Measurement and kinetic study of the formation of adrenaline sulphate in vitro.

The sulpho-conjugation of [14C]adrenaline form inorganic sulphate and ATP or preformed adenosine 3'-phosphate 5'-sulphatophosphate was demonstrated in the high-speed supernatant prepared from the liver and small intestine of various animals. Hydrolysis with sulphatase indicated the sulphate nature of the conjugate. The overall sulphation reaction has a pH optimum of 9.0. Maximal activity was obtained with a ratio of ATP/Mg2+ of 1 at 4--6mM. Above their optimal concentrations, ATP and Mg2+, separately or in combination, were inhibitory. Dithiothreitol at 3 mM stimulated the reaction by about 30%. The Km for adrenaline, determined by the sulphotransferase reaction and by the three-step (sulphate-activating and sulphotransferase) reactions was 125 micrometer. The rate of synthesis of [14C]-adrenaline sulphate, expressed in pmol/min per mg of protein for the livers of dog, monkey, rat, guinea pig and rabbit were, respectively, 144, 77, 47, 11 and 6. The corresponding values for the small intestines of dog and monkey were 60 and 62. Brain and heart tissues showed no measurable activity.     

THE BIOSYNTHESIS OF 3-METHOXY-4-HYDROXYPHENYLGLYCOL SULPHATE BY LIVER AND BRAIN

—3-Methoxy-4-hydroxyphenylglycol (MHPG) formed a sulphate conjugate when incubated with ATP, Mg²⁺ ions, Na₂³⁵SO₄ and the high-speed supernatant preparations of rabbit or rat brain. The same reactions could be catalysed by similar enzyme preparations from liver. The sulphated product was separated and identified by paper chromatography. On acid hydrolysis, it released both Na₂³⁵SO₄ and the free glycol. The measurement of this labelled sulphate was used as a specific assay procedure for determining the overall sulphoconjugatory process. The pH optimum of the reaction is 7.8. For rabbit brain, the K_m for Na₂SO₄ determined for the activating system is 3.6 × 10⁻⁴m , and that for MHPG for the sulphotransferase reaction is 1.05 × 10⁻⁴m . The specific enzyme activity, expressed as nmol ³⁵SO₄ incorporated/h/mg protein for a 30-min assay is as follows: rat brain, 2.8; rabbit brain, 1.6; rat liver, 33.4and rabbit liver, 15.0. Dithiothreitol at 3 mm concentration had no significant effect on the sulphation of MHPG in all these preparations.     

OCCURRENCE OF A SEROTONIN SULPHOTRANSFERASE IN THE BRAIN

Abstract— —An enzyme catalysing the transfer of sulphate from 3'-phosphoadenylsulphate to serotonin was purified from rabbit brain. The purification procedure involved ammonium sulphate fractionation of the 200,000 g supernatant of rabbit brain homogenate, treatment with alumina Cγ, and chromatography on DEAE-cellulose. The enzyme was purified 67-fold from the 200,000 g supernatant of the brain homogenate. The intracranial distribution of the sulphotransferase was investigated and the cerebellum found to have rather high activity. The sulphotransferase activities of rabbit, dog, rat and bovine brains were compared; rabbit brain had the highest activity, followed by dog, rat and bovine brain.     

BIOGENIC AMINES AND THEIR METABOLITES AS SUBSTRATES FOR PHENOL SULPHOTRANSFERASE (EC 2.8.2.1) OF BRAIN AND LIVER

Phenol sulphotransferase activity in homogenates of rat liver and brain was determined spectrophotometrically. Rat liver had about 100-fold more phenol sulphotransferase activity than brain; however, both tissues showed about the same spectrum of activity towards the phenolic compounds tested. Dopamine and its acidic and neutral metabolites and the neutral metabolites of norepinephrine were the compounds most readily sulphury-lated in vitro. They were also the compounds most readily sulphurylated in vivo when they were injected intraventricularly together with labelled Na₂SO₄. When labelled Na₂SO₄ was injected alone, we detected conjugation of endogenous phenols. One of the compounds formed was identified by its chromatographic characteristics as 3-methoxy-4-hydroxy-phenylethyleneglycol sulphate. We detected other conjugates which appeared to be the sulphate esters of 3,4-dihydroxyphenylethyleneglycol; 3,4-dihydroxyphenylacetic acid; and homovanillic acid. In brain, sulphate conjugation may be a major route of metabolism for many of the phenolic compounds related to the biogenic amines and possibly for phenolic drugs which enter the brain.     

Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C₁₈ columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.     

The utilization of alanine, glutamic acid, and serine as amino acid substrates for glycine N-acyltransferase

The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs.     

Comparison in different species of biliary bilirubin-IX alpha conjugates with the activities of hepatic and renal bilirubin-IX alpha-uridine diphosphate glycosyltransferases.

The bilrubin-IXalpha conjugates in bile and the activities of bilirubin-IX alpha--UDP-glycosyltransferases in liver and kidney were determined for ten species of mammals and for the chicken. 1. In the mammalian species, bilirubin-IX alpha glucuronide was the predominant bile pigment. Excretion of neutral glycosides was unimportant, except in the cat, the mouse, the rabbit and the dog, where glucose and xylose represented 12--41% of total conjugating groups bound to bilirubin-IX alpha. In chicken bile, glucoside and glucuronide conjugates were of equal importance. They probably represent only a small fraction of the total bile pigment. 2. The transferase activities in liver showed pronounced species variation. This was also apparent with regard to activation by digitonin, pH optimum and relative activities of transferases acting on either UDP-glucuronic acid or neutral UDP-sugars. 3. Man, the dog, the cat and the rat excrete bilirubin-IX alpha largely as diconjugated derivatives. In general, diconjugated bilirubin-IX alpha could also be synthesized in vitro with liver homogenate, bilirubin-IX alpha and UDP-sugar. In contrast, for the other species examined, bilirubin pigments consisted predominantly of monoconjugated bilirubin-IX alpha. Synthesis in vitro with UDP-glucuronic acid, UDP-glucose or UDP-xylose as the sugar donor led exclusively to the formation of monoconjugated bilirubin-IX alpha. 4. The transferase activities in the kidney were restricted to the cortex and were important only for the rat and the dog. No activity at all could be detected for several species, including man. 5. Comparison of the transferase activities in liver with reported values of the maximal rate of excretion in bile suggests a close linkage between conjugation and biliary secretion of bilirubin-IX alpha.     

Kinetics and mechanism of the rat brain phenol sulphotransferase reaction.

Some properties of rat brain phenol sulphotransferase were investigated in in vitro at pH7.4. The enzyme was purified 10-fold by chromatography on DEAE-Sephadex -50. It can be assayed with 4-hydroxy-3-methoxyphenylethylene glycol or 4-methylumbelliferone as the sulphate acceptor. The partially purified enzyme is stable for at least 1 week when stored at 4 degrees C. It is, however, additionally activated (10--20%) and stabilized by 1 mM-dithiothreitol. The activity of the enzyme does not depend on the addition of exogenous Mg2+. The pH optima for the sulphation of 4-hydroxy-3-methoxyphenylethylene glycol and 4-methylumbelliferone are 7.8 and 7.4 respectively. Substrate inhibition by the sulphate acceptor is apparent at concentrations over 0.05mM. Initial-velocity studies in the absence and presence of product and dead-end inhibitors suggested that the mechanism of the rat brain sulphotransferase reaction is sequential ordered Bi Bi with a dead-end complex of enzyme with adenosine 3',5'-biphosphate and sulphate acceptor. The sulphate donor adenosine 3'-phosphate 5'-sulphatophosphate is the first substrate that adds to the enzyme, and the sulphate acceptor is the second substrate. The dissociation constant for the complex of enzyme with sulphate donor is 21 micron. The sulphated substrate is the first product and adenosine 3',5'-biphosphate is the second product that leaves the enzyme.     

The conjugation of tyramine with sulphate by liver and intestine of different animals.

