A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle

Abstract

We describe the development of a preparative method to isolate molluscan catch muscle, calponin. This method is based on the ability of calponin to interact with actin in a temperature-dependent manner. After extracting thin filaments, as previously described, the extract was ultracentrifuged at 2 °C. While other surface proteins of thin filaments co-precipitated with actin, calponin, along with some minor contaminants, remained in the supernatant. Calponin was purified through cation-exchange chromatography. The yield of pure protein was four-fold higher than that achieved through high-temperature extraction. To evaluate functionally isolated proteins, we determined the effect of calponin on Mg2+-ATPase activity of hybrid and non-hybrid actomyosin. The degree of ATPase inhibition was consistent with previously published data but strongly dependent on the environmental conditions and source of actin and myosin used. Furthermore, at low concentrations, calponin could induce the ATPase activity of hybrid actomyosin. This result was consistent with data indicating that calponin can modulate actin conformation to increase the relative content of “switched on” actin monomers in thin filaments. We assume that calponin obtained by the isolation method proposed herein is a fully functional protein that can both inhibit and induce the ATPase activity.

Role of the Neuroendocrine System of Marine Bivalves in Their Response to Hypoxia

“…This occurs following the initial activation of the muscle. This state is characterized by prolonged force maintenance in the face of low Ca 2+ , high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage [ 124 , 125 , 126 ]. Tension is maintained until the serotonergic fibers release 5-HT, which stimulates the AC/cAMP/PKA system.…”

Short Overview of Not a Short Way to Reveal the Molecular Mechanisms of Molluscan Catch Muscles Contraction

“…Along with these results, important achievements of the laboratory have been the development of a new method for obtaining Ca 2 +regulated thin filaments from the smooth muscles of bivalve mollusks; the discovery of calponin in the thin invertebrate muscle filaments and a detailed study of its physico-chemical properties [7] and a preparative method to isolate molluscan catch muscle calponin [8]; study of the proteins of the catch muscles of the mussel Crenomytilus grayanus providing Ca 2 + sensitivity to thin filaments, isolation of these proteins and their identification [9]. The laboratory's pioneering achievement is the development of an original method for isolating "natural", "non-Straub-type" fibrillar actin and comparison of its properties with those of skeletal and smooth muscle actins obtained by the classical method [10].…”