Stimulation of Bacterial Arylsulfatase Activity by Arylamines:

Transcription

1 JOURNAL OF BACTIOLOGY, July 1981, p /81/ $02.00/0 Vol. 147, No. 1 Stimulation of Bacterial Arylsulfatase Activity by Arylamines: Evidence for Substrate Activation JAMES R. GEORGEt AND JOHN W. FITZGERALD* Department ofmicrobiology, University of Georgia, Athens, Georgia Received 12 January 1981/Accepted 8 April 1981 A number of arylamines (including tyramine and tryptamine) increased the in vitro activity of arylsulfatase from Pseudomonas sp. strain C12B. Amino acid analogs of these amines (e.g., tyrosine and tryptophan) failed to exert an effect. Stimulation of activity by tyramine could not be accounted for in terms of sulfotransferase activity for this phenol, and no shift in the ph optimum for the enzyme occurred in the presence of tryptamine. Increased V.. due to these amines was independent of enzyme concentration but varied significantly with substrate concentration. Evidence is presented which suggests that arylamines enhance arylsulfatase activity by forming a salt linkage with the substrate and rendering it more susceptible to enzymatic and acid-catalyzed hydrolyses. The recrystallized tryptamine salt of the substrate exhibited a reduced affinity for the enzyme but was hydrolyzed more rapidly than the potassium salt, which is nornally employed as the assay substrate. Arylsulfatase (aryl-sulfate sulfohydrolase, EC ) catalyzes the hydrolysis of sulfate esters of simple phenols such as p-nitrophenol and p- nitrocatechol. The physiological function of this enzyme, which comes from microbial sources, has been questioned (12, 13), and it has been suggested by numerous authors that esters such as those mentioned above are not in vivo substrates for the enzyme. Indeed, there is now convincing evidence that arylsulfatase enzymes of mammalian origin utilize more complex sulfate esters, such as cerebroside 3-sulfate, as physiological substrates (15, 36). In a study of the physiological function of arylsulfatase from Aspergillus oryzae, Burns and Wynn (4) obtained evidence that one chromatographic form of the enzyme (arylsulfatase II) possesses sulfotransferase activity for tyramine as well as a number of other phenols. In the absence of a suitable phenolic acceptor, the enzyme functions as a sulfatase, releasing equimolar concentrations of p-nitrocatechol and S042- from p-nitrocatechol sulfate. With tyramine, little free S042- is detected in reaction mixtures despite recoveries of substantial amounts of the catechol, indicating that hydrolysis of the ester has occurred. Since tyramine o- sulfate is also detected as a product of the reaction, it has been suggested that S042- liberated by hydrolysis is transferred to the hydroxyl group of tyramine. The kinetics of the reaction (3) and the observation that tyramine enhances t Present address: Centers for Disease Control, Atlanta, GA in vitro arylsulfatase activity when assays are made for phenol release (4) are consistent with a sulfotransferase function for this enzyme. As discussed previously (17), the above findings are of considerable importance, since they suggest that some arylsulfatase enzymes of microbial origin may be involved in the generation of sulfate esters in natural environments. It is a well established fact that the sulfur content of terrestrial soils is largely in the form of ester sulfate (16, 23, 39). Moreover, because tyramine o-sulfate is an intemally compensated salt, the formation of this ester from acidic phenolic esters could represent a mechanism for the storage of soil sulfur in a neutral form. A similar stimulation by tyramine of in vitro arylsulfatase activity was noted when crude or 60-fold-purified extracts of Pseudomonas sp. strain C12B were incubated with tyramine (24). To ensure that the effect of tyramine could be related to arylsulfatase protein, the enzyme from this bacterium was purified to homogeneity (26) subequent to the current study of the effect of this as well as related amines on enzyme activity. The major objective of this work was to determine whether the observed stimulation of activity could be accounted for in terms of a sulfotransferase function for this enzyme. A brief account of some aspects of this work has already been given (25). MATERIALS AND METHODS Enzyme and substrates. Unless otherwise indicated, a 1:200 dilution (0.37,Lg of protein ml-') of the step 9 preparation described by George and Fitzgerald 69