Tyramine was conjugated with sulphate by extracts of monkey intestine and livers of monkey, rat, mouse, guinea pig and man. The activity measured in monkey intestine was almost three times that of monkey liver. Labelled tyramine sulphate synthesized from [14C] tyramine, [3H] tyramine or Na235SO4, on acid hydrolysis, released its radioactive precursor. Liver extracts of monkey, rat, mouse and guinea pig synthesized respectively 145,66,21 and 6 pmol of [14C] tyramine sulphate/min per mg of protein. Except with the monkey, intestine exhibited very low activity. trans-2-Phenylcyclopropylamine, a monoamine oxidase inhibitor, was added as a routine to the enzyme preparation, as its omission resulted in the production of p-hydroxyphenylacetic acid in appreciable amounts. This oxidative deamination of tyramine, however, did not decrease the sulpho-conjugation of tyramine. The low Km (9.1 muM) of sulphotransferase for tyramine is probably responsible.     

The distribution of phosphofructokinase isoenzymes in the liver of camel, rat and rabbit

1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 2. Only a single phosphofructokinase isoenzyme is present in the camel liver which has shown different physical and regulatory properties from the isoenzymes of rat and rabbit liver. 3. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 4. Rabbit liver phosphofructokinase was slightly more anodic than the rat enzyme, whereas the camel enzyme was the least anodic as shown by the techniques of DEAE-cellulose chromatography and cellulose acetate electrophoresis. 5. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent Km values were 0.58, 0.45 and 0.82 mM respectively.     

Studies on the kynurenine adminotransferase activity in rat liver and kidney.

The kynurenine aminotransferase activity of supernatant and mitochondrial fractions obtained from rat liver and kidney was studied with L-kynurenine and L-3-hydroxykynurenine as substrates. A substrate inhibition with L-kynurenine at concentrations higher than 6-7mM was observed with all four enzyme preparations. This did not happen with L-3-hydroxykynurenine as a substrate. Moreover, the liver mitochondrial enzyme shows a Km for pyridoxal phosphate 2-4 times smaller than the other preparations when assayed with L-3-hydroxykynurenine as a substrate. Therefore, the accumulation of xanthurenic acid and not of kynurenic acid in B6 deficiency could be related both to this high activity of liver mitochondrial kynurenine aminotransferase with L-3-hydroxykynurenine, even at small concentrations of B6, and to substrate inhibition observed with L-kynurenine and not with L-3-hydroxykynurenine.     

A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.

1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Studies on the meta and para O-sulphation of the catechol compound 3,4-dihydroxybenzoic acid by rat liver sulphotransferase in vitro.

The enzymic meta and para O-sulphation of 3,4-dihydroxybenzoic acid was investigated in vitro with a dialysed high-speed supernatant from rat liver. The O-sulphated products were identified by comparison with the reference compounds. The chemical synthesis and identification of the reference O-sulphate esters is described in detail. The sulphotransferase activity of the dialysed supernatant from rat liver towards 3,4-dihydroxybenzoic acid was 580 pmol of 3-O-sulphate and 120 pmol of 4-O-sulphate formed/min per mg of protein at the optimal pH of 7.4. The meta/para ratio of O-sulphation was independent of pH, time of incubation, concentration of enzyme and presence of dithiothreitol. The O-sulphate esters of 3,4-dihydroxybenzoic acid were found to be good substrates for the arylsulphatase reaction at pH 5.6. The arylsulphatase activity of a dialysed preparation from rat liver was 4.0 nmol of 3-O- and 5.7 nmol of 4-O-sulphate ester hydrolysed/min per mg of protein, respectively. Arylsulphatase from Helix pomatia had an activity of 620 pmol of 3-O-sulphate and of 16.6 nmol of 4-O-sulphate ester hydrolysed/min per unit (mumol/h) of sulphatase.     

THE NATURE OF SULPHATION OF URONIC ACID-CONTAINING GLYCOSAMINOGLYCANS CATALYSED BY BRAIN SULPHOTRANSFERASE

—A sulphotransferase system of rat brain catalyses the transfer of sulphate from 3′-phosphoadenosine 5′-phosphosulphate to the low-sulphated glycosaminoglycans isolated from normal adult human brain. These were shown to be precursors of higher-sulphated glycosaminoglycans by DEAE-Sephadex column chromatography and paper electrophoresis. Nitrous acid degradation and mild acid hydrolysis of enzymically-sulphated fractions further confirmed the presence of heparan sulphate in human brain. A partially purified sulphotransferase preparation was obtained from neonatal human brain using chondroitin-4-sulphate as sulphate acceptor. This sulphotransferase catalyses the transfer of sulphate to the various uronic acid containing glycosaminoglycans. Heparan sulphate was the best sulphate acceptor followed by dermatan sulphate, N-desulphoheparin, chondroitin-4-sulphate and chondroitin-6-sulphate in decreasing order. Sulphotransferase obtained from 1-day-old rat, rabbit and guinea pig brain also had the same pattern of specificity towards various sulphate acceptors. This sulphotransferase catalyses both N-sulphation and O-sulphation. Studies on the sulphotransferase obtained from both rat and human brain of various age groups indicate that the ratio of N-sulphation: O-sulphation decreases as the brain matures.     

