Amino acid sequence of chicken gizzard smooth muscle SM22 alpha

The complete amino acid sequence of SM22 alpha, a novel and abundant 22-kDa protein from chicken gizzard smooth muscle, was determined by a combination of automated and manual Edman degradation methods on fragments produced by suitable chemical and proteolytic cleavages. The protein consists of a single polypeptide chain of 197 residues, has a Mr of 21, 978, and a net charge of +4.5 at neutral pH. The pattern of alternating hydrophilic and hydrophobic regions throughout the length of SM23 alpha is typical of a globular protein. The overall secondary structural analysis, using several algorithms based on the sequence, predicts approximately 31% alpha-helix, 24% beta-sheet, 18% beta-turn, and 27% random coil. A search against the National Biomedical Research Foundation Protein Sequence Databank (Washington) and GenBank (Los Alamos) failed to demonstrate significant similarity with any other protein of known sequence.     

The effects of troponin T fragments T1 and T2 on the binding of nonpolymerizable tropomyosin to F-actin in the presence and absence of troponin I and troponin C

Using a nonpolymerizable form of tropomyosin (NPTM) we have investigated the interactions between the T1 (residues 1-158) and T2 (residues 159-259) regions of troponin T and the other components of the thin filament at 50 mM KCl +/- Ca2+. Under these conditions the binding of NPTM to F-actin is fully restored by whole troponin (+/- Ca2+), and in each case, retains a residual degree of cooperativity as demonstrated by Scatchard and Hill plots. Fragment T2 alone had a small inductive effect on the interaction of NPTM with F-actin. In the presence of troponin I, this interaction is increased to a level which exceeds that observed with either component alone. The effects of T2 and troponin I are moderately (-Ca2+) and markedly (+Ca2+) reduced by troponin C. While fragment T1 alone did not promote induction, it accentuated the effects of T2 and troponin I. Since T1 does not interact with T2 or troponin I but does interact weakly with the NH2 terminus of tropomyosin and can be expected to bind weakly at the residual interaction site(s) at the COOH terminus of NPTM, the observed effects of T1 have been ascribed to the linking of neighboring NPTM molecules at their ends.     

Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase.

The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.     

Repetition structure of mammalian nuclear DNA

We have used Fragmentation Sequencing logic to analyse the repetition structure of several large human genomic genes. The method, based on a proposed laboratory scheme for DNA sequencing, detects short sequences which are repeated near, but not necessarily adjacent, to each other (cryptically simple DNA). We find a low frequency of such repeats. There is a slight excess of such repeats in introns over exons, and a slight but significant excess in genomic DNA over random DNA, confirming that cryptically simple sequences are over-represented in the genome. The analysis suggests that Fragmentation Sequencing will be a suitable method for sequencing large mammalian genes.     

Amino acid sequence of pilin isolated from pseudomonas aeruginosa PAK

A polar metabolite of leukotriene C4 was formed by sequential conversions with soybean lipoxygenase I and liver peroxidase. The structure of this product was found to be 5(S), 15(S)-dihydroxy-6(R)-S-glutathionyl-7,9,13-trans-11-cis-eicosatetraenoic acid (15-hydroxy-delta 13-trans-leukotriene C3. The HPLC behaviour, the molar extinction coefficient and the biological activity of the metabolite are reported. Preliminary evidence suggests that this product is formed by mammalian tissues.