2 70 GEORGE AND FITZGERALD (26) was used throughout. The specific activity of the undiluted preparation (in 0.05 M Tris-hydrochloride buffer, ph 7.5) was 6.57 umol of p-nitrocatechol released per mg of arylsulfatase protein in 1 min. This preparation was maintained at -20 C without loss in activity. p-nitrocatechol sulfate (di-k+ p-ncs: 2-hydroxy-5-nitrophenyl sulfate, dipotassium salt),p-nitrophenyl sulfate (K+ p-nps, potassium salt), and, L- ascorbic acid 2-sulfate (barium salt) were purchased from Sigma Chemical Co. The last ester was converted to the potassium salt before it was used as a potential substrate. Tyramine o-sulfate (internally compensated salt) was a gift from F. A. Rose. PotassiUM L-tyrosine o-sulfate was prepared as previously described (20), whereas sulfate esters of p-acetylphenol and 4-hydroxy-3-nitrophenol were obtained from K. S. Dodgson and C. H. Wynn, respectively. Enzyme assay. Arylsulfatase activity was determined by measuring the release of S042- (19) or the anionic form of the liberated phenol (18). In some cases, determinations for both products were based on the same reaction mixture. Although SO4'- is usually measured at an absorbance at 360 nm (A3M0) (10), interference by p-acetylphenol and p-nitrophenol was observed when the esters of these phenols were used as substrates. Interference was overcome by assaying for S042- release in these reaction mixtures at As. The amount of product released was calculated by constructing lines of best fit (least-squares method) for a series of standards prepared in the presence of 10 mm substrate. The r2 value for each standard line was ;0.98. Substrate hydrolysis was a linear function of incubation time (up to 9 min) at 30 C, provided that dilutions of the step 9 preparation '1:100 were employed. Under no condition was the enzyme permitted to hydrolyze more than about 10% of the substrate. Determination ofkm and Vm... Km and V. were obtained from double-reciprocal plots (30) of data based upon phenol release after incubation at 30 C at ph values optimal for the hydrolysis of each substrate. Lines of best fit relating substrate concentration to initial velocity were constructed by the least-squares method and gave r2 values of and when K' p-nps and di- K' p-ncs were substrates, respectively. Equilibrium dialysis. Arylsulfatase protein (1.32 mg) in 0.01 M Tris-hydrochloride buffer (ph 7.5; 3 ml) containing 0.2 M NaCl (to eliminate possible Donnan effects) was dialyzed at 4 C against the same buffer (4 ml) containing salt and ['4C]tyramine-hydrochloride. Based upon a molecular weight of 52,318 for this protein (26), the enzyme concentration in the dialysis bag was 8.3 nmol ml-'. The concentration of the label in the external buffer was adjusted so that a 50-pl sample gave 1.84 kcpm. The initial concentration of tyramine was 0.3 rmol ml-'. Dialysis to equilibrium was established by monitoring the radioactivity of the external buffer. About 28% of the label was taken up after 4 h, and the remainder (about 3% more) was taken up within 28 h, after which the radioactivity in the external buffer remained constant. After dialysis for 57 h, radioactivity of the enzyme solution and external buffer was determined. This procedure did not result in a detectable loss of enzyme activity. Controls which consisted of the same quantity of J. BACTERIOL. denatured protein (10 min at 100 C) and dialysis buffer alone were dialyzed in parallel and assayed for radioactivity as for native enzyme. Dialysis of this latter protein in the absence of salt yielded similar results. Chromatography and electrophoresis. Reaction mixtures and substrate-phenylamine solutions lacking the enzyme were examined by descending chromatography on Whatman no. 1 papers developed separately with the following solvents: (i) n-butyl alcohol-acetic acid-water (25:4:10, vol/vol/vol), (ii) n- butyl alcohol-ethanol-water (4:1:1, vol/vol/vol), and (iii) ethanol-methanol-water (9:9:2, vol/vol/vol). In some cases, samples (applied to the centers of paper strips) were also subjected to electrophoresis for 1 h at 400 V in M Veronal buffer (ph 8.6; ionic strength, 0.05) or in 0.1 M sodium acetate-acetic acid buffer (ph 4.5). Phenylamines