phosphoramidon and zincov

A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes. It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov. The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.

Topics: Amino Acid Sequence; Ammonium Sulfate; Animals; Chemical Fractionation; Chromatography; Glycopeptides; Hemagglutination; Humans; Hydroxamic Acids; Isoelectric Point; Metalloproteases; Molecular Sequence Data; Molecular Weight; Phenanthrolines; Protease Inhibitors; Sequence Homology, Amino Acid; Vibrio; Zinc

The effects of the metalloproteinase inhibitors thiorphan and R-94138 on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. The inhibitor constants (K(i)) of thiorphan and R-94138 for matrilysin at pH 7.5, 25 degrees C were determined to be 11.2 and 7.65 microM, respectively. From the temperature dependence of the K(i) values at pH 7.5, the standard enthalpy change (Delta H degrees ') values for the binding of matrilysin with thiorphan and R-94138 were determined to be -(18.2 +/- 0.9) and (1.65 +/- 1.07) kJ x mol(-1), respectively. The binding of matrilysin to thiorphan is exothermic and the free energy change in the complex formation depends mainly on the change in enthalpy, while the binding to R-94138 is endothermic and typically entropy-driven. Hydrophobic interactions are suggested to contribute significantly to the binding of matrilysin to R-94138 as well as to the substrate. The pH dependence of the K(i) value suggests that at least two ionizing groups with pK(a) values of 4.5 and 9.1--9.3 are involved in the binding. The matrilysin activity is regulated by ionizing groups with pK(a) values of 4.3 and 9.6. Both inhibition and hydrolysis are suggested to be controlled by the same residues in matrilysin, most likely Glu 198 and Tyr 219, respectively.

Topics: Acetamides; Drug Design; Enzyme Stability; Glycopeptides; Humans; Hydrogen-Ion Concentration; Hydroxamic Acids; Kinetics; Matrix Metalloproteinase 7; Matrix Metalloproteinase Inhibitors; Protease Inhibitors; Protein Binding; Static Electricity; Structure-Activity Relationship; Temperature; Thermodynamics; Thiorphan

Intracisternal (ic) injection of the neutral endopeptidase-24.11 inhibitor phosphoramidon (1-100 nmol) produced a dose-dependent inhibition of gastric acid secretion in 2-h pylorus-ligated rats. The response resulted from a reduction in acid concentration and volume. Likewise, ic injection of another neutral endopeptidase-24.11 inhibitor Zincov (200 nmol) produced a 63% inhibition in gastric acid output. In contrast, neither intravenous injection of phosphoramidon (100 nmol) nor ic injection of the aminopeptidase inhibitor amastatin (100 nmol) produced any change in gastric acid secretion. The inhibitory effect of ic phosphoramidon (10 nmol) was not reversed by a dose of naloxone sufficient to antagonize the acid inhibitory effects of ic [D-Ala2-D-met5]enkephalinamide (8.5 nmol). Moreover, phosphoramidon-induced inhibition of acid was not reduced by the centrally effective bombesin antagonist N-acetyl-GRP(20-26)-O-CH3 or by reserpine pretreatment at a dose effective to antagonize ic neurotensin-induced inhibition in acid secretion. These results suggest that an endogenous neutral endopeptidase-24.11 sensitive substrate may act in the brain to inhibit gastric acid output by mechanisms independent of CNS opiate, bombesin or neurotensin activity.

Topics: Animals; Anti-Bacterial Agents; Drug Interactions; Gastric Acid; Gastrointestinal Hormones; Glycopeptides; Hydroxamic Acids; Injections, Intravenous; Injections, Intraventricular; Male; Naloxone; Neprilysin; Oligopeptides; Peptides; Rats; Rats, Sprague-Dawley; Reserpine