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V J Hruby

Publications and source records attributed to V J Hruby.

At least 433 records · Page 24Linked to original sources
PubMed1980

Synthesis and structure-function studies of melanocyte stimulating hormone analogues modified in the 2 and 4(7) positions: comparison of activities on frog skin melanophores and melanoma adenylate cyclase.

The synthesis and purification of several analogues of the melanotropins with amino acid substitutions at the tyrosine-2 and methionine-4(7) positions are reported. The compounds synthesized included [4-norleucine]-alpha-MSH, [7-norleucine]-beta p-MSH, [2-3',5'-diiodotyrosine]-alpha-MSH, [2-D-tyrosine]-alpha-MSH, and [2-phenylalanine,4-norleucine]-alpha-MSH. The biological activities of these derivatives were measured and compared on normal melanocytes (frog skins) and on transformed melanocytes (mouse melanoma adenylate cyclase), over the entire dose-response range. All compounds tested were full agonists in both assay systems but varied considerably in potency. The relative potencies in the frog skin assay (alpha-MSH = 1.0) were as follows: [Nle7]-beta p-MSH (5.2) > [Nle4]-alpha-MSH (2.3) > alpha-MSH (1.0) > [Phe2,Nle4]-alpha-MSH (0.80) > beta p-MSH (0.55) > [I2-Tyr2]-alpha-MSH (0.12) > [D-Tyr2]-alpha-MSH (0.04). The relative potencies in the melanoma adenylate cyclase system were [Nle7]-beta p-MSH (4.2) > beta p-MSH (2.2) > [Nle4]-alpha-MSH (2.0) > alpha-MSH (1.0) approximately equal to [Phe2,Nle4]-alpha-MSH (0.9) > [I2-Tyr2]-alpha-MSH (0.40) > [D-Tyr2]-alpha-MSH (0.20). There appears to be some differences in structural specificity at the melanotropin receptors of the two cell systems.

Adenylyl Cyclases↗
PubMed1980

4-Norleucine, 7-D-phenylalanine-alpha-melanocyte-stimulating hormone: a highly potent alpha-melanotropin with ultralong biological activity.

alpha-Melanocyte-stimulating hormone (alpha-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of alpha-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4(methionine) and 7(phenylalanine) positions. [Nle4]-alpha-MSH, a synthetic analogue of alpha-MSH, reversibly darkens frog skins and also exhibits prolonged activity after heat-alkali treatment. Synthesis of [Nle4, D-Phe7]-alpha-MSH provided an analogue with prolonged biological activity identical to that observed with heat-alkali-treated alpha-MSH or [Nle4]-alpha-MSH. [Nle4, D-Phe7]-alpha-MSH was resistant to enzymatic degradation by serum enzymes. In addition, this peptide exhibited dramatically increased biological activity as determined by frog skin bioassay, activation of mouse melanoma adenylate cyclase, and stimulation of mouse melanoma cell tyrosinase activity. This Nle4, D-Phe7 synthetic analogue of alpha-MSH is a very porent melanotropin, 26 times as potent as alpha-MSH in the adenylate cyclase assay. The resistance of the peptide to enzymatic degradation and its extraordinarily potent and prolonged biological activity should make this analogue of alpha-MSH an important molecular probe for studying the melanotropin receptors of both normal and abnormal (melanoma) melanocytes.

Animals↗
PubMed1980

Synthesis, pharmacological, conformational, and dynamic studies of the potent hormone antagonists [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin. Conformational and dynamic considerations in the design of antagonists.

The solid phase synthesis of [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin is reported. The two compounds have no in vitro milk ejecting activity and no in vivo or in vitro oxytocic activity, but both are potent antagonists in these three assay systems. In the in vitro oxytocic assay, [1-penicillamine, 4-threonine]- and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin have pA2 values of 7.55 +/- 0.04 and 7.67 +/- 0.02, respectively, and both inhibit the uterine contractile response to oxytocin in nonpregnant and pregnant rats. [1-Penicillamine, 2-phenylalanine, 4-threonine]-oxytocin has a weak antipressor activity and at high doses, consistently caused a weak and transient fall in blood pressure in the rat. Carbon-13 nuclear magnetic resonance chemical shift parameters and spin-lattice relaxation times (T1) indicate that these two new oxytocin antagonists have very similar conformation and dynamic properties to oxytocin inhibitors which have previously been examined. These results are discussed in terms of conformational and dynamic models of oxytocin antagonism at the uterus. It is suggested that conformational restrictions at the 2- and 4-positions of penicillamine-1 analogues of oxytocin are important to antagonist activity and potency.

