To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.

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Based on two criteria, the tightness of packing of myosin rods within the backbone of the filament and the degree of order of the myosin heads, thick filaments isolated from a control group of rat hearts had three different structures. Two of the structures of thick filaments had ordered myosin heads and were distinguishable from each other by the difference in tightness of packing of the myosin rods. Depending on the packing, their structure has been called loose or tight. The third structure had narrow shafts and disordered myosin heads extending at different angles from the backbone. This structure has been called disordered. After phosphorylation of myosin-binding protein C (MyBP-C) with protein kinase A (PKA), almost all thick filaments exhibited the loose structure. Transitions from one structure to another in quiescent muscles were produced by changing the concentration of extracellular Ca. The probability of interaction between isolated thick and thin filaments in control, PKA-treated preparations, and preparations exposed to different Ca concentrations was estimated by electron microscopy. Interactions were more frequent with phosphorylated thick filaments having the loose structure than with either the tight or disordered structure. In view of the presence of MgATP and the absence of Ca, the interaction between the myosin heads and the thin filaments was most likely the weak attachment that precedes the force-generating steps in the cross-bridge cycle. These results suggest that phosphorylation of MyBP-C in cardiac thick filaments increases the probability of cross-bridges forming weak attachments to thin filaments in the absence of activation. This mechanism may modulate the number of cross-bridges generating force during activation.

Schistosomes are blood-dwelling trematode parasites that infect 200 million people in developing countries. The critical role served by the tegument in immune evasion and parasite homeostasis suggests that a detailed knowledge of tegumental components would be helpful in the design of new drugs and the production of vaccines. We demonstrate here, by immunoelectron microscopy, that the cytoskeletal proteins actin and paramyosin are organized into major tegumental structures of Schistosoma mansoni. The surface spines are composed of paracrystalline arrays of actin filaments. Actin is also present in areas recovering from damage, implying an important role for this structural protein in tegumental repair. Paramyosin exists predominantly in the tegument in a non-filamentous form, the membrane-bounded elongate bodies. The localization of this protein to the tegument of the parasite is the likely basis for resistance to S. mansoni observed in mice immunized with paramyosin (refs 1, 2 and T. P. Flanigen et al., in preparation).

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles: Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles: Limulus telson, Homarus slow claw, Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.The paramyosin content of molluscan muscle has been observed to vary (a) with the structural organization of fibers, and (b) with the dimensions of the thick filaments. A paramyosin:myosin ratio greater than 1:1 has been reported for lamellibranch smooth adductors (30, 32). Obliquely and cross-striated adductors have been reported to contain proportionally less (1:2 and 1:3, respectively) of this protein (30, 32). In such molluscan smooth "catch" muscles as lamellibranch opaque adductors (6-8, 10-12, 20, 27) and Mytilus anterior byssus retractor muscle (ABRM) (18,20,21,26,28, 32), thick filaments range from 500 to 1,500 /~ in diameter and from 10 to 40 tzm in length. They are oriented parallel to the cells, but are not organized into identifiable ordered, repeating sarcomeric units (33). The cross-striated adductors of the lamellibranch Aequipecten, on the other hand, resemble vertebrate striated muscle with respect to both filament dimensions and sarcomere organization (24).In previous papers (1, 5, 19) we and others reported the identification of paramyosin as a component of striated arthropod muscles, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunodiffusion. We also showed that the arthropodan and molluscan paramyosins in homogenates of glycerinated muscle have identical chain weights, and further, that this protein is similar enough in these different muscles to cross-react immunologically across phyletic lines.Here we report the paramyosin:myosin heavy chain ratios in the following arthropodan muscles:Limulus telson levator, Hornarus slow claw muscle, Balanus scutal depressor, and Lethocerus air tube retractor; as well as in the following molluscan muscles: Aequipecten striated adductor, Mytilus ABRM, and Mercenaria opaque adductor.The correlation between paramyosin content, filament dimensions, and maximum active tension development is discussed in relation to the functional role of paramyosin in these muscles.