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Biomedical subjects

D E Wright

Publications and source records attributed to D E Wright.

At least 73 records · Page 4Linked to original sources
PubMed1981

Fluorometric assay for adenosine 3',5'-cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities.

A novel peptide substrate for adenosine 3',5'-cyclic monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), Leu-Arg-Arg-Trp-Ser-Leu-Gly, was synthesized. Phosphorylation of the peptide causes a 20% increase in the peptide fluorescence intensity at 358 nm. Values of Km and kcat for the phosphorylation reaction at pH 7.0 (25 degrees C), were determined to be 2.7 +/- 0.5 microM and 5.5 +/- 0.4 sec-1, respectively. The phosphorylated peptide was shown to be an effective substrate for phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) with a Km of 113 +/- 10 microM and a kcat of 2.4 +/- 0.2 sec-1 in the presence of 2.5 mM MnCl2. Changes in the peptide fluorescence intensity as a function of its phosphorylation state provide a highly sensitive assay of cyclic AMP-dependent protein kinase and phosphoprotein phosphatase activities.

Amino Acid Sequence↗
PubMed1980

Preparation of 2-thioltryptophan-glucagon and (tryptophan-S-glucagon)2. Differences in binding to the glucagon receptor in the hepatic adenylate cyclase system.

The synthesis of 2-thioltryptophan-glucagon is described. Oxidation of this compound gives the dimer (Trp-S-glucagon)2. Both monomer and dimer are equi-potent on a molar basis with native glucagon as activators of adenylate cyclase in hepatic plasma membranes. However, from the ability to compete with 125I-glucagon for binding at the glucagon receptor, the dimer has one-fourth the binding affinity for the receptor as does native glucagon, 2-thiol-Trp-glucagon, and Trp-(2,4-dinitrophenylsulfenyl)-glucagon which have equal affinities for the receptor. Addition of GTP, which converts the receptor from a tight-binding to a lower affinity form and which activates adenylate cyclase in the presence of glucagon, allows (Trp-S-glucagon)2 to bind equally with native glucagon and the other thiol derivatives of the hormone. This effect of GTP on the binding of the glucagon dimer and the uses of 2-thiol-Trp-glucagon in the semisynthesis of new glucagon derivatives are discussed.

Adenylyl Cyclases↗
PubMed1980

Preparation and properties of glucagon analogs prepared by semi-synthesis from CNBr-glucagon.

The semi-synthetic approach has been used to obtain new analogs of the peptide hormone glucagon. Using the highly purified 27 amino acid fragment of cyanogen bromide-treated glucagon, we have prepared, by nucleophilic addition to the lactone ring, the following derivatives: CNBr-Gly28-glucagon, CNBr-glucagon hydrazide, CNBr-glucagon n-butylamide and CNBr-glucagon biotinamide. Direct aminolysis of the lactone was successful only with sterically unhindered primary amines. Addition of an amino acid could be accomplished by formation of the peptide hydrazide followed by azide coupling. All these analogs were full agonists with decreased potency relative to the native hormone. Examination of the structure-function relationships of these new C-terminal glucagon derivatives suggests that the hydrophobic side-chain of methionine is important to the binding of glucagon to its receptor and that the C-terminal portion of glucagon is only involved in the binding of the hormone to the receptor and not in the transduction process.

Adenylyl Cyclases↗
PubMed1980

Properties of amidinated glucagons.

Porcine glucagon has been reacted with a series of alkyl imidates. The epsilon-amino group and both the alpha and epsilon-amino groups were modified and the subsequent glucagon derivatives were purified by ion-exchange chromatography and characterized. The modified glucagons were compared with native glucagon in their ability to activate hepatic adenylate cyclase and to compete with 125I-glucagon for binding to sites specific for glucagon in hepatic plasma membranes. N epsilon-acetamidino-glucagon was as biologically potent, in both activity and binding, as native glucagon, whereas N epsilon-4-hydroxyphenylamidinoglucagon required a twofold higher concentration to obtain similar levels. These findings suggest that modification through the epsilon-amino group with alkyl imidates possessing reporter groups should result in glucagon derivatives with significant biological potency, thus providing a new approach to the study of this peptide hormone. Amidination of both epsilon and alpha-amino groups resulted in glucagon derivatives which were agonists with respect to adenylate cyclase activation and which displayed unexpected anomylous behavior on chromatography.