were located by spraying with 0.25% ninhydrin in acetone, and the location of tyramine was confirmed in some cases by including [14C]tyramine in the initial reaction or substrate mixture and as a standard in the buffer alone. Intact sulfate esters and phenolic hydrolysis products were detected by the characteristic colors of the anionic form of the parent phenols. Paper strips were exposed to HCI vapor (to hydrolyze the esters) and to NH40H vapor to allow color development. Preparation of arylsulfatase esters as tryptamine salts. Tryptamine-hydrochloride (5 mmol in 25 ml of water) was added dropwise to 15 ml of an aqueous solution containing 5 mmol of K+ p-nps. After it stood at -2 C for 30 min, the precipitate was collected by filtration and washed once with ice-cold water. The material was recrystallized twice from hot water to produce yellow crystals which were dried in vacuo over CaCl2. Electrophoresis at high ph of a sample of the product revealed a ninhydrin-positive cathodic component and an acid-hydrolyzable anodic component. The existence of a salt linkage in the product was confirmed by the absorption spectra of the product and its constituents (14), all of which exhibited a XAm of 278 nm. If one assumes a molecular weight for the monotryptamine salt of p-nps, a 0.1 mm aqueous solution of the product exhibited an absorbance of 1.43, which represented the approximate sum of that for separate 0.1 mm solutions of K+ p-nps (A278, 0.879) and tryptamine (A278, 0.533). The typtamine salt of p-ncs was prepared and analyzed by electrophoresis as described above, except that the amount of tryptamine-hydrochloride added to an aqueous solution of the dipotassium salt of this ester was doubled. If one assumes a molecular weight for the ditryptamine salt of p-ncs, the absorption spectrum for a 0.1 mm aqueous solution of the final product revealed a X.,B of 278 (A278, 1.44) and a Xn,,, of 402 (A402, 1.10). Dipotassiump-NCS (0.1 mm) also absorbs light at 278 nm (A278, 0.34; An., 402 nm; A402, 1.14). If the A278 of the product is corrected by subtracting from it that due to p-ncs, then the expected value for the ditryptamine salt of p-ncs (A278, 1.10) is obtained. Acid-catalyzed hydrolysis. Rates of hydrolysis for the K' and tryptamine salts ofp-nps and p-ncs were determined by incubating 5 mm aqueous solutions of each ester at 30 C and ph 2.5 for time intervals

3 VOL. 147, 1981 up to 40 min. A linear relationship (r2-0.98) between incubation time and phenol (anionic form) release was established for each ester. Both esters (either salt form) were stable at ph 10 to 4.5. Only trace hydrolyses of p-nps and p-ncs (either salt form) were evident after 40 min at ph 4.0 and 3.5, respectively. RESULTS Arylsulfatase activity toward di-k+ p-ncs has been found to be stimulated by tyramine (2 to 50 mm) when phenol release was measured, with maximum enhancement at 40 mm tyramine (24). At this concentration, the amine also stimulated activity (by S042- release) toward other arylsulfatase esters except tyrosine o-sulfate (Table 1). As with the enzyme from Enterobacter aerogenes (35), arylsulfatase from Pseudomonas sp. strain C12B did not hydrolyze ascorbic acid sulfate (a substrate for mammalian arylsulfatase A). The observed stimulation was not due to a transfer of S042- to tyramine, since approximately equimolar quantities of both hydrolysis products were recovered from reaction mixtures containing di-k+ p-ncs or K+ p-nps and 40 mm tyramine (Table 2). Moreover, tyramine o-sulfate was not detected in reaction mixtures by chromatography (three solvents) or by electrophoresis at low and high ph (see above). Tryptamine and other arylamines were also found to stimulate the hydrolysis of di-k+ p- NCS and K+ p-nps (Table 3). No requirement existed for a phenolic hydroxyl group, since f8- phenethylamine was as effective as tyramine and more effective than either octpamine or dopamine. An absolute requirement for a sterically (or electrostatically) unhindered amine group did exist, since 2-phenyl ethanol (replacement of the amine group with an hydroxyl TABLE 1. Increased hydrolysis of sulfate esters by arylsulfatase from Pseudomonas sp. strain C12Ba Inorganic sulfate released (nmol/min) Sulfate esterb of: With Fold intyra- With- crease mine out ty- (40 ramine mm) p-nitrophenol p-acetylphenol