ABSTRACT
3'-Phosphoadenosine 5'-phospho[35S]sulfate (PAPS) biosynthesized from inorganic [35S]sulfate and ATP was separated from its radiolabeled precursor by reversed-phase paired-ion HPLC and quantified by on-line radiometric detection. This single-step procedure circumvents several problems inherent in conventional sulfotransferase-coupled assays employed in the measurement of PAPS formation. A good correlation was observed between the rate of PAPS generation assayed in several mammalian tissues measured by direct HPLC-radiometry and by coupling to the sulfation of minoxidil or 4-methylumbelliferone. Both AMP and ADP inhibited the rat liver sulfate-activating enzymes competitively with respect to MgATP2-, and the rate of PAPS production was decreased with decreasing ratios of [ATP]:[ADP] and [ATP]:[AMP]. It is possible that these adenine nucleotides regulate sulfate activation by kinetic control and by negative feedback modulation.
Subject(s)
Phosphoadenosine Phosphosulfate/analysis , Phosphoadenosine Phosphosulfate/biosynthesis , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Chromatography, High Pressure Liquid/methods , Guinea Pigs , Haplorhini , Humans , In Vitro Techniques , Kinetics , Liver/enzymology , Mice , Nucleotides/pharmacology , Radiometry/methods , Rats , Sulfates/metabolism , SulfotransferasesABSTRACT
1. Amine N-sulphotransferase (NST) activity with desipramine (DMI) as substrate was assayed in vitro in various areas of the rat brain. Biosynthesis of 3'-phosphoadenosine-5'-phospho35sulphate (PAPS) from sodium 35sulphate and ATP was also measured by coupling it to the sulphation of minoxidil by minoxidil sulphotransferase (MST). 2. For the DMI-NST reaction, an apparent Km = 0.5 mM was obtained for DMI and two apparent Kms = 0.3 and 1.7 microM for PAPS, whereas in the PAPS-generating reactions, Km for sodium 35sulphate = 20 microM. 3. Both the enzyme activities were widely distributed in rat brain. The rate of NST activity was 2-3 orders of magnitude lower than that of PAPS generation. N-sulphoconjugation of DMI, which is proposed as a possible biotransformation pathway of DMI in the rat brain, could conceivably be supported adequately by the 'active sulphate' generated within the same areas of the brain.
Subject(s)
Antidepressive Agents, Tricyclic/pharmacokinetics , Brain/metabolism , Desipramine/pharmacokinetics , Adenosine Triphosphate/metabolism , Animals , Biotransformation , Dopamine/pharmacology , Kinetics , Male , Minoxidil/pharmacokinetics , Nerve Tissue Proteins/metabolism , Nitrophenols/pharmacology , Phosphoadenosine Phosphosulfate/metabolism , Rats , Rats, Wistar , Sulfates/metabolism , Sulfotransferases/metabolism , Sulfur Radioisotopes , Vasodilator Agents/pharmacokineticsABSTRACT
A rapid and sensitive radiometric assay was developed for the measurement of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis in rat skin extract. The formation of PAP35S from sodium 35sulfate and ATP was quantified by the transfer of the 35sulfate to minoxidil by rat liver minoxidil sulfotransferase (MST). The assay is sensitive enough for the detection of as little as 2 pmol of PAP35S. The PAPS-generating system showed a pH optimum of 8.6, with an apparent Km value of 1 mM for the ATP-Mg2+ complex and 68 microM for sodium 35sulfate. ATP and Mg2+, present individually or together in equimolar concentrations, were inhibitory above 8 mM. Excess (or free) ATP was a competitive inhibitor with respect to the ATP-Mg2+ complex; the apparent Ki measured was 0.32 mM. The specific activity of the PAPS-generating system, measured in rat skin cytosol was 0.15 nmol PAPS/min/mg protein. The importance of PAPS generation in detoxification and bioactivation of xenobiotics in skin is discussed.
Subject(s)
Phosphoadenosine Phosphosulfate/biosynthesis , Skin/metabolism , Adenosine Triphosphate/pharmacology , Animals , Arylsulfotransferase/metabolism , Kinetics , Liver/enzymology , Rats , Rats, Wistar , Sulfotransferases/metabolism , Sulfur RadioisotopesABSTRACT
Minoxidil sulphotransferase (MST) activity was determined in the cytosolic fraction of rat skin and liver. MST of rat skin is similar to the P (phenol)-form of phenosulphotransferase (PST) of human tissues with respect to thermostability and inhibition by 2,6-dichloro-4-nitrophenol (DCNP). p-Nitrophenol, a prototype substrate of human P-PST form, inhibits MST at micromolar concentration while millimolar concentrations of dopamine and tyramine, substrates of human M-(monoamine)-PST, are required to elicit a similar degree of inhibition. The enzymatic transfer of 35S from sodium 35sulphate to minoxidil was also demonstrated suggesting that the rat skin is potentially capable of synthesizing 3'-phosphoadenosine-5'-phosphosulphate (PAPS) from inorganic sulphate and utilizing it for the biosynthesis of minoxidil sulphate, its active metabolite. Thus, it is conceivable that the pharmacological action of minoxidil as a promoter of hair growth could be carried out by the cutaneous tissues without the contribution of hepatic or other extrahepatic organs.