Adenylyl Cyclases↗
PubMed1980

Synthesis of protected secretin 16-27 on a Merrifield resin. Examination of ammonolysis conditions for preparing carboxamide terminal protected peptides suitable for fragment condensation.

The partially protected dodecapeptide to secretin, H-Ser(Bzl)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-NH2 (protected secretin 16-27) was prepared using a standard Merrifield resin and solid phase synthesis methods. For comparative purposes the unprotected peptide also was prepared on a benz-hydrylamine resin. Contrary to previous reports, the valine C-terminal peptide can be cleaved from the resin and the amide obtained in high yield. A variety of conditions were examined to accomplish the cleavage of the peptide from the resin in its carboxamide terminal form. The best conditions found were trans-esterification followed by ammonolysis in a mixed solvent system. A thin-layer chromatography system which clearly separates the methyl ester and carboxamide terminal secretin 16-27 was developed.

Amino Acid Sequence↗
PubMed1979

Glucagon1-6 binds to the glucagon receptor and activates hepatic adenylate cyclase.

A fragment of glucagon encompassing its first six NH2-terminal residues (His-Ser-Gln-Gly-Thr-Phe) binds to the glucagon receptor and stimulates adenylate cyclase activity in rat liver plasma membranes. Glucagon1-6 is a partial agonist since it stimulates, at saturating concentrations, to the extent of 75% of the maximal activity given by the native hormone. The binding affinity and potency of glucagon1-6 are 0.001% the native hormone. Discussed are the implications of these findings on the structure-function relationships required for the action of glucagon and for preparing clinically useful analogs of the hormone.

Adenylyl Cyclases↗
PubMed1979

Nuclear-substituted styryl ketone analogs: effects on neoplasms, microorganisms, and mitochondrial respiration of tumorous and normal cells.

Analogs of some antineoplastic and cytotoxic Mannich bases derived from conjugated styryl ketones were prepared and evaluated for activity in the P-388 lymphocytic leukemia screen. Most of the new compounds had lower antineoplastic and murine toxicity than the parent compounds. Antimicrobial evaluation of some oximes and alcohols related to the Mannich bases revealed activity against certain Gram-positive bacteria and fungi. Primary pharmacological evaluation showed that some compounds containing a dimethylaminomethyl group displayed analgesic and antihistaminic properties. Five of the Mannich bases were evaluated as respiratory inhibitors in mitochondria derived from hepatic tumors, liver tissue from tumor-bearing animals, and normal rat liver. No statistical difference between the sensitivity of the three tissues to the compounds was obtained.

Amines↗
PubMed1978

A reassessment of structure-function relationships in glucagon. Glucagon1-21 is a full agonist.

Glucagon1-21 has been prepared by treating native glucagon with carboxypeptidase A. Purified glucagon1-21 did not contain detectable methionine (less than 0.001 residue/mol) and the activity of the compound did not change after treatment with cyanogen bromide as has been shown with native glucagon. Glucagon1-21 stimulates hepatic adenylate cyclase activity to the same extent as native glucagon but with 0.1% the potency. Glucagon1-21 also displayed 0.1% the binding affinity of native glucagon to the glucagon receptor in hepatic membranes. Glucagon22-29 alone or in combination with glucagon1-21 did not activate adenylate cyclase or displase 125I-glucagon from its receptor. The finding that glucagon1-21 is a full agonist on adenylate cyclase is discussed in relation to the structure-function relationships required for the biological action of glucagon.