p-nitrocatechol Hydroxy 3-nitrophenol Tyrosine (o-sulfate) L-Ascorbic acid (2-sulfate) a Esters (10 mm) were incubated with enzyme (1:100 dilution, step 9 preparation) in 0.05 M Tris-hydrochloride buffer (ph 7.0). All as potassium salts. ARYLAMINE STIMULATION 71 TABLE 2. Hydrolysis of di-k+ p-ncs and K+ p- NPS by arylsulfatase in the presence and absence of tyraminea Incu- Product reccovered (inol) bation With tyramine time (min) Phenol S (0.41) 0.54 (0.45) (0.65) 0.78 (0.60) (0.76) 1.00 (0.73) Without tyramine Phenol S (0.06) 0.27 (0.06) 0.33 (0.10) 0.37 (0.11) 0.50 (0.14) 0.49 (0.14) a Esters (25 mm) were incubated with enzyme and 40 mm tyramine in 0.05 M Tris-hydrochloride buffer (ph 7.8 or ph 8.8 when p-ncs or p-nps was the substrate, respectively). Values for the hydrolysis of the latter ester are given in parentheses. TABLE 3. Activators and nonactivators of arylsulfatase from Pseudomonas sp. strain C12B0 Fold increase in hydrolysis of.b Effector (1 mm) di-k+ p- K+ p- NCS NPS Tryptamine fl-phenethylamine Tyramine L-Phenylephrine Octpamine Dopamine Phenylalanine Phenylethanol Tyrosine Tryptophan a Hydrolysis of 1 mmp-ncs andp-nps determined in 0.05 M Tris-hydrochloride buffer (ph 7.8 and 8.8, respectively). b Based upon SO42- release. Except for coloration due to dopamine which rendered assays for phenol release inaccurate, similar values were obtained in all cases when phenol release was measured. group) failed to stimulate arylsulfatase activity toward either substrate. The failure of the amino acid analogs of tyramine (tyrosine), phenethylamine (phenylalanine), and tryptamine (tryptophan) to stimulate arylsulfatase activity suggests that a carboxyl group on the carbon atom bearing the amino group prevents stimulation of arylsulfatase activity (Table 3). It is well established that anions (9, 21) and increasing substrate concentration (8, 11) cause a shift in the optimum ph for arylsulfatase activity from various sources. Indeed, Cl- activation of the enzyme from Proteus vulgaris could be accounted for on this basis (9). Stimulation of activity in Pseudomonas sp. strain C12B cannot be explained in terms of a ph shift, since the ph profiles for arylsulfatase with tryptamine were almost identical to those obtained without tryptamine. Optimum ph values of about 8.8

4 72 GEORGE AND FITZGERALD and 7.8 were obtained by using K+ p-nps (Fig. 1) and di-k+ p-ncs, respectively (data not shown), with broad optima evident for both substrates. Increased activity toward K+ p-nps with tryptamine was essentially independent of ph between about 7 and 10.5, and no increase in activity occurred at ph values below 6.5 (Fig. 1). With di-k+ p-ncs as the substrate, similar increases were independent of ph between 6.2 and 9.2, but activity rose sharply with tryptamine at ph >9.2 or <5.2. Similar results were obtained previously (24) by using 60-fold-purified arylsulfatase with this substrate and tyramine, and again this amine did not alter the ph optimum (24). Increased activity due to tryptamine at ph extremes was not a result of low enzyme activity at these ph values, since assays which used higher enzyme concentrations produced similar results (data not shown; 24). The possibility that arylamines stimulate arylsulfatase activity by interacting with this protein was investigated by determining the uptake of [I4C]tyramine by the enzyme after dialysis to equilibrium. Even though enzyme protein was present in sufficient concentration to bind 27 times the initial concentration of amine in the external buffer (see above), almost identical levels of radioactivity were detected in the dialyzand and dialyzate, regardless of whether the dialysis bag contained native enzyme, heattreated enzyme, or buffer alone (data not shown). These results suggest that a binding site on the enzyme for tyramine does not exist in the absence of substrate. The possibility that substrate might promote a tyramine-binding site *E , 40, t35 in's 25 c s a. i1 30-0t_ ph FIG. 1. Influence of ph on arylsulfatase activity toward K' p-nps in the presence (0) and absence (-) of 4 mm tryptamine. Release of p-nitrophenol (pnp) was determined with 4 mm KR p-nps in the following buffers: citric acid-na2hpo4 (ph 4 to 7); This-hydrochloride (ph 7.2 to 9.2); andglycine-naoh (ph 9.2 to 10.5). J. BACTERIOL. has not been ruled out as