Search PubMed⌕ Search

Biomedical subjects

K F Fok

Publications and source records attributed to K F Fok.

47 records · Page 3Linked to original sources
PubMed1984

Conformational analysis of COOH-terminal segments of human C3a. Evidence of ordered conformation in an active 21-residue peptide.

Human C3a, a 77-residue fragment released during activation of the serum complement system, is a potent spasmogen that contracts a variety of smooth muscle tissues and enhances vascular permeability. Previous studies have suggested that a 5-residue, COOH-terminal segment of C3a constitutes the essential active site of this molecule; however, the pentapeptide is 1000-fold less active than C3a. Human C3a 57-77, a synthetic segment containing the 21 COOH-terminal residues of C3a, exhibits potencies nearly equivalent to those of natural C3a in several biologic assay systems. The circular dichroism spectra of synthetic peptides corresponding to sequences 57-77, 65-77, and 73-77 in human C3a were measured in water and trifluoroethanol. The CD spectra in the far-UV region indicate that each C3a peptide assumes a random coil conformation in aqueous solution with little evidence of alpha-helical structure. However, C3a peptide 57-77 assumes predominantly an alpha-helical conformation (47%) in 25% trifluoroethanol, while the shorter tridecapeptide 65-77 and pentapeptide 73-77 appear by CD to contain beta-turn conformations only Crystallographic analysis of human C3a indicated that the NH2-terminal portion of peptide 57-77 adopts an alpha-helical structure and that the COOH-terminal portion, including residues 73-77, contains an irregular fold much like a beta-turn. Since C3a peptide 57-77 exhibits activities qualitatively and quantitatively similar to natural C3a, we propose that this synthetic peptide adopts a helical conformation when bound to its cellular receptor which corresponds to that in the intact C3a molecule. Consequently, the NH2-terminal portion (residues 1-21) and the disulfide-linked core region (residues 22-57) in intact C3a serve primarily to stabilize ordered conformation in the COOH-terminal region (residues 58-77) and thereby orient side chains at the essential active site for optimal receptor interaction.

Amino Acid Sequence↗
PubMed1984

Atriopeptins: a family of potent biologically active peptides derived from mammalian atria.

Extracts of rat atria are potent stimulators of sodium and urine excretion, and relax vascular and intestinal smooth muscle preparations. The structures of six biologically active peptides obtained from atrial extracts are reported here. Ion exchange chromatography of a low molecular weight fraction obtained by gel filtration of atrial extracts produced two natriuretic fractions: the first induced relaxation of intestinal smooth muscle strips only, whereas the second also relaxed vascular strips as well. From the first fraction four pure biologically active peptides obtained by reverse phase HPLC have been sequenced: the 21 amino acid peptide, designated atriopeptin I, and three homologs (des- ser1 -, des- ser1 -ser2-, and des- ser21 - atriopeptin I). From the second fraction two pure biologically active peptides were obtained, which had C-terminal extensions of atriopeptin I: atriopeptins II (23 amino acid residues) and III (24 residues), having respectively phe-arg and phe-arg-tyr C-termini. These results suggest that this family of six peptides, sharing the same 17 membered ring formed by an internal cystine disulfide, is derived from a common high molecular weight precursor.

Amino Acid Sequence↗
PubMed1984

Purification and sequence analysis of bioactive atrial peptides (atriopeptins).

Mammalian cardiac atria have several biologically active peptides that exert profound effects on sodium excretion, urine volume, and smooth muscle tone. In the present study two such peptides of low molecular weight were purified and separated from each other on the basis of differences in charge, hydrophobicity, and biological profile. The first peptide, designated atriopeptin I, exhibits natriuretic and diuretic activity and selectivity relaxes intestinal smooth muscle but not vascular smooth muscle strips. The second peptide, atriopeptin II, is a potent natriuretic and diuretic that relaxes both intestinal and vascular strips. Sequence analysis of atriopeptin I indicates that it is composed of 21 amino acids, of which serine and glycine residues predominate. The amino terminal sequence of atriopeptin II up to residue 21 is the same as that of atriopeptin I, with the addition of the Phe-Arg extension at the carboxyl terminus. Both peptides appear to be derived from a common high molecular weight precursor (designated atriopeptigen); their biological selectivity and potency may be determined by the site of carboxyl terminal cleavage.

Amino Acid Sequence↗
PubMed1984

Towards a molecular and atomic anatomy of calmodulin and calmodulin-binding proteins.

