Claw Muscle Research Articles

Ca2+-dependent proteolytic activity in crab claw muscle. Effects of inhibitors and specificity for myofibrillar proteins.

The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. We describe here the role of Ca2+-dependent proteinases in the turnover of myofibrillar protein in normal anecdysial (intermolt) claw muscle. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 X g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The Ca2+-dependent proteinase is a sarcoplasmic cysteine proteinase that shows maximal activation at 1 mM Ca2+ at neutral pH. Since approximately 28% of the activity remains at 1.5 microM Ca2+, the enzyme is partially active at physiological Ca2+ concentrations. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: 1) heavy chain (Mr = 200,000) is cleaved into four large fragments (Mr = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (Mr = 18,000) is cleaved to a 15,000-Da fragment; 2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.

Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D. L. Mykles and D. M. Skinner, 1981, J. Ultrastruct. Res., 75, 314–325) . The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca 2+ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of cysteine proteinases, leupeptin and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.

Field and experimental studies on cadmium in the edible crab Cancer pagurus

The distribution of cadmium within captive crabs (Cancer pagurus) exposed experimentally to cadmium-contaminated food and water is described and illustrated by triangular diagrams. Crabs from the Orkney Islands (Scotland) are known to contain relatively high levels of cadmium (up to 62 μg g-1 wet wt) in the hepatopancreas. The distribution of cadmium between the hepatopancreas, gonad, gill, carapace, claw muscle, heart, and haemolymph, is described in crabs collected during 1978, and compared with similar data from crabs exposed to cadmium for ca. 300 d (September 1978 – June 1979) experimentally. It is concluded that the dominant uptake route of cadmium to Orkney crabs is through their diet.

Physiological Identification and Asymmetry of Lobster Claw Closer Motoneurones

ABSTRACT The somata of the fast closer exciter (FCE) and slow closer exciter (SCE) motoneurones to the dimorphic claw muscles of the lobster Homarus americanas were physiologically identified in an isolated claw-ganglion preparation, by recording from a cell body simultaneously with recording from an identified axon. The FCE, SCE, and opener exciter (OE) somata are the largest cells in the anterior ventral region of the ganglion and appear consistently as a triad. A marked asymmetry occurred in the responsiveness of the identified FCE and SCE somata to sensory stimulation via the 2nd root. The SCE soma produced a longer burst with higher spike frequencies than the FCE soma in either cutter or crusher claw. When homologous somata were compared, the crusher FCE and SCE produced higher frequencies and longer bursts of spikes than their cutter counterparts. Since this occurred in response to both sensory stimulation via the 2nd root and depolarization of the soma it suggests that differences in motor output between homologous motoneurones have an extrinsic and intrinsic origin. Furthermore, asymmetry sometimes occurred within an individual motoneurone with a larger number of spikes in the soma than in the axon. Thus the homologous FCE and SCE motoneurones are differentiated into physiologically different types.

Preferential loss of thin filaments during molt-induced atrophy in crab claw muscle

The closer muscle of the claw of a land crab Gecarcinus lateralis consists of slow, or tonic, fibers that undergo a sequential atrophy and restoration during each molting cycle. We have examined the ultrastructural organization of claw fibers during the process of protein degradation that takes place in proecdysial muscle atrophy. The myofibril cross-sectional area is reduced fourfold by dissolution of peripheral myofilaments and by focal erosion within the fibrils. As degradation continues, there is a preferential loss of thin filaments relative to thick filaments: (1) the average distance between thick filaments decreases from 59 to 45 nm, causing a 72% increase in packing density of thick filaments in cross section and (2) the relative numbers of thin and thick filaments decrease from an average ratio of 9:1 to 6:1. Despite these significant structural changes, the general organization of the sarcomere is unaltered; the membrane surface density of the sarcoplasmic reticulum and the A-band length remain unchanged. Although there is autophagy of some mitochondria, others retain their normal appearance, as do the nuclei. These data suggest that the specific degradation of contractile proteins in procedysial atrophic muscle includes the differential breakdown of thin versus thick filaments.

Two types of tonic fibers in lobster muscle based on enzyme histochemistry

AbstractTonic fibers are differentiated into two types based on enzyme histochemistry in the claw opener and closer muscle of the lobster Homarus americanus. Some of the fibers have a much lower myofibrillar ATPase activity and much higher oxidative capacity (indicated by NADH diaphorase) than the remainder. These slower‐contracting, more fatigue‐resistant fibers are regionally distributed in the claw muscles. The occurrence of two types of tonic fibers particularly in the opener muscle, which is innervated by a single slow excitor axon, shows that the oxidative properties of these fibers are not controlled simply by the presence of this motoneuron as previously proposed.

