Does muscle-type myosin have ADPase activity?

Adenosine diphosphate (ADP) is a nucleotide that is structurally very similar to ATP but lacks one of the two high-energy bonds due to hydrolysis. In muscle studies, ADP is usually considered exclusively as a product formed during myosin cross-bridge cycling and is not otherwise involved in this process. In our study, we question the widely held view of ADP as a final product formed during muscle contraction. Using biophysical and biochemical methods, we managed to show that ADP can act as a substrate for myosins in at least three types of muscles: smooth and striated adductor muscles of bivalves (Mytilidae and Pectinidae), and also vertebrate skeletal muscles. According to our data, the differences in the effect of ATP and ADP on the optical, biochemical, and structural properties of actomyosins are exclusively quantitative. We explain the previous ideas about ADP as a compound capable of inhibiting the ATPase activity of actomyosin by the ability of ATP and ADP to depolymerize the polymeric myosin when the concentration in the medium reaches more than 0.3 mM.

Gelsolin from mussel's catch muscle.

Proteins of the gelsolin family are Ca2+-dependent, multifunctional, actin-binding proteins containing three (S1-S3, about 40 kDa) or six (S1-S6, about 80 kDa) highly conserved repeats in the amino acid sequence. The pattern of interaction of these proteins with actin is complex: they can sever actin filaments; promote polymer nucleation after binding to two actin monomers; and cap the growing barbed end of actin filaments. In the present study, an actin polymerizing factor (46 kDa) from the adductor muscle of a bivalve mollusc has been discovered and identified for the first time. This protein has turned out to belong to the gelsolin family of actin regulatory proteins. The expression of gelsolin-like proteins in the tissues of bivalves was predicted after analyzing their proteome, but this is the first study where an actually expressed protein has been found. A primary determination of its physicochemical properties such as molecular weight, charge, resistance to urea, influence on actin polymerization by viscosity, and light scattering is carried out and the molecular structure analyzed.

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

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.

Modeling of the mussel catch muscle contractile apparatus in actomyosin suspension.

In this paper, we tried to create a contractile model from proteins of the catch muscle of the Gray mussel, similar to the well-described suspension contractile model of vertebrate skeletal muscles. This model makes it possible to characterize the processes in the reconstructed contractile apparatus with the help of monitoring the two characteristics of muscle suspensions - the optical density and the particle size. Contractile model of the catch muscle we constructed was the simplest model consisting of two proteins, actin and myosin. During this work we compared the optical manifestations of the contraction and relaxation states of constructed model with earlier data on the actomyosin suspension of skeletal muscles. It appeared that the approach used in the study of skeletal muscle actomyosin relaxing - the use of an increased amount of ATP - cannot be applied to the contractile model of the molluscan catch muscle. Nevertheless we managed to reach relaxed state of this model with modifying calcium concentration. As a result, we laid the foundation for further reconstruction of the third state of the catch muscle - the catch tone.

Protein composition of thick filaments from molluscan catch muscle and the role of twitchin in the catch-state formation.

In the work, we performed densitometry of thick filaments of the Gray's mussel catch muscle; densitometry included determination of electrophoretic dye binding constants of proteins. The results of densitometry showed that the content of twitchin in thick filaments is significantly (10 times) lower than the content of myosin. We performed an in vitro simulation of the contractile apparatus of the catch muscle and showed that with such content, links formed by twitchin cannot stop "relaxation". So, we doubt that the role of twitchin in the formation of the catch state is to form load-bearing links between thin and thick filaments that keep the muscle in the contracted state. At the same time, densitometry has shown that the content of the unique catch-muscle protein - myorod - significantly exceeds the content of twitchin and reaches the level of myosin. Like twitchin, myorod is capable of forming regulated cross-links between thick and thin filaments. Such a high content of this protein may indicate that it is myorod, and not twitchin, that is responsible for the formation of catch load-bearing cross-links.

Troponin-like regulation in muscle thin filaments of the mussel Crenomytilus grayanus (Bivalvia: Mytiloida).

Muscles of bivalve molluscs have double calcium regulation--myosin-linked and actin-linked. While the mechanism of myosin-linked regulation is sufficiently studied, there is still no consensus on the mechanism of actin-linked regulation. Earlier we showed a high degree of Ca2+-sensitivity of thin filaments from the adductor muscle of the mussel Crenomytilus grayanus (Mytiloida). In order to elucidate the nature of this regulation, we isolated the fraction of minor proteins from the mussel thin filaments, which confers Ca2+-sensitivity to reconstituted actomyosin-tropomyosin. Proteins of this fraction, ABP-19, ABP-20, and ABP-28, were chromatographically purified and identified. According to the results of mass spectrometry and Western blot analysis, as well as by their functional properties, these mussel actin-binding proteins appeared to correspond to the troponin components from the skeletal muscles of vertebrates (TnC, TnI and TnT). The reconstituted mussel troponin complex confers to actomyosin-tropomyosin more than 80% Ca2+-sensitivity. The in vivo molar ratio of actin/tropomyosin/troponin was calculated to be 7:1:0.5, i.e., the content of troponin in mussel thin filaments is two times lower than in thin filaments of skeletal muscles of vertebrates. These data demonstrate that troponin-like regulation found in the catch muscle of the mussel C. grayanus is present at least in two suborders of bivalves: Pectinoida and Mytiloida.

Hybrid and non-hybrid actomyosins reconstituted with actin, myosin and tropomyosin from skeletal and catch muscles.

In this study, we investigated hybrid and non-hybrid actomyosin models including key contractile proteins: actin, myosin, and tropomyosin. These proteins were isolated from the rabbit skeletal muscle and the catch muscle of the mussel Crenomytilus grayanus. Our results confirmed literature data on an unusual ability of bivalve's tropomyosin to inhibit Mg-ATPase activity of skeletal muscle actomyosin. We have shown that the degree of inhibition depends on the environmental conditions and may vary within a wide range. The inhibitory effect of mussel tropomyosin was not detected in non-hybrid model (mussel myosin + mussel actin + mussel tropomyosin). This effect was revealed only in hybrid models containing mussel tropomyosin + rabbit (or mussel) actin + rabbit myosin. We assume that mussel and rabbit myosins have mismatched binding sites for actin. In addition, mussel tropomyosin interacting with actin is able to close the binding sites of rabbit myosin with actin, which leads to inhibition of Mg-ATPase activity.

Catch muscle myorod modulates ATPase activity of Myosin in a phosphorylation-dependent way.

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. Myorod is an alternatively spliced product of the myosin heavy-chain gene that contains the C-terminal rod part of myosin and a unique N-terminal domain. The unique domain is a target for phosphorylation by gizzard smooth myosin light chain kinase (smMLCK) and, perhaps, molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod and its phosphorylation in the catch muscle, the effect of chromatographically purified myorod on the actin-activated Mg2+-ATPase activity of myosin was studied. We found that phosphorylation at the N-terminus of myorod potentiated the actin-activated Mg2+-ATPase activity of mussel and rabbit myosins. This potentiation occurred only if myorod was phosphorylated and introduced into the ATPase assay as a co-filament with myosin. We suggest that myorod could be related to the catch state, a function specific to molluscan muscle.