb against 1,200 KDa peptide of a-connectin (P1200) (Matsuura et al, 1991) and mAb 4C9 (Matsuno et al, 1989); anti-myosin pAb (Transformation Research Inc., Framingham, MA) and mAb MF20 (Masaki et al, 1982); anti-a-actinin pAb (Endo and Masaki, 1984) and mAb BM-75.2 (BioMakor, Rehovot); and anti-troponin C (TnC) pAb (Toyota and Shimada, 1981). Secondary antibodies were FITC-or rho-labeled anti-rabbit, anti-mouse, or antigoat IgG (Cappel Laboratories) and rho-labeled antimouse IgG (Tago, Inc., Burlingame, CA).…”

“…6 ) . Previous studies have shown that the membrane areas where actin filaments of myofibrils and stress fibers anchor exhibit proteins such as vinculin, a-actinin, and talin Using two Abs that recognize distant domains of connectin filaments, mAb 4C9 which binds the edges of the A band domain of connectin filaments (Matsuno et al, 1989) and pAb P1200 which binds the I band near the Z line (Matsuura et al, 1991), we monitored the devel-…”

“…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.…”