Hybrid formation between scallop myofibrils and foreign regulatory light-chains

Scallop myofibrils ( Placopecten magellanicus) from which regulatory light-chains had been completely removed by EDTA treatment at 30 °C were hybridized with regulatory light-chains of different myosins. Pure hybrids, containing only foreign regulatory light-chains with a stoichiometry of two moles per myosin, were readily formed with all the light-chains tested. Some of the regulatory light-chains restored regulatory functions to desensitized myofibrils by selectively inhibiting the actin activated Mg-ATPase in the absence of calcium. Light-chains from Mercenaria, Spisula Loligo and Urechis behaved as scallop regulatory light-chains, were inhibitory in the absence of calcium, and restored high-affinity calcium binding sites. Regulatory light-chains of Limulus, cricket, chicken gizzard and platelet were also inhibitory; however, calcium binding was restored with a lowered affinity and the hybrids required higher calcium concentrations for ATPase activation. Hybrids formed with the regulatory light-chains of vertebrate striated (rabbit, chicken, skate), bovine cardiac and lobster tail and claw muscles remained insensitive to calcium, their ATPase activity was not selectively depressed in the absence of calcium and specific high-affinity calcium binding sites were not restored. Phosphorylation of the light-chains (rabbit, cardiac and gizzard) has no effect on ATPase activity. The behaviour of the hybrids supports the interpretation that in vertebrate striated muscles myosin does not function as a regulatory switch. Foreign regulatory light-chains ( Spisula, Loligo, Mercenaria, rabbit) bind to desensitized myofibrils with a similar or slightly higher affinity as scallop regulatory light-chains. The two light-chain binding sites of myosin are equivalent and differences in affinity appear to be the result of an interaction between the two halves of the myosin molecules.

Detection of the membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline.

The distribution of membrane-associated calcium has been determined at various stages of mitosis in Haemanthus endosperm cells with the fluorescent chelate probe chlorotetracycline (CTC). CTC fluorescence in Haemanthus has two components: punctate, because of mitochondrial and plastid membrane-Ca++; and diffuse, primarily because of Ca++ associated with endoplasmic reticulum membranes. Punctate fluorescence assumes a polar distribution throughout mitosis. Cones of diffuse fluorescence in the chromosomse-to-pole regions of the metaphase spindle appear to coincide with the kinetochore fibers; during anaphase, the cones of fluorescence coalesce and this region of the spindle exhibits uniform diffuse fluorescence. Perturbation of the cellular Ca++ distribution by treatment with lanthanum, procaine, or EGTA results in a loss of diffuse fluorescence with no accompanying change in the intensity of punctate fluorescence. Detergent extraction of cellular membranes causes a total elimination of CTC fluorescence. CTC fluorescence of freshly teased crayfish claw muscle sarcoplasmic reticulum coincides with the A bands and is reduced by perfusion with lanthanum, procaine, and EGTA in a manner similar to that for diffuse fluorescence in the endosperm cells. These results are consistent with the hypothesis that a membrane system in the chromosome-to-pole region of the mitotic apparatus functions in the localized release of sequestered Ca++, thereby regulating the mechanochemical events of mitosis.

Studies on the influence of synthetic adipokinetic hormone and some analogs on cyclic AMP levels in different arthropod systems

Injection of synthetic adipokinetic hormone (AKH) into adult Locusta migratoria resulted in an increase of cyclic AMP level in the fat body. No differences in response between male or female insects could be observed. the accumulation of cyclc AMP was time dependent with a maximum effect 6 min after injection. The effect was dose dependent; approximately 2 pmol of synthetic AKH were needed to produce a singificant elevation of cyclic AMP. Studies with AKH analogs revealed that only peptides with a minimum of the first eight amino acids were able to elevate the cyclic AMP level. No analog, however, was as active as the true hormone. Although synthetic AKH did elevate the glucose concentration in the hemolymph as well as the cyclic AMP content of the claw muscle of the fiddler crab, this response was always less by comparison with purified hyperglycemic hormone of Cancer magister.

Comparison of glycolytic and associated enzyme activities in the claw and tail muscles of the lobster with those of the mantle of Mytilus edulis

In the mussel Mytilus edulis, the activities of phosphoenolpyruvate car☐ykinase (PEP-CK) and of malate dehydrogenase (MDH) are greater than those of pyruvate kinase (PK) and lactate dehydrogenase (LDH). The activities of PEP-CK are very low in the lobster (H. vulgaris.) and the ratio of MDH/LDH activities are 0·041 and 1·83 in the tail and the claw muscles respectively. Intracellular L-lactate and L-alanine concentrations suggest a different carbohydrate utilization in the tail and the claw muscle of the lobster. Consistent with this finding is the fact that L-lactate concentration is higher in the tail muscle than in the claw muscle; the opposite is true for L-alanine concentrations.

