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.     

Artifacts produced by acidic hydrolysis of lipids containing 3-hydroxyalkanoic acids

After acidic hydrolysis of lipid A preparations from pseudomonads, products containing both hydroxy and nonhydroxy fatty acids were obtained. The major products were alkanoate esters of the hydroxy acids. Similar compounds were formed when a 3-hydroxy acid was heated with a nonhydroxy acid under the same conditions.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

The human FA2H gene encodes a fatty acid 2-hydroxylase

2-Hydroxysphingolipids are a subset of sphingolipids containing 2-hydroxy fatty acids. The 2-hydroxylation occurs during de novo ceramide synthesis and is catalyzed by fatty acid 2-hydroxylase (also known as fatty acid alpha-hydroxylase). In mammals, 2-hydroxysphingolipids are present abundantly in brain because the major myelin lipids galactosylceramides and sulfatides contain 2-hydroxy fatty acids. Here we report identification and characterization of a human gene that encodes a fatty acid 2-hydroxylase. Data base searches revealed a human homologue of the yeast ceramide 2-hydroxylase gene (FAH1), which we named FA2H. The FA2H gene encodes a 372-amino acid protein with 36% identity and 46% similarity to yeast Fah1p. The amino acid sequence indicates that FA2H protein contains an N-terminal cytochrome b5 domain and four potential transmembrane domains. FA2H also contains the iron-binding histidine motif conserved among membrane-bound desaturases/hydroxylases. COS7 cells expressing human FA2H contained 3-20-fold higher levels of 2-hydroxyceramides (C16, C18, C24, and C24:1) and 2-hydroxy fatty acids compared with control cells. Microsomal fractions prepared from transfected COS7 cells showed tetracosanoic acid 2-hydroxylase activities in an NADPH- and NADPH: cytochrome P-450 reductase-dependent manner. FA2H lacking the N-terminal cytochrome b5 domain had little activity, indicating that this domain is a functional component of this enzyme. Northern blot analysis showed that the FA2H gene is highly expressed in brain and colon tissues. These results demonstrate that the human FA2H gene encodes a fatty acid 2-hydroxylase. FA2H is likely involved in the formation of myelin 2-hydroxy galactosylceramides and -sulfatides.     

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg₂⁺ and purported to involve residues D₈₇GD₈₉N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D₈₇ and D₈₉ did not hamper ATPS activity however abolished APSK activity severely. Thus, D₈₇GD₈₉N region is required for stabilization of Mg²⁺-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma³²P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg²⁺ and APS of wild type and mutants supports the experimental results.     

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.     

THE SULPHATION OF VANILYLMANDELIC ACID AND HOMOVANILLIC ACID BY LIVER, SMALL INTESTINE AND BRAIN

Abstract— The sulpho-conjugates of vanilylmandelic acid (VMA) and homovanillic acid (HVA) were biosynthesized in vitro from ATP and sodium sulphate and from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) obtained commercially or generated in vitro . Hydrolysis with sulphatase indicated the sulphate nature of these conjugates. In decreasing order of activity, the liver, small intestine and brain of various animals exhibited this sulpho-conjugatory ability. The K _m, values of VMA and HVA for the sulphotransferase of mouse liver were, respectively, 1740 and 93 μM. Competition studies suggested that the same enzyme may be involved in the sulphation of these two acids.     

Influence of thyroid on formation of myelin lipids

—The formation of lipids found primarily in the myelin sheath was investigated in rats made hypothyroid, at birth. A marked reduction in cerebroside, sulphatide, and cholesterol of brains was found in 18-day-old hypothyroid animals. These lipids were also reduced, although to a lesser degree, in 30-day-old ex-hypothyroid animals allowed to return to the euthyroid state at age 18 days. The onset of formation of sulphatide in vivo was delayed in the hypothyroid animals. Sulphatide formation reached a peak at a later time and was greatly reduced in comparison to control animals. The activity in vitro of galactolipid sulphotransferase, which forms sulphatide from cerebroside and PAPS, was reduced only when exogenous PAPS was not added to the assay medium. This finding suggests a defect in the formation of PAPS from ATP and sulphate in the hypothyroid brain. In addition, T₃ (tri-iodothyronine) had a stimulatory effect in vivo of formation brain sulphatide when administered to rats during the first 5 days of life.     

Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

High resolution nuclear magnetic resonance spectroscopy of glycosphingolipids. I: 360 MHz 1H and 90.5 MHz 13C NMR analysis of galactosylceramides

The high resolution ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of galactosylceramides containing n-fatty acids and α-hydroxy fatty acids were recorded in dimethylsulfoxide solution with and without addition of D₂O. From the coupling constants of the sugar ring protons, a ⁴C₁ conformation can be deduced. In contrast to the conformation in aqueous solution, the C⁶ hydroxymethylene group is freely rotating around the C⁶C⁵ bond. In the ceramide residue all signals produced by protons linked to carbons bearing electronegative substituents could be attributed. The large difference in coupling constants of the methylene protons of C^1′ to the C^2′ methine proton of the sphingosine indicates a restricted rotation around the C^1′C^2′ bond. The assignments of the hydroxy and amino protons follow from the decoupling of the corresponding methine protons.     

FATTY ACIDS AND HYDROXY FATTY ACIDS IN THREE SPECIES OF FRESHWATER EUSTIGMATOPHYTES

The monocarboxylic fatty acids and hydroxy fatty acids of three species of freshwater microalgae—Vischeria punctata Vischer, Vischeria helvetica (Vischer et Pascher) Taylor, and Eustigmatos vischeri (Hulbert) Taylor, all from the class Eustigmatophyceae— were examined. Each species displayed a very similar distribution of fatty acids, the most abundant of which were 20:5n-3, 16:0, and 16:1n-7; C₁₈ polyunsaturated fatty acids were minor components. These fatty acid distributions closely resemble those found in marine eustigmatophytes but are quite distinct from those found in most other algal classes. These microalgae also contain long-chain saturated and unsaturated monohydroxy fatty acids. Two distinct types of hydroxy fatty acids were found: a series of saturated α-hydroxy acids ranging from C₂₄ to C₃₀ with a shorter series of monounsaturated α-hydroxy acids ranging from C₂₆ to C₃₀ together with a series of saturated β-hydroxy acids ranging from C₂₆ to C₃₀. The latter have not previously been reported in either marine or freshwater microalgae, although C₃₀ to C₃₄ midchain (ω-18)-hydroxy fatty acids have been identified in hydrolyzed extracts from marine eustigmatophytes of the genus Nannochloropsis, and C₂₂ to C₂₆ saturated and monounsaturated α-hydroxy fatty acids have been found in three marine chlorophytes. These findings have provided a more complete picture of the lipid distributions within this little studied group of microalgae as well as a range of unusual compounds that might prove useful chemotaxonomic markers. The functions of the hydroxy fatty acids are not known, but a link to the formation of the lipid precursors of highly aliphatic biopolymers is suggested.     