Adenylyl Cyclases↗
PubMed1978

Effect of antineoplastic and cytotoxic Mannich Bases derived from conjugated styryl ketones on mitochondrial respiration in rat liver cells.

Five cytotoxic Mannich bases (5-dimethylamino-1-substituted phenyl-1-penten-3-ones), three having antineoplastic activity, were evaluated for respiratory-inhibiting properties in rat liver mitochondria in the presence of four substrates: succinate, glutamate, 3-hydroxybutyrate, and palmitylcarnitine. Four compounds (Ib--Ie) showed significant inhibiting properties which, on occasion, were reversed partially by coenzyme Q10. Evaluation of the spectra of the mitochondrial cytochromes indicated that Ib--Ie blocked the electron transport chain prior to the sequence of cytochromes. Since inhibition occurred when different substrates were used, a common site of action for Ib--Ie is likely; competition of Ib--Ie with coenzyme Q10 probably occurs. Compounds Ia--Ie inhibited RNA polymerase from Swiss mouse kidney cells but were virtually bereft of activity versus RNA polymerase from L-1210 leukemia cells. Polarography of the Mannich bases and the related styryl ketones showed that antineoplastic activity was associated with higher half-wave potentials.

Amines↗
PubMed1977

Studies on the generation of B lymphocytes in fetal liver and bone marrow.

With the use of immunofluorescence techniques, cells containing cytoplasmic IgM (cIgM+), but lacking detectable surface IgM (sIgM+), have been identified in mouse fetal liver and adult bone marrow as a distinct cell population to sIgM+ B lymphocytes. We have shown that there is a considerable difference in the rate of entry of cIgM+ and sIgM+ cells into DNA synthesis in these locations. Moreover, within the cIgM+ population, the largest cells are the main group entering DNA synthesis. Our results are compatible with the notion that a pool of rapidly proliferating, large cIgM+ cells is present in fetal liver and adult bone marrow and that these cells give rise to populations of smaller cIgM+ cells, which move out of cell cycle, and convert to sIgM+ B lymphocytes. However, we recognize that this interpretation is speculative. Finally, we have shown that fetal bone marrow is a site of generation of sIgM+ B lymphocytes, but the question as to whether these cells are derived from Ig- precursors within marrow itself remains open.

Animals↗
PubMed1975

Structure-function relationships in glucagon: properties of highly purified des-His-1-, monoiodo-, and (des-Asn-28, Thr-29)(homoserine lactone-27)-glucagon.

We have compared the ability of glucagon and three highly purified derivatives of the hormone to activate hepatic adenylate cyclase (an expression of biological activity of the hormone) and to compete with [125]glucagon for binding to sites specific for glucagon in hepatic plasma membranes. Relative to that of glucagon, biological activity and affinity of [des-Asn-28,Thr-29](homoserine lactone-27)-glucagon, prepared by CNBr treatment of glucagon, were reduced equally by 40- to 50-fold. By contrast, des-His-1-glucagon, prepared by an insoluble Edman reagent and highly purified (less than 0.5% contamination with native glucagon), displayed a 15-fold decrease in affinity but a 50-fold decrease in biological activity relative to that of the native hormone. At maximal stimulating concentrations, des-His-1-glucagon yielded 70% of the activity given by saturating concentrations of glucagon. Thus, des-His-1-glucagon can be classified as a partial weak agonist. Highly purified monoiodoglucagon and native glucagon displayed identical biological activity and affinity for the binding sites. Our findings suggest that the hydrophilic residues at the terminus of the carboxy region of glucagon are involved in the process of recognition at the glucagon receptor but do not participate in the sequence of events leading to activation of adenylate cyclase. The amino-terminal histidyl residue in glucagon plays an important but not obligatory role in the expression of hormone action and contributes to a significant extent in the recognition process.

Adenylyl Cyclases↗