Amino Acid Sequence↗
PubMed1980

Synthesis of alpha- and beta-melanocyte stimulating hormones.

The solid phase synthesis of alpha-melanocyte stimulating hormone (alpha-MSH) using the benzhydrylamine resin and a number of recently described side chain protecting groups is given. The carboxamide terminal peptide was obtained directly by treatment of the peptide resin with liquid hydrogen fluoride at 0 degrees in the presence of carbonium ion scavengers. The solid phase synthesis of the C-terminal carboxylate hormone, porcine beta-melanocyte stimulating hormone (beta p-MSH), using a 1% cross-linked Merrifield resin and the same side chain protecting groups as in the alpha-MSH synthesis also is presented. Purification of both peptides was carried out by conventional chromatographic techniques. Both hormones were fully active and beta p-MSH was slightly more potent than previously reported in the literature.

Animals↗
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

Investigation of the interactions of oxytocin with neurophysins at low pH using carbon-13 nuclear magnetic resonance and carbon-13-labeled hormones.

The specifically 13C-labeled (90% 13C-enriched) peptide hormone derivatives [1-hem[2-13C]cystine]oxytocin, [1-hemi[1-13C]cystine]oxytocin, and [2-[-2-13C]tyrosine[-oxytocin and the analogue [3-[2-13C]leucine]oxytocin were prepared by total synthesis and used to study the interactions of the neurohypophyseal hormones with the bovine neurophysins as a function of pH and temperature. Under all conditions, whether high or low pH, the chemical shifts of the labeled carbon atoms of the bound hormones are the same, but they are shifted significantly from their positions in the free hormone. These results indicate that interactions of the side chain and disulfide moieties of the hormone with the neurophysins do not change as a function of pH. At neutral pH and 20--35 degrees C, the labeled atoms of the hormone are in slow exchange (1--5 s-1) with the neurophysins for the above hormone derivatives, but at low pH they are in intermediate or fast exchange depending upon the pH and temperature. At low pH, the dissociation rate constant (koff) is about 100-fold greater than the value at neutral pH, and this increase appears to be due exclusively to the breaking of the salt bridge involving the N-terminal amino group of oxytocin and a side-chain carboxyl group of neurophysin. Since the dissociation constant (Kd) also increases by about 100-fold in going from neutral to low pH, the association rate constant is deduced to be the same at neutral and low pH. In contrast to the low pH results, an increase in pH (from 6.6 to 10.5) leads to a continual decrease in the binding constant but to no apparent change in the dissociation rate constant. The bound hormone is always in slow exchange at high pH, even when the binding constant has been reduced by 2 or 3 orders of magnitude. At high pH, the decrease in binding affinity is due solely to the deprotonation of the alpha-amino group of the free hormone. Thus, at high pH the apparent association rate constant decreases, while the dissociation rate constant remains unchanged.

Animals↗
PubMed1979

Thermodynamics of glucagon aggregation.

Heats of dilution of concentrated glucagon solutions have been measured calorimetrically at 10 and 25 degrees C in 0.2 M potassium phosphate buffer of pH 10.6. Analysis of the data in terms of a monomer-trimer equilibrium gives the following thermodynamic parameters for the association reaction at 25 degrees C: delta G degrees = 7.34 kcal/mol of trimer, delta H degrees = -31.2 kcal/mol, deltaS degrees = -80 cal/(K mol), deltaCp = 430 cal/(K mol). The sensitivity of heat of dilution data to the association constant and stoichiometry of the reaction is discussed.

Calorimetry↗
PubMed1979

Location by paper chromatography of compensatory ovarian hypertrophy (COH) inhibiting activity in isobutanol extracts of bovine pineals.