The molecular mechanisms by which calcium regulates cellular processes such as metabolic and mechanochemical events probably involve interactions with a variety of molecules. A large body of evidence suggests that the targets of calcium's regulatory effects inside the cell are calcium-binding proteins. Our work attempts to correlate calcium-binding protein structure with activities. In this chapter we have presented some of our recent studies on these functional domains in calmodulin and calmodulin-binding proteins. Selected chemical modifications of known amino acid sequence positions have demonstrated the presence of multiple functional domains on calmodulin, have allowed the dissociation of calmodulin functions, and have provided the necessary tools for further investigations of the molecular basis of calmodulin action. One of these modifications, iodination of tyrosine-99, has allowed us to develop procedures to reproducibly detect calmodulin-binding proteins by a binding technique. This method is technically simple. It allows us to detect and study calmodulin-binding proteins (e.g., myosin heavy chain and membrane gap junction proteins) that would be difficult to study with immobilized calmodulin. We have developed a library of antibodies to calmodulin and related proteins such as troponin C and S100 beta. Some of these antisera appear to be site-specific gamma globulins. We have demonstrated that reactivity can be contained in an amino acid sequence as short as seven residues. We have demonstrated the feasibility of using an immunochemical mapping approach to study calmodulin and calmodulin-binding proteins. Comparative sequence analyses combined with functional analyses have allowed correlation of function and structure and have suggested logical candidates for functional domains on calmodulin and calmodulin-binding proteins. Although not discussed in detail in this chapter, these calmodulin-binding proteins appear to contain amino acid sequence homologies. This suggests, analogous to the approach used for calmodulin and related proteins, a logical starting point for domain analyses of calmodulin-binding proteins. Interestingly, structural homologies among calmodulin-binding proteins are reminiscent of the different phosphorylation sites found in many of the physiological substrates for protein kinases. Since a number of calmodulin-binding proteins are themselves substrates for protein kinases, these results suggest another possible point of interrelationships between calcium and cyclic nucleotide regulation.

Amino Acid Sequence↗
PubMed1983

Radioimmunoassays for the thymic hormone serum thymic factor (FTS).

Four radioimmunoassays (RIA) are described for the quantitation of serum thymic factor (facteur thymique serique, FTS), a thymic peptide hormone. Each assay employs an antibody specific for FTS, synthetic FTS (Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn) as the hormone standard, and a radioiodinated FTS analogue as the tracer. Since FTS lacks a tyrosine residue, 2 FTS analogues were synthesized by the solid-phase method with tyrosyl-alanyl or 3-(2,6-dichlorobenzyl)tyrosyl-alanyl in place of the amino-terminal pyroglutamyl residue (Glp). They showed full FTS immunoreactivity and their radioiodinated derivatives served as FTS tracers. Two assays used the antiserum from a rabbit immunized with an FTS-protein conjugate. Two other assays used a monoclonal antibody against FTS produced by a hybridoma derived from mouse myeloma cells and splenocytes from a BALB/c mouse immunized with an FTS-mouse IgG conjugate (Ohga et al., 1982). All 4 RIAs were specific for FTS. The more sensitive rabbit antiserum can detect as little as 1 pg of FTS in a 50 microliters sample, which may allow quantitation of the FTS circulating in human peripheral blood.

Amino Acid Sequence↗
PubMed1983

Elucidation of a minimal immunoreactive site of vertebrate calmodulin.

The heptapeptide Asn-Tyr-Glu-Glu-Phe-Val-Gln-NH2 corresponding to residues 137-143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem. 256, 4205-4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127-144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only seven-residue segment in the 135-145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.

Animals↗
PubMed1983

Engineering of site-directed antisera against vertebrate calmodulin by using synthetic peptide immunogens containing an immunoreactive site.

Site-directed antisera against vertebrate calmodulin were elicited in rabbits by injection of a synthetic immunogen containing the pentadecapeptide Gly-Gln-Val-Asn-Tyr-Glu-Glu-Phe-Val-Gln-Met-Met-Thr-Ala-Lys-OH, which corresponds to residues 134-148 of vertebrate calmodulin. A major immunoreactive region (residues 127-144) of calmodulin is found in the COOH-terminal structural domain and an immunoreactive site for one antiserum is contained in the heptapeptide Asn-Tyr-Glu-Glu-Phe-Val-Gln-NH2, which corresponds to residues 137-143 of vertebrate calmodulin. This immunoreactive heptapeptide was conjugated to a carrier protein by adding a cysteine residue to the NH2 terminus of the peptide and coupling the Cys-heptapeptide to the carrier through the thiol group of the cysteine residue. Injection of this Cys-heptapeptide-protein conjugate into rabbits yielded antisera that react with the heptapeptide but not with native calmodulin. Thus, the immunoreactive heptapeptide that is exposed on the surface of calmodulin is immunogenic, but it is not sufficient to elicit antibodies that react with native calmodulin. However, when the Cys-pentadecapeptide corresponding to residues 134-148 and containing the immunoreactive heptapeptide sequence was conjugated to a carrier protein and injected into rabbits, antisera were elicited that react with the intact calmodulin molecule. The affinities and specificities of these antisera for calmodulin are similar to those of antisera elicited by injection of the intact protein and are sufficient for their use in radioimmunoassays. These results indicate that the successful engineering of site-directed antisera against proteins by using synthetic peptide immunogens may require an appropriate intramolecular environment that allows the peptide region to closely approximate the spatial orientation it adopts in the intact protein.

Amino Acid Sequence↗
PubMed1983

Suppression of humoral immune responses by synthetic C3a peptides.

Synthetic oligopeptides based on the COOH-terminal sequence of native human C3a were examined for the ability to suppress antigen-specific and polyclonal antibody responses. The synthetic peptides were found to qualitatively mimic the suppressive actions of human C3a, but proved to be less active on a molar basis. Comparison of the activity of the peptides with native C3a indicates that the most potent peptide is C3a 57-77 with C3a 65-77, C3a 70-77, C3a[Ala 71,72]70-77, and C3a 73-77 being less active, respectively. These results indicate that the region of the C3a molecule responsible for immunosuppression is located in the COOH region.

Anaphylatoxins↗