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Conformational transitions in the myosin head induced by temperature, nucleotide and actin. Studies on subfragment-1 of myosins from rabbit and frog fast skeletal muscle with a limited proteolysis method.
Eur J Biochem 165, 353-62.
Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (S1) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides. The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its beta,gamma-imido analog AdoP P[NH]P) at 25 degrees C were observed when the digestion was carried out at 0 degrees C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoP P[NH]P at 0 degrees C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.
Specific high-affinity binding sites for a synthetic gliadin heptapeptide on human peripheral blood lymphocytes.
Life Sci 40, 1229-36.
The synthetic peptide containing residues 43-49 of alpha-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. We now demonstrate using radiolabeled alpha-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (KD) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of alpha-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of [125I]alpha-gliadin(43-49) than T-lymphocytes. The lymphocyte alpha-gliadin(43-49) receptor may play an important role in mediating the immunological response to alpha-gliadin.
Interaction of 2,3-benzodiazepines with peripheral benzodiazepine receptors.
Pharmacol Res Commun 19, 1-14.
2,3-Benzodiazepines (BZs), such as tofizopam (TP) and GYKI-51 189 have anxiolytic potency accompanied by moderate sedative action, but no anticonvulsant and muscle relaxant activities. These compounds show relatively low affinity to the peripheral benzodiazepine (PBZ) receptors, nevertheless, they decrease the binding of (3H)Ro5-4864 to its receptors in heart, kidney and brain membranes. This diminution in the binding is due to a decrease in the affinity for the ligand (Kd) without any change in the maximal number of binding sites (Bmax). This interaction of 2,3-BZs with PBZ binding sites may explain their pharmacological profile.
Effect of two synthetic alpha-gliadin peptides on lymphocytes in celiac disease: identification of a novel class of opioid receptors.
Neuropeptides 9, 113-22.
Two synthetic peptides containing residues 43-47 and 43-49 of alpha-gliadin were tested for inhibition of leukocyte migration in 47 patients with celiac disease. In nineteen patients, all on a normal diet, leukocyte migration was inhibited by the peptides and naloxone blocked this effect. In twenty-eight patients (24 of whom were on strict gluten-free diet) leukocyte migration was not affected by the peptides. Our results suggest that alpha-gliadin-(43-49), Tyr-Pro-Gln-Pro-Gln-Pro-Phe, is closely related to the active fragment, or to one of the active fragments of alpha-gliadin, and that it interacts with receptors that are similar to but not identical with the known opiate receptors.
Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin.
Biochemistry 26, 2616-23.
To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.