Glycolytic activity of the claw and tail muscle homogenates of the lobster, Homarus vulgaris

The fate of D-glucose-6-phosphate and phosphoenolpyruvate in homogenates of tail and claw muscles of the lobster (H vulgaris) has been studied. With both substrates oxygen utilization is higher for the claw than for the tail muscle. Succinate is not an end product of anaerobic D-glucose-6-phosphate-U- 14C degradation in either muscle but L-lactate is the major product of such catabolism in tail muscle whereas both L-lactate and L-alanine are produced in claw muscle. Dihydroxyacetone phosphate production was observed both tissues: this can be related to the known high lipid content of these tissues.

Properties of pyruvate kinase in the lobster

Pyruvate kinase from the tail muscle and claw muscle of a lobster (H. vulgaris) has been purified by ammonium sulfate precipitation, chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 200-fold purification with a yield of 70%. The molecular weights of the enzymes were found to be 254 000 ± 10% (tail muscle) and 266 000 ± 10% (claw muscle). Moreover both enzymes have a tetrameric structure with a molecular mass of 65 000 ± 10% for the subunit. Kinetic properties of the enzymes are such that a regulation of anaerobic glycolysis through allosteric modulation of pyruvate kinase activity appears to be non-functional for the two tissues investigated.

Effects Of Chemicals In Thermal Effluent On Homarus americanus Maintained In Aquaculture Systems1

ABSTRACTEffects of chemicals in thermal effluent on American lobsters (Homarus americanus) maintained in aquaculture systems were evaluated. Atomic absorption analysis of intake and effluent water samples from three fossil‐fuel generating stations in southern California indicated that chemical additions did not affect concentrations of Cu, Zn, Cd, Co, Cr, Pb, and As in the thermal effluent. Concentrations of these metals in the intake and effluent water at the San Diego Gas & Electric Company's Encina Power Plant were not significantly different than their concentrations in seawater from the Scripps Institution, and were well within the reported ranges for levels of those metals in normal seawater. The amounts of heavy metal chelating agents in Encina effluent were significantly greater than in Encina intake and Scripps seawater.No significant differences were observed in mean concentrations of Cu, Zn, Cd, Pb, Cr, and Co in larval, juvenile and adult lobsters maintained in three water types (Encina effluent, Encina intake and Scripps). Lobsters maintained in Scripps seawater were dissected and metal levels in the gills, hepatopancreas, tail muscles, claw muscles, digestive tract, and exoskeleton were analyzed by atomic absorption. The highest concentrations of Cu, Zn, Co and Cr were found in the gills, while Cd was found in equally high concentrations in the gills and hepatopancreas. The lowest concentrations of Cu, Zn, Cd, and Cr were found in the exoskeleton, while Pb and Co were found in the lowest concentrations in the tail muscle. Regressions of metal levels in tissues of lobsters on carapace length indicated that Hamericanus does not accumulate metals in the tissues, but rather regulates the levels.The 24, 48, 72, and 96 hour median lethal limits for Cu++, Zn++, Cr6+, Co+++, Cd++, Pb++, Cl−, and acids were established for lobster larvae held in static systems. Median effective times also were determined. Median lethal limits for these eight chemicals were all far above levels of the chemicals encountered in the generating station effluents. In most cases they were at least an order of magnitude higher. Longterm bioassays in static systems indicated that growth and survival of larval and juvenile lobsters were similar in Encina effleunt, Encina intake, and Scripps seawater at constant temperature.All of these studies indicate that the thermal effluent from typical generating stations in southern California provides an essentially pollution‐free heated water source for the culture of H. americanus.

Cytolytic enzymes in relation to the breakdown of the chelae muscle of the land crab, Gecarcinus lateralis

1. 1. In the land crab, Gecarcinus lateralis, the claw muscle undergoes degeneration and regeneration during specific stages of the molt cycle. 2. 2. Correlative in time with the degeneration, there is a three-fold increase in latent cathepsin D activity and a two-fold increase in latent acid phosphatase activity, where latency is defined as activity released by Triton X-100. 3. 3. The activity may be in either muscle cells themselves or connective tissue elements, including macrophages.

