Connectin (titin) is a large filamentous protein (single peptide) with a molecular mass of approximately 3 MDa, contour length approximately 900 nm, and diameter approximately 4 nm, and resides in striated muscle. Connectin links the thick filaments to the Z-lines in a sarcomere and produces a passive elastic force when muscle fiber is stretched. The aim of this study is to elucidate some aspects of physical properties of isolated beta-connectin (titin 2), a proteolytic fragment of connectin, by means of dynamic light-scattering (DLS) spectroscopy. The analysis of DLS spectra for beta-connectin gave the translational diffusion coefficient of 3.60 x 10(-8) cm2/s at 10 degrees C (or the hydrodynamic radius of 44.1 nm), molecular mass little smaller than 3.0 MDa (for a literature value of sedimentation coefficient), the root-mean-square end-to-end distance of 163 nm (or the radius of gyration of 66.6 nm), and the Kuhn segment number of 30 and segment length of 30 nm (or the persistence length of 15 nm). These results permitted to estimate the flexural rigidity of 6.0 x 10(-20) dyn x cm2 for filament bending, and the elastic constant of 7 dyn/cm for extension of one persistence length. Based on a simple model, implications of the present results in muscle physiology are discussed.
A simplified procedure to isolate alpha-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate alpha-connectin is introduced. Separation of alpha-connectin from beta-connectin is introduced. Separation of alpha-connectin from beta-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of alpha-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of alpha-connectin (MW, approximately 3 x 10(6)) were similar to those of its proteolytic product, beta-connectin (MW, approximately 2 x 10(6)). Circular dichroic spectra suggested that both alpha- and beta-connectin consist of 60% beta-sheet and 30% beta-turn. It thus appears that the whole elastic filament of connectin has a folded beta-strand structure. Proteolysis of alpha-connectin by calpain resulted in formation of beta-connectin and smaller peptides. The alpha-connectin interacted with both myosin and actin filaments similarly to beta-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with alpha-connectin (titin 1, TI) but only weakly with beta-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SM1 whose position depends on sarcomere length. It thus appears that beta-connectin extends from the edge of M-line to the above epitope region in the I-band.
Internal fertilization ensures successful reproduction of tetrapod vertebrates on land, although how this mode of reproduction evolved is unknown. Here, we identified a novel gene encoding sperm motility-initiating substance (SMIS), a key protein for the internal fertilization of the urodele Cynops pyrrhogaster by Edman degradation of an isolated protein and subsequent reverse transcription polymerase chain reaction. The SMIS gene encoded a 150 amino-acid sequence including the cysteine knot (CK) motif. No gene with substantial similarity to the SMIS was in the data bank of any model organisms. An active site of the SMIS was in the C-terminal region of the 2nd loop of CK motif. A synthetic peptide including the active site sequence bound to the midpiece and initiated/enhanced the circular motion of C. pyrrhogaster sperm, which allows penetration of the egg jelly specialized for the internal fertilization of this species. The synthetic peptide bound to whole sperm of Rhacophorus arboreus and enhanced the rotary motion, which is adapted to propel the sperm through egg coat matrix specialized for arboreal reproduction, while it bound to the tip of head and tail of Bufo japonicus sperm, and enhanced the vibratory motion, which is suited to sperm penetration through the egg jelly specialized for the reproduction of that species in freshwater. The polyclonal antibody against the active site of the SMIS specifically bound to egg coat matrix of R. arboreus. These findings suggest that diversification of amphibian reproductive modes accompanies the specialization of egg coat and the adaptation of sperm motility to penetrate the specialized egg coat, and SMIS acts as the sperm motility enhancer of anurans and urodeles that might facilitate to adaptively optimize sperm motility for allowing the establishment of internal fertilization.
Sperm storage is supposed to influence sperm quality, although the details remain unclear. In the present study, we found that sperm stored in a sperm storage site, the vas deferens of Cynops pyrrhogaster, spontaneously undergo acrosome reaction following incubation in Steinberg's salt solution (ST). Percentages of acrosome-reacted sperm increased time-dependently to about 60% in 24 hr. The concentration of cyclic adenosine monophosphate (cAMP) was elevated after incubating sperm in ST, while dibutylyl cAMP induced an acrosome reaction. Chelating of extracellular Ca suppressed the dibutylyl cAMP-induced acrosome reaction as well as spontaneous acrosome reaction in ST. These results suggest that cAMP elevation driven by Ca influx can be a cue for spontaneous acrosome reaction. Relatively low Ca concentration and pH in the vas deferens were sufficient to suppress spontaneous acrosome reaction within 1 hr. In addition, the cysteine rich secretory protein 2 gene was expressed in the vas deferens, indicating that it may be involved in the continuous suppression of spontaneous acrosome reaction. Sperm that underwent spontaneous acrosome reaction in ST was significantly increased when stored in the vas deferens for longer periods, or by males experiencing temperatures in excess of 12°C during hibernation conditions. Percentages of the spontaneously acrosome-reacted sperm were found to differ among males even though they were of identical genetic background. Taken together, C. pyrrhogaster sperm possess the potential for spontaneous acrosome reaction that does not become obvious in the vas deferens, unless promoted in correlation with sperm storage.
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