Bovine pineal glands were extracted according to the methods reported by Bensinger et al. (1973) and Cheesman and Fariss (1970). Isobutanol soluble COH-inhibiting activity was further separated by chromatography on Sephadex G-15 and paper chromatography. With the Bensinger method, different active pineal fractions were obtained from Sephadex G-15 columns. Certain of those fractions were further separated by paper chromatography in butanol : acetic acid : water (4 : 1 : 1) and the COH-inhibitor was localized. The pineal COH-inhibitor could also be localized by high pressure, reverse phase liquid chromatography. More COH-inhibiting activity was extracted with the Bensinger method than with aqueous and acetic acid extraction methods used earlier by us. The Cheesman extraction method for arginine vasotocin gave less regular results in our hands than the Bensinger extraction method.

Animals↗
PubMed1979

Localization and chemical characterization of a partially purified bovine pineal antigonadotropin.

An antigonadotropic substance was partially purified from aqueous extracts of bovine pineal glands by methods of gel filtration, ultrafiltration and ion exchange chromatography. Two biological tests, viz. inhibition of compensatory ovarian hypertrophy and reduction of ventral prostate weight, were used to guide the purification. The partially purified antigonadotropin was characterized chemically using techniques of UV and fluorescence spectrometry, thin layer and paper chromatography, paper electrophoresis and amino acid analysis. The results reveal a diversity of ninhydrin-positive components present in the preparations, including free and peptide-bound amino acids, as well as other unidentified components, but not including any of the commonly occurring indoles, indoleamines or catecholeamines. One peptide, oxidized glutathione, was identified in the most purified material containing the biologically active principle yet pure, synthetric glutathione has no antigonadotropic activity in the biological tests utilized. Although the chemical nature of the bovine pineal antigonadotropin remains in question it may be purified by the methods described. The activity is thought to reside in the extremely small, perhaps trace quantities of residues derived. It is believed that large scale, preparative studies will be required for structural determination.

Amino Acids↗
PubMed1979

[1-Penicillamine,2-leucine]oxytocin. Synthesis and pharmacological and conformational studies of a potent peptide hormone inhibitor.

[1-Penicillamine,2-leucine]oxytocin was synthesized by the solid-phase method of peptide synthesis and purified by partition chromatography on Sephadex G-25, followed by gel filtration. The peptide was found to be a very potent competitive inhibitor of oxytocin in the oxytocic assay with a pA2 of 7.14 and an inhibitor of oxytocin in the milk-ejecting assay. The compound showed no agonist activity in either of these assays, and its inhibitory activity at the uterus was of prolonged duration. The 13C nuclear magnetic resonance spectral properties and the 13C T1 (spin-lattice) relaxation times of [Pen1,Leu2]oxytocin were determined, and the results were compared with previous studies of [Pen1]oxytocin, a related competitive inhibitor, and oxytocin, the native hormone agonist. These studies indicated that the hormone inhibitors [Pen1,Leu2]oxytocin and [Pen1]oxytocin have similar conformational and dynamic properties which are different than those of the agonist, oxytocin.

Animals↗
PubMed1979

The separation of peptide hormone diastereoisomers by reverse phase high pressure liquid chromatography. Factors affecting separation of oxytocin and its diastereoisomers--structural implications.

Experimental conditions and parameters involved in high performance liquid chromatography (HPLC) separations of the peptide hormone oxytocin and seven of its diastereoisomers, namely [1-hemi-D-cystine]-, [2-D-tyrosine]-, [4-D-glutamine]-, [5-D-asparagine]-, [6-hemi-D-cystine-], [7-D-proline]-, and [8-D-leucine]-oxytocin, on reverse phase columns were investigated. The effects of solvent, pH, and salt concentration were studied. Using the solvent systems 10% tetrahydrofuran-ammonium acetate buffer or 18% acetonitrile-ammonium acetate buffer and the muBondapak C18 support, oxytocin was separated from each of its diastereoisomers under all conditions studied, but the order of elution of diastereoisomers was highly dependent on solvent and to a lesser extent on pH. Separations of the hormone and its diastereoisomers on reverse phase HPLC and on classical partition chromatography on Sephadex G-25 were compared. The results are discussed in terms of the interactions of the solute with the reverse phase column and the solvent system. Implications of these findings in terms of the different solution conformations of the peptides are discussed.

Amino Acid Sequence↗
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↗