yet, since we have been unable to find a nonhydrolyzable substrate analog. All known substrates for the enzyme were hydrolyzed substantially even at dialysis temperature. Attempts to block the catalytic site of the enzyme with antibody (to reveal a second site on the enzyme for tyramine in the presence of substrate) have yielded equivocal results, because activity was not completely inhibited by antibody (95% inhibition; cf. 80% inhibition of the enzyme from Klebsiella aerogenes, [31]). Furthermore, tyramine-promoted conformational changes in the enzyme could not be detected by changes in the fluorescence emission maximum of the protein (38), because tyramine caused a quenching of fluorescence (data not shown). The possibility that tyramine and related arylamines bind the enzyme in the presence of substrate cannot be ruled out, although kinetic data on substrate hydrolysis with and without tyramine or tryptamine and chromatographic analysis of reaction mixtures suggest that these effectors activate the substrate rather than the enzyme. Thus, the amount of tryptamine required for the V with K+ p-nps as the substrate was independent of enzyme concentration when 0.15 to 0.37 jig of arylsulfatase protein was added to the reaction mixtures. Moreover, when reaction mixtures contained 0.37,g of enzyme, the amount of either tryptamine or tyramine required for the V,. varied considerably with the amount of either K+ p-nps or di-k+ p-ncs present as the substrate (data not shown). Chromatographic analysis of reaction mixtures containing enzyme, K+ p-nps and ['IC]- tyramine revealed two ninhydrin-positive radioactive components (Rf, 0.59, 0.79; solvent i). The first component corresponded in mobility to tyramine, whereas the second did not correspond with the enzyme (Rf, 0), tyramine o-sulfate (Rf, 0.19), K+p-NPS (Rf, 0.50), orp-nitrophenol (p- NP'-; Rf, 0.28). Both radioactive components were also found in reaction mixtures lacking enzyme. The second component was most likely a tyramine salt ofp-nps, since exposure to acid and then to base revealed the presence of p- NP'-. Elution of the component and subsequent electrophoresis (high ph, see above) showed that it consisted of tyramine and p-nps. Identical results were obtained after chromatography in solvent ii or -iii with K+ p-nps, K+ p-acetylphenyl sulfate, and di-k+ p-ncs, but only one radioactive component (Rf for tyramine) was detected when tyrosine o-sulfate was the substrate. The hydrolysis of the latter ester was not stimulted by tyramine (Table 1), probably because the carboxyl group of the amino acid prevented salt fonnation with the amine. Mix-

5 VOL. 147, 1981 tures of K+ p-nps and various activating and nonactivating effectors (Table 3) were chromatographed separately (solvents i and ii). In every instance, an additional ninhydrin-positive component yieldingp-np'- after acid hydrolysis was detected in mixtures containing an effector (e.g., tryptamine) which stimulated arylsulfatase activity (Table 3). This second component was not detected in reaction mixtures containing effectors (e.g., tryptophan) which failed to increase the hydrolysis of this substrate (Table 3). The involvement oftryptamine as an activator of arylsulfatase activity was investigated further by determining Km and V.. values for the enzyme acting on K+ p-nps and di-k+ p-ncs in the absence and presence of the amine at various molar ratios with respect to substrate (Table 4). As the amount of tryptamine was increased, the Km for the enzyme increased regardless of the substrate employed. The V.. of the reaction also increased in response to increasing concentrations of tryptamine, and maximum values were observed at molar ratios of amine to substrate of 2.5:1 for K' p-nps and 5:1 for di-k' p- NCS. The recrystallized monotryptamine and ditryptamine salts ofp-nps and p-ncs respectively also served as substrates for the enzyme, with Km and Vmx values identical to those obtained when the amine was present in reaction mixtures at molar ratios of 1:1 for K' p-nps (Table 4) and 2:1 for di-k' p-ncs (data not shown). Thus, arylsulfatase exhibited a reduced affinity for the tryptamine salt compared with the K' salt of either substrate, but hydrolyzed the former salt forms at a more rapid rate. Since a tendency exists for tryptamine salt formation in reaction