IN VIVO METABOLISM OF [G-3H]LIGNOCERIC ACID IN DEVELOPING RAT BRAIN1

Abstract— [G-³H]Lignoceric acid (tetracosanoic acid) was injected into the brains of 20-day-old rats, and the animals were killed after 8, 24, or 72 h. Various lipids were isolated from these brains, and the distribution of radioactivity was determined. The injected free acid rapidly disappeared, and the radioactivity was incorporated into varying chain-length nonhydroxy- and hydroxy saturated fatty acids of sphingolipids and phospholipids. Little radioactivity was found in unsaturated acids, sphingo-sine, and cholesterol. A time-dependent shift of the label among various fatty acids was relatively small 8 h after injection, probably because of the metabolic stability of the brain sphingolipids. In cerebrosides, the radioactivity was equally distributed between nonhydroxy and x-hydroxy fatty acids of all chain lengths. C₂₃ and C₂₂ fatty acids contained equal total radioactivities; C₂₃ and C₂₄ fatty acids contained similar specific activities. These results confirm the significant role of a-hydroxylation and 2-oxidation in the synthesis of very long-chain fatty acids in brain. In total lipid fatty acids, docosanoic acid (22:0) contained more radioactivity than its α-oxidation precursor, α-hydroxytricosanoic acid (23h:0) at all times. In sphingolipid fatty acids, the specific activity of 21:0 was always higher than that of its ct-oxidation precursor 22:0. These observations indicate that part of the 22:0 and 21:0 was derived by β-oxidation from the injected lignoceric acid or its α-oxidation product, respectively.     

The enzymatic sulphation of heparan sulphate by hen's uterus

A sulphotransferase preparation from hen's uterus catalysed the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to N-desulphated heparan sulphate, heparan sulphate, N-desulphated heparin and dermatan sulphate. Heparin, chondroitin sulphate and hyaluronic acid were inactive as substrates for the enzyme. N-desulphated heparin was a much poorer substrate for the enzyme than N-desulphated heparan sulphate suggesting that properties of the substrate other than available glucosaminyl residues influenced enzyme activity. N-acetylation of N-desulphated heparin and N-desulphated heparan sulphate reduced their sulphate acceptor properties so it was unlikely that the N-acetyl groups of heparan sulphate facilitated its sulphatiion. Direct evidence for the transfer of [³⁵S]sulphate to amino groups of N-desulphated haparan sulphate was obtained by subsequent isolation of glucosamine $\text{N-[}{}^{35}\text{S}\text{]}\text{sulphate}$ from heparan [³⁵S]sulphate product. This was made possible through the use of a flavobacterial enzyme preparation which contained “heparitinase” activity but had been essentially freed of sulphatases. Attempts to transfer [³⁵S]sulphate to glucosamine or N-acetylglucosamine were unsuccessfull.     

A method for fractionation of cerebrosides into classes with different fatty acid compositions

A method is described for the separation of beef brain cerebrosides into three fractions containing different classes of fatty acids: nonhydroxy (I), unsaturated nonhydroxy (II), and hydroxy fatty acid cerebrosides (III). The procedure consists of benzoylation of either crude or purified cerebrosides, followed by column chromatographic separation of benzoylated derivatives containing nonhydroxy acids from those containing hydroxy fatty acids. The benzoyl groups are removed by sodium methoxide-catalyzed transesterification; from the reaction mixtures, fractions I and III precipitate. The fraction II present in mother liquor of I was shown to contain mainly short-chain and unsaturated nonhydroxy fatty acid cerebrosides. The fatty acid composition of each fraction was obtained by gas-liquid chromatography.     

Structure and composition of sulfatides isolated from livers of patients with metachromatic leukodystrophy: galactosyl sulfatide and lactosyl sulfatide

The livers of four patients with metachromatic leukodystrophy contained galactosyl sulfatide and lactosyl sulfatide, whereas these substances were undetectable in normal human liver. On the basis of methanolysis and permethylation studies, both sulfatides were shown to be substituted with sulfate at the C-3 position of the galactose moiety. Examination of the fatty acid compositions of these sulfatides showed that C(22:0) and higher 2-hydroxy and nonhydroxy fatty acids predominated in both. Both sulfatides contained the same long-chain bases, predominantly sphingosine, dihydrosphingosine, and phytosphingosine. Using as criteria the proportion of lactosyl sulfatide to galactosyl sulfatide, and the fatty acid and long-chain base compositions, the liver sulfatides from subjects with metachromatic leukodystrophy closely resemble those in the kidney and differ from those in brain and peripheral nerve.     

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.     

Hydroxy fatty acids in Bacteroides species: D-(--)-3-hydroxy-15-methylhexadecanoate and its homologs.

Acid hydrolysates of 140 strains, representing 11 species of the genus Bacteroides, were analyzed by capillary gas-liquid chromatography for total cellular fatty acid. All samples contained components which appeared to be hydroxy fatty acid. The relative amount and chain length distribution of the hydroxy fatty acids, as well as the nonhydroxy fatty acids, varied according to species. To characterize the presumed hydroxy acids, a composite of some 40 of these samples was analyzed by thin-layer and capillary gas-liquid chromatography, mass spectrometry, infrared spectrophotometry, and polarimetry. The hydroxy acids were shown to be of the D-(--)-3-hydroxy acid family. The predominant component was the iso-branched D-(--)-3-hydroxy-15-methylhexadecanoic acid. Lesser amounts of the iso-branched 15-carbon, straight-chain 16-carbon, and anteiso-branched 17-carbon acids were also found.     

THE DISTRIBUTION OF LIPIDS IN THE HUMAN NERVOUS SYSTEM-V. GANGLIOSIDES AND ALLIED NEUTRAL GLYCOLIPIDS OF INFANT BRAIN

—Gangliosides and allied neutral glycosylceramides were isolated from human infant (2-24 months of age) cerebral cortex and white matter. The individual glycolipids were separated quantitatively by a combination of column and thin-layer chromatographic methods on silica gel, DEAE-cellulose and Sephadex G-25. In cerebral cortex G_D1a and G_M1 were the major fractions and constituted more than 70 per cent of the total gangliosides. The concentrations of neutral glycolipids, except for galactosylceramides, were very low: lactosylceramide and glucosylceramide comprised 30 and 5 nmol/g wet weight, respectively. In white matter their concentrations were 10 times higher. The ganglioside concentration was only 50 per cent of that in cerebral cortex: the difference was accounted for mainly by the much lower content of the major di- and trisialogangliosides. Stearic acid was the predominant fatty acid of all brain gangliosides. G_M3, and G_D3 had a considerable content of the very long-chain fatty acids, C₂₂-C₂₄, particularly in the white matter. Glucosylceramide and lactosylceramide had almost identical fatty acid patterns between each other in cerebral cortex and white matter. In the cerebral cortex stearic acid and in the white matter the very long-chain acids predominated. d20:1 Sphingosine comprised more than 20 per cent of total sphingosine in all the gangliosides of the G_l- and G₂-series. G_M3, and G_D3 like lactosylceramide contained significantly less of d20:1 sphingosine. The findings suggest the existence of separate compartments for the biosynthesis of the gangliosides. Glucosylceramides and lactosylceramides of white matter have the same ceramide composition as the galactosylceramides with normal fatty acids and are thus unlikely to be intermediates in the metabolism of the major brain gangliosides which have a completely different fatty acid composition.     

A convenient procedure for the synthesis of ceramides

A procedure for the preparation of ceramides by direct coupling of long-chain bases and fatty acids in the presence of a mixed carbodiimide is described. This method has been used to prepare ceramides containing sphing-4-enine or sphinganine and various saturated and unsaturated fatty acids as well as saturated 2-hydroxy acids. Ceramides containing 4-hydroxy sphinganine and saturated nonhydroxy acids have also been prepared. The yields were 60-75%. The characterization of these compounds by gas-liquid chromatography-mass spectrometry as trimethylsilyl derivatives has been previously reported. Some of the ceramides are further characterized in this report by infrared spectroscopy and one compound, in addition, by elementary analysis. Use of racemic constituents for 2-hydroxy acid ceramide syntheses leads to the formation of diastereoisomers which separate by thin-layer chromatography. These were characterized by gas-liquid chromatography-mass spectrometry as the trimethylsilyl derivatives and by infrared spectroscopy. Their configurations were established by syntheses with optically active constituents.     

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.