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Invertebrate mini-titins are members of a class of myosin-binding proteins belonging to the immunoglobulin superfamily that may have structural and/or regulatory properties. We have isolated mini-titins from three molluscan sources: the striated and smooth adductor muscles of the scallop, and the smooth catch muscles of the mussel. Electron microscopy reveals flexible rod-like molecules about 0.2 micron long and 30 A wide with a distinctive polarity. Antibodies to scallop mini-titin label the A-band and especially the A/I junction of scallop striated muscle myofibrils by indirect immunofluorescence and immuno-electron microscopy. This antibody crossreacts with mini-titins in scallop smooth and Mytilus catch muscles, as well as with proteins in striated muscles from Limulus, Lethocerus (asynchronous flight muscle), and crayfish. It labels the A/I junction (I-region in Lethocerus) in these striated muscles as well as in chicken skeletal muscle. Antibodies to the repetitive immunoglobulin-like regions and also to the kinase domain of nematode twitchin crossreact with scallop mini-titin and label the A-band of scallop myofibrils. Electron microscopy of single molecules shows that antibodies to twitchin kinase bind to scallop mini-titin near one end of the molecule, suggesting how the scallop structure might be aligned with the sequence of nematode twitchin.

Electron microscope images of frozen-hydrated crystals of a proteolytically modified fibrinogen show excellent preservation of the structure. An electron density map of the key centric projection of the crystal at 18 A resolution has been obtained by combining the phases derived from cryo-electron microscopy with X-ray amplitudes. Simulation methods developed in earlier studies have been used to interpret the map. In contrast to the earlier images, the map allows us to visualize the coiled-coil region of the molecule and possible substructure in the beta domains. The map also shows that there is a marked difference in density in the two regions corresponding to the molecular ends where the gamma domains interact. A possible interpretation of this finding is provided by assuming substructure in the gamma domains and the breaking of molecular symmetry where these domains interact. Some additional constraints useful for the determination of the three-dimensional structure were obtained from cryo-electron micrographs of a perpendicular view at 25 A resolution. Implications of this working model for the molecular length and contacts in the filaments in both the crystal and fibrin are described. The data used here will be valuable as a starting point for obtaining the three-dimensional structure.

Myosin from a molluscan catch muscle displays unusual properties: when phosphorylated in the rod by an endogenous heavy-chain kinase, myosin solubility is enhanced and the molecule folds (Castellani & Cohen, Proc. natn. Acad. Sci. U.S.A. 84, (1987) 4058-62). We have now localized the sites of phosphorylation to the carboxy-terminal end of the rod by selective proteolytic cleavage. Two major stretches of sequence, 18 and 21 residues long, have been identified, each containing a single residue of phosphoserine. Analysis of the amino-acid sequence of these two peptides indicates that they form a non-helical tailpiece. We discuss how phosphorylation of this tailpiece might influence enzymatic activity in catch muscle thick filaments.

The immunoglobulins of the bullfrog Rana catesbeiana are unusual in that, in all classes, the light chains are not disulfide bonded to heavy chains or to other light chains. Moreover, the light chains contain six, rather than the usual five, residues of half-cystine. As none of these half-cystines is in the sulfhydryl form or is alkylated after mild reduction, we suggested that the light chains probably contain three intrachain disulfide bridges. We have now carried out experiments to confirm the existence of an extra intrachain disulfide bridge in Rana catesbeiana light chains and to determine its location. Disulfide bridge assignments were based on 1) isolation and sequence analysis of S-(carboxymethyl)cysteine-containing peptides and 2) isolation, from unreduced light chains, of peptides containing a disulfide bridge. Half-cystine residues were found at positions 134 and 194, and these were shown to be joined in the conserved intradomain disulfide bridge. In addition, we found that a residue of half-cystine, located at the third position from the carboxy-terminus, forms a disulfide bridge with a half-cystine at position 119, near the amino-terminus of the domain, the latter residue replacing a proline that has been found at this position in all other light chains. An intrachain disulfide bridge has not been found at this location in any other light chain.