mixtures containing substrate (added initially as the K' salt), the results of Table 4 can be interpreted as indicating that an excess of amine (2.5:1, p-nps; 5:1, p-ncs; Table 4) is required to maintain total substrate in the tryptamine salt form. At lower amine-tosubstrate ratios, more of the K' salt of the substrate is available in the reaction mixture. Since this forn of the substrate has a greater affinity for the enzyme but is hydrolyzed less rapidly, the V.. will decrease in response to decreasing tryptamine concentration. That the formation and subsequent hydrolysis of the ditryptamine salt ofp-ncs accounts for the stimulation of arylsulfatase activity by this amine is suggested by the observation that twice as much tryptamine was required for maxrimal hydrolysis ofp-ncs compared with p-nps (Table 4). In contrast to the hydrolysis of certain alkylsulfate esters (2), the enzymic (37) and acidcatalyzed hydrolysis (29) of arylsulfate esters involves the cleavage of the 0-S bond in the ester sulfate linkage. Since the reaction cata- ARYLAMINE STIMULATION 73 TABLE 4. Influence of tryptamine on Km and Vm. for arylsulfate ester hydrolysisa Molar ra- Km (10-5 M) V. (10-"tmol/ tio (trypt- min) subatrate) amine) K+ p- di-k+ p- K+ p- di-k+ p- NPS NCS NPS NCS Substrate alone 1: : : : : a Based upon phenol release. lyzed by arylsulfatase from Pseudomonas sp. strain C12B proceeds according to this mechanism (6), it is possible that the tryptamine salts of these esters were hydrolyzed more rapidly compared with K+ salt forms because salt formation with tryptamine was more stable and presumably weakened the 0-S bond. It is interesting that the tryptamine salts ofp-nps and p-ncs were also about 25% more susceptible to acid-catalyzed hydrolysis than their K+ counterparts (data not shown). DISCUSSION Although tyramine can increase the bacterial synthesis of arylsulfatase (1, 27, 28, 32, 33), the sitmulatory effect of this amine on in vitro arylsulfatase activity in A. oryzae (4) and Pseudomonas sp. strain C12B might be considered unique, since others have noted substantial inhibition of the enzyme with this and related arylamines (32, 34; F. H. Milazzo, personal communication). Unlike arylsulfatase II from A. oryzae (4), the Pseudomonas enzyme lacks sulfotransferase activity for tyramine and functions only as a true sulfohydrolase. A study of the physical properties of this enzyme (26) showed that it is similar to other bacterial arylsulfatases with respect to molecular weight and lack of subunit association (7, 22, 34), and its frictional ratio suggests that it may be nearly spherical in shape (26). It is likely that tyramine salts of p-nps (5) andp-ncs (4) were also formed, at least to some extent, in the A. oryzae reaction mixtures employed by Burns and co-workers, but it is not known to what extent this salt serves as substrate for any of the three arylsulfatase isozymes (5) in this fungus. It is also possible that the competitive inhibition which was noted when tyramine was added to assay mixtures for other arylsulfatase enzymes may be due to the tyramine salt of the substrate rather than to tyramine alone.

6 74 GEORGE AND FITZGERALD ACKNOWLEDGMENTS We thank C. H. Wynn (Department of Biochemistry, University of Manchester), F. A. Rose, and K. S. Dodgson (Department of Biochemistry, University College, Cardiff) for providing sulfate esters. We are also particularly indebted to K. S. Dodgson for invaluable advice throughout this investigation. LITERATURE CITED 1. Adachi, T., Y. Murooka, and T. Harada Derepression of arylsulfatase synthesis in Aerobacter aerogenes by tyramine. J. Bacteriol. 116: Bartholomew, B., K. S. Dodgson, G. W. J. Matcham, D. J. Shaw, and G. F. White A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent degrading microorganisms. Biochem. J. 165: Burns, G. R. J., E. Galanopoulou, and C. H. Wynn Kinetic studies of the phenol sulphate-phenol sulphotransferase of Aspergillus oryzae. Biochem. J. 167: Burns, G. R. J., and C. H. Wynn Studies on the arylsulphatase and phenol sulphotransferase activities of Aspergillus oryzae. Biochem. J. 149: Burns, G. R. J., and C. H. Wynn A rapid direct assay for the determination of the separate activities of the three arylsulphatases of Aspergillus oryzae. Biochem. J. 166: Cloves, J. M., K. S. Dodgson, D. E. Games, D. J. