Sulfation Of P-nitrophenol Research Articles

P-Nitrophenol sulfation in rat liver cytosol: multiple forms and substrate inhibition of aryl sulfotransferase.

The p-nitrophenol (PNP) sulfate conjugation rate in rat liver cytosol at pH 7.4 under 10-30 microM 3'-phosphoadenosine-5'-phosphosulfate (PAPS) was decreased at 10-200 microM PNP and increased again at more than 200 microM PNP. The higher the PAPS concentration, the more remarkable such substrate inhibition. These results indicate that the substrate inhibition was due to the direct interaction of PNP with the aryl sulfotransferase (phenol sulfotransferase, PST). The PST reaction was thermolabile at less than 20 microM PNP but thermostable at more than 200 microM PNP. The Km values for PAPS obtained in the PST reactions where the inhibition was not observed was about 23 microM at 2.5-5 microM PNP and 11-14 microM at more than 200 microM PNP. And the Km values for PNP at more than 500 microM PNP was 2.2-2.6 mM. On the other hand, the inhibition in the PST reaction at pH 5.6 was observed at much higher PNP concentration, approximately 200 microM, than that at pH 7.4. Based on these characteristic results, the correspondence of the PST catalyzing the PNP sulfate conjugation (pH 7.4) at the low and high concentrations of PNP in the liver cytosol with the multiplicity obtained in the purified preparation of the liver previously reported by another researchers was compared and discussed. Finally the selection of an endogeneous substrate for estimating such complexed sulfate conjugation in advance in each individual man was also proposed.

P-Nitrophenol sulfate conjugation with substrate inhibition in rat liver cytosol fraction.

p-Nitrophenol (PNP) sulfate conjugation in rat liver cytosol fraction was investigated over a wide PNP concentration range (1.25 μM-5 mM). The PNP sulfate synthesis rate in the liver cytosol decreased in the low PNP concentration range (10-500 μM) and increased again at concentrations above approximately 500 μM, as was previously found in isolated liver cells (J. Pharm. Dyn., 5, 811 (1982)). This apparent substrate at low PNP concentration was found in the sulfotransferase reaction when the cosubstrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), had been generated in advance as well as in the overall reaction (i.e., including the generation process of PAPS), suggesting that the inhibition was due to direct interaction between PNP and aryl sulfotransferase, and not to inhibition of PAPS generation by PNP.

Correlation of drug conjugative metabolism rates between in vivo and in vitro: glucuronidation and sulfation of p-nitrophenol as a model compound in rat.

The correspondence of conjugative metabolism rates in vivo and in vitro was studied in rat using p-nitrophenol (PNP) as a model compound. In PNP-glucuronide conjugation, the hepatic intrinsic clearance calculated from Km and Vmax obtained in the isolated liver cells was approximately three times larger than that calculated by the in vivo blood concentration on the basis of the linear pharmacokinetic concepts, but this difference was not considered essential. On the other hand, in PNP-sulfate conjugation, some inhibition in the isolated liver cells, which was not expected in the in vivo blood concentration, was found at more than 5 microM PNP. Such inhibition mechanism could not be elucidated by the inorganic sulfate concentration in the reaction medium. Accordingly, it was suggested that some unknown reaction mechanism still remained to be studied for the applicability of the conjugation rates in the isolated liver cells, especially sulfation rates, to the pharmacokinetic study in vivo.

Purification and properties of aryl sulfatases from rabbit sperm acrosomes.

SummaryThe extraction of rabbit spermatozoa with the MgCl2 treatment, followed by chromatography of extracts on a DEAE-cellulose column, yield highly purified aryl sulfatases. The enzyme preparation showed one major band with the possibility of a minor contaminant by acrylamide gel electrophoresis. The single band had two activities, that of aryl sulfatases A and B. Aryl sulfatase C was not detectable. The purified aryl sulfatases had optima at pH's 4.8, 5.6, and 6.0. The enzymes exhibited no activity at 4° but exhibited. higher activity at 50° than at 37°. The pure aryl sulfatases had higher affinity for p-nitrocatechol sulfate than for p-nitrophenol sulfate. Phosphate and sulfite ions inhibited the enzymes, but NaCl, KCl, and sulfate did not. Rabbit, ram, bull, and boar sperm acrosomal extracts showed high aryl sulfatase activities.The authors thank Mrs. Diane Fitzgerald for her skillful technical assistance.

The effect of some cultural conditions on the arylsulfatase of Proteus rettgeri.

The influence of temperature, pH, and sulfur source on arylsulfatase formation in P. rettgeri was investigated. The highest level of enzyme activity was found when the organism was grown at 28 °C in a glycerol–salts medium (pH 7.0) containing 2.7 mM methionine. Low levels of enzyme activity were found when the organism was grown in the presence of 0.8 and 1.5 mM sulfate but 3.0 and 9.0 mM sulfate repressed enzyme formation.Conditions optimal for the formation of enzyme active against p-nitrophenol sulfate were not identical with those optimal for formation of enzyme active against nitrocatechol sulfate.The findings of this work are compared to similar findings in other bacterial systems.