The transfer of estrone glucuronide to bile in an isolated perfused rat liver system.

An isolated rat liver perfusion system has been utilized in a study of the biliary excretion of estrone glucuronide. The estrogen was initially shown to be excreted without prior metabolism. Disappearance from the medium was rapid and biliary concentrations exceeded that in the medium by more than a thousand-fold. Disappearance rates were decreased when medium estrone glucuronide concentrations exceeded 0.29 mM. Inhibition by other steroidal conjugates, testosterone glucuronide, 2-methoxyestrone (3-hydroxy-2-methoxy-estra-1,3,5(10)-trien-17-one glucuronide and 2-hydroxyestrone (2,3-dihydroxyestra-1,3,5(10)-trien-17-one) glutathione, was also demonstrated. Phenolphthalein glucuronide, at 10 times the molar concentration of estrone glucuronide, did not affect the medium clearance of the latter compound. These findings indicate the possibility of utilizing this system for further studies of possible interactions by other organic compounds for excretion via the biliary route.     

Androgen glucuronides: I. Direct formation in rat accessory sex organs

A simple one-step procedure is described on the isolation of androgen glucuronides from various rat tissues. This procedure uses polyacrylamide gel electrophoresis, and permits a quantitative isolation of a single band containing the total androgen glucuronides without the contamination of free androgens and androgen sulfates. This procedure was used to determine the ability of various tissues of the rat to form androgen glucuronides directly when they were incubated with 1,2-[³H]-testosterone (0.1 μM) $\text{in}$$\text{vitro}$. Of eleven organs studied, only the accessory sex organs (ventral prostate, seminal vesicle, and coagulating gland), liver, and kidney were capable of forming androgen glucuronides. At the end of a one-hour incubation period, approximately 1% of the total radiolabeled steroids in the prostatic tissue minces were in the form of glucuronide conjugates. The predominant androgen glucuronide formed in the accessory sex organs was 5α-androstane-3α,17β-diol 17β-d-glucuronide. This is in contrast to the rat liver and kidney in which testosterone glucuronide was the predominant conjugate.A similar amount of labeled glucuronide conjugates was formed from either [³H]-testosterone, [³H]-dihydrotestosterone or [³H]-androstenedione, whereas negligible amount of steroid conjugates was formed from [³H]-cortisol. The formation of androgen glucuronides requires metabolically active tissues; furthermore, the conjugation process was inhibited by the antiandrogen, cyproterone acetate, or by metabolic inhibitors, such as oligomycin or N-ethylmaleimide.     

Studies on the sulphation of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds by rat tissues

The formation of sulpho-conjugates of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds was examined in preparations of rat tissues. Liver high-speed-supernatant preparations readily transferred sulphate from adenosine 3'-phosphate 5'-sulphato-phosphate to dopamine under standard conditions. The main product was identified as the 3-O-sulphate. The preparation also sulphated the 3- and 4-methoxy derivatives but to a lesser extent (44% and 95% respectively) relative to dopamine. Brain preparations possessed only half the activity of liver but formed both the 3- and 4-O-sulphates in the molar ratio of 1.7:1. l-3,4-Dihydroxyphenylalanine (l-dopa) in both tissue preparations did not yield any significant amount of sulpho-conjugate when the dopa decarboxylase present was inhibited. The sulphotransferase activity of preparations was doubled in the presence of dithiothreitol and it was concluded that l-tyrosine methyl ester sulphotransferase was the enzyme involved. A method for the preparation of authentic dopamine 3-O-sulphate and 4-O-sulphate was developed.     

A Spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH₂ and oxygen. Activity with NADH₂ was one-third of that with NADPH₂. 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at −12°. Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2′-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.