The lambda-chains of immunoglobulins from BALB/c mice constitute the simplest system presently available for studying patterns of variable-region diversity. The limited number of V lambda and J lambda germ-line gene segments facilitates comparison of expressed and germ-line sequences. We report here the complete amino acid sequence of the variable regions of three lambda 2 chains and of one chain representing a V lambda 2----J lambda 3 rearrangement. Together with the previously determined sequence of the lambda 2 chain from myeloma MOPC-315, the results illustrate the following types of variable-region diversification: expression of a single V gene segment with more than one J segment, variability at the V-J junction, and presumably, somatic mutation in V and in J. The extent of somatic diversification in these lambda 2 chains is limited, consistent with results obtained previously with lambda 1 chains.

Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain.

We report here a procedure which results in the purification of an extracellular protease (designated Ps-1) from Pseudomonas aeruginosa. This enzyme cleaves fibrinogen so that the modified molecules form microcrystals and large single crystals. Precise knowledge of the Ps-1 cleavage sites is essential for the interpretation of the structural information provided by these crystals (Weisel, J. W., Stauffacher, C. V., Bullitt, E., and Cohen, C. (1985) Science 230, 1388-1391). Ps-1 is a single-chain polypeptide of Mr 30,000 which appears to function as a monomer. The pH optimum is 8-9. The activity of the protease is not decreased by inhibitors of thiol, carboxyl, or metallo proteases; the abolishment of activity by N alpha-p-tosyl-L-lysine chloromethyl ketone and the partial inhibition obtained with serine-reactive inhibitors suggests that Ps-1 may be a serine protease with an unusual active-site conformation. Studies with synthetic peptide substrates show that Ps-1 exhibits one of the most restricted specificities known for an endoproteinase: only peptide, ester, and amide bonds containing the carbonyl group of lysine are hydrolyzed. The limited specificity of Ps-1 should make it useful for other applications requiring the selective cleavage of proteins, such as sequence analysis and the isolation of domains.

The reaction of sIgA with 5,5'dithiobis (2-nitrobenzoic acid) or with 14C-iodoacetamide revealed an average of 1 mol -SH per mol sIgA under nondenaturing conditions, whereas an average of 7.5 mol -SH was found under denaturing conditions. These -SH groups were found in both the sIgA-f and sIgA-g subclasses; in the sIgA-g subclass, the free -SH groups were found in the Fab alpha portion but not in the Fc alpha portion of the molecule. To determine whether the noncovalently bound polypeptide chains in sIgA contained free -SH groups, sIgA was alkylated with 14C-iodoacetamide and subjected to gel filtration in 6 M guanidine-HCl. This treatment liberated several different polypeptides that were identified as secretory component (SC), L chain dimers, and L chain monomers; measurement of radioactivity incorporated in each of these noncovalently bound chains showed that SC and the L chain dimers did not contain free -SH groups but that an average of 1 -SH group per L chain monomer was found. Of the covalently bound polypeptide chains, J chain and SC did not have detectable -SH groups, whereas the L chains and alpha-chains had 0.3 and 1.2 mol -SH per mol protein, respectively. The identification of free -SH groups in the Fab portion of the sIgA-g subclass and their preferential localization within only some of the L chains reflect the structural heterogeneity of sIgA; these -SH groups presumably arise during biosynthesis.

The actin-binding property of the myosin head 20 K (K = 10(3) Mr) fragment has been examined by a structural assay. A new fragment is produced by digestion of scallop myosin synthetic filaments with a lysine-specific protease. This fragment consists of the rod together with two "nubs" corresponding to the 20 K fragment, which retain both the regulatory and essential light chains. Myosin filaments, digested for different lengths of time, were mixed with F-actin and visualized by electron microscopy after negative staining. When the head is cleaved, but the head fragments remain associated, the filaments bind actin in an ATP-sensitive manner. Filaments made primarily of the nub-containing fragments, however, bind actin very poorly. In addition, electron microscopic characterization of actin-binding by the isolated tryptic 20 K fragment from chicken myosin indicates that binding of this fragment to actin is probably non-specific. These results suggest that interactions between the 20 K region and the other peptides in the head are essential for actin-binding.