The Effect of Temperature and Salinity on Cadmium Uptake by the Blue Crab, Callinectes sapidus

Adult blue crabs were exposed to various concentrations of cadmium in sea water at different thermosaline regimes. Concentration and rates of uptake of cadmium were greatest at low salinities and higher temperatres with major sites of localization in gill, hepatopancreas and carapace. Claw muscle was comparatively low in cadmium.

Comparative Study of the Muscle Catheptic Activity of Some Marine Species

The hemoglobin-splitting activity and the autolytic- (endogenous) catheptic activity of the white skeletal muscle of 13 Atlantic fish species were measured and found to be low relative to the catheptic activity of other tissues. On the other hand, catheptic activities of lobster and crab claw muscles were high. Lobster tail muscle showed little activity, a factor perhaps contributing to its tougher texture relative to the claw muscle. The individual variations between samples of the same species were great and the ranges of the species overlapped each other with no evident phylogenetic distinction. The hemoglobin-splitting activity was always higher and was always directly correlated with the autolytic activity for each individual sample. Using an urea-free hemoglobin substrate pH optima were found in the range 3.0–3.6. Urea shifted the pH optima upwards approximately 0.4 unit. Fish muscle cathepsins were found to have the same level of activity as chicken muscle.

Structural features of the tarsal claw muscles of the spider Eurypelma marxi Simon

Structural features of the two tarsal claw muscles (levator pretarsi and depressor pretarsi) of the tarantula spider Eurypelma marxi Simon were studied using both the light and electron microscopes. The principal purpose of the study was to determine if these arachnid skeletal muscles differ in any of their structural features. Although the fibers in the two claw muscles are qualitatively similar, they differ quantitatively in several important respects. In comparison with the levator muscle, fibers in the depressor are larger, fewer in number, have longer A bands and sarcomeres, and larger myofibril bundles. They also appear to have more dyads per sarcomere and a greater number of nerve endings per synaptic region. In general, both muscles are similar in structure to other arthropod skeletal muscles.

Electron microscope observations on thick filaments in striated muscle from the lobster Homarus americanus

The size and appearance of thick filaments in lobster claw and tail muscle have been determined from EM observations on thin sections and isolated filaments. The filaments from both types of tissue are about 180 Å in diameter, but those from claw muscle are approximately 6 μ long, whereas the filaments from tail muscle are only ca. 3.4 μ long. The ratio of thin to thick filaments in the two muscles is 12/1 and 3/1, respectively. Also noted was an apparent variation in cross-sectional density of the filaments. In tail muscle, the filaments appear to be solid in the central region whereas in the A—I overlap region they appear to be hollow.

Structural and contractile properties of lobster leg-muscle fibers.

AbstractThree leg muscles of the American lobster, namely, the extensor muscle of the carpopodite in the walking legs, the closer muscle of the “cutter” claw, and the closer muscle of the “crusher” claw, were examined to see what types of muscle fiber they contain. In all three muscles, slow and fast fibers, as well as intermediate types, were present. Contraction rates of both types increased with the amplitude of the applied depolarization; slow fibers showed more lability in this respect, but they did not contract as rapidly as the fast fibers even when strongly stimulated. Membrane potential threshold for contraction was positively correlated with resting membrane potential in both types of fiber. Slow fibers differed from fast fibers structurally in having longer sarcomeres and A‐bands, smaller numbers of diads per unit length of muscle fiber, larger thin:thick filament ratios, and more connective tissue and sarcolemmal elements. The two claw closer muscles differed markedly in their proportions of slow and fast fibers, the “cutter” claw having a preponderance of short‐sarcomere, fast fibers. This finding helps to explain the difference in contraction speed of these two claw muscles.

A comparative study on striated muscle fibers of the first antenna and the claw muscle of the crab Pinnixia sp.

Two types of muscle fibers, here called Type-A and Type-B muscle fibers, are found in the peduncle of the first antenna of Pinnixia sp. One type is found in the abductor claw muscle. Type-A muscle fibers are reminiscent of invertebrate fast-acting muscle: straight Z-lines, prominent M-lines, hollow tubule-shaped thick myofilaments, hexagonally arranged thin and thick myofilaments, and a thin/thick myofilament ratio of 3/1. Dyadic contacts occur at mid-sarcomere levels. Type-B antenna and abductor claw muscle fibers are reminiscent of some invertebrate slow-acting muscle fibers. They lack M-lines, dyadic contacts occur at A/I junctions, thin and thick myofilaments are non hexagonally arranged, and thin/thick myofilament ratios are 6–7/1. A distinct feature of abductor claw muscle is the presence of plasmalemmal invaginations associated with Z-lines and cisternae of sarcoplasmic reticulum. This evidence is discussed in terms of a comparison with other invertebrate muscles with known speeds of activity.