Enzyme assay

About: Enzyme assay is a research topic. Over the lifetime, 25406 publications have been published within this topic receiving 690706 citations.

Influence of age, hormones and germ cells on glutathione S-transferase activity in cultured Sertoli cells.

Abstract: Glutathione S-transferase (GSH-S-T) activity was measured, using 1-Cl-2,4-dinitrobenzene as substrate, in Sertoli cell cultures obtained from rats aged 10, 18, and 26 days. The GSH-S-T activity showed a significant increase with age of the Sertoli cell donor. When cultures were treated with hypotonic solution, in order to eliminate residual contaminating germ cells, the age dependent increase in enzyme activity was less pronounced. FSH, but not testosterone, increased enzyme activity in all cultures. Addition of freshly isolated germ cells (mainly pachytene spermatocytes) to hypotonic-treated Sertoli cell monolayers enhanced GSH-S-T activity at all ages. It is concluded that GSH-S-T activity can be measured in cultured Sertoli cells during the period of onset of spermatogenesis (10-26 days). This enzyme activity is dependent on age of the Sertoli cell donor and is influenced by FSH and germ cells. Since GSH-S-Ts are actively engaged in cell detoxificative functions through conjugation of xenobiotics with glutathione, the present findings suggest that this enzyme may have a relevant protective role during the critical period when spermatogenesis is being established.

Red blood cell glucose metabolism in human chronic fluoride toxicity.

Abstract: Fluoride is a well known inhibitor of many enzyme systems in vitro. The most widely studied classic example of fluoride inhibition is its potent inhibition of glycolysis, specifically its action on the enzyme enolase. Despite the plethora of in vitro studies on the effects of fluoride on the enzyme activity, there is a paucity of information concerning the in vivo metabolic lesions caused by the chronic toxic doses of fluoride in humans. The present study has been undertaken with a view to assess the changes in glucose metabolism and related enzymes in erythrocytes of humans consuming toxic doses of fluoride for prolonged periods.

Characterization of a novel, high-molecular weight, acidic, endothelin-1 inactivating metalloendopeptidase from the rat kidney.

Abstract: OBJECTIVE To characterize endothelin-1 inactivating peptidase (ET-1 peptidase) recently isolated from rat kidney. METHODS ET-1 peptidase was purified from the membranes of whole Wistar-Kyoto (WKY) rat kidneys using differential centrifugation, detergent solubilization, ion-exchange chromatography, ultrafiltration and preparative electrophoresis. The enzyme activity in the presence of increasing concentrations of unlabelled peptides, inhibitors and other substances was determined at pH 5.5 and 37 degrees C using fixed amounts of [125I]-ET-1 as the substrate. RESULTS On non-denaturing gels, the purified enzyme migrated in the form of a compact, low-mobility (Rf 0.07), high relative molecular mass (approximately 250,000) protein band. During denaturing polyacrylamide gel electrophoresis this protein separated into three fractions with apparent relative molecular masses 158,000, 110,000 and 61,000. Using different buffers, the optimum pH for this enzyme was found to be 5.5. Zinc (3.7 mmol/l), nickel (4.0 mmol/l), citrate (0.6 mmol/l), phosphate (1.3 mmol/l) and barbital ions (2.5 mmol/l) inhibited ET-1 peptidase activity by 50%, whereas magnesium, calcium, cobalt, manganous, sodium and borate ions were without effect. The most powerful inhibitors of the enzyme included: phenanthroline [median inhibitory concentration (IC50) 28 mumol/l], phosphoramidon (IC50 8.0 nmol/l), thiorphan (IC50 32 nmol/l) and N-carboxymethyl-Phe-Leu (IC50 12 mumol/l). Also, bacitracin (25 mumol/l), cyclosporine A (20 mumol/l) and sodium dodecyl sulphate (0.5%) inhibited enzyme activity by 50%, whereas bestatin, puromycin, aprotinin, phenylmethylsulphonyl fluoride, amanitin (50-100 mumol/l) and cardiotoxin (25 micrograms/assay) had no effect. The Michaelis constant (Km) values of 70 and 66 nmol/l were found towards ET-1 and the ET(16-21) fragment, respectively, whereas the Km values in respect to big-ET-1, sarafotoxin S6b, sulphated cholecystokin octapeptide, gastrin, glucagon, insulin, gastric inhibitory peptide and growth hormone ranged from 1.5 to approximately 50 mumol/l. The enzyme showed no apparent affinity for enkephalins, bradykinin, angiotensins, cholecystokinin tetrapeptides and kyotorphin. CONCLUSIONS The present data suggest that the ET-1 peptidase that we isolated from rat kidney displays inhibitory characteristics similar to that of other known metalloendopeptidases. However, this enzyme exhibits several unique properties such as high molecular mass, an apparent complex subunits structure, pH optimum at 5.5, and very high substrate specificity towards ET-1 and the ET(16-21) fragment compared with other peptides either related or unrelated to endothelin.

Phenyl and pyridyl LTA4H modulators

Abstract: Leukotrfene A4 hydrolase (LTA4H) inhibitors, compositions containing them, and methods of use for the inhibition of LTA4H enzyme activity and the treatment, prevention or inhibition of inflammation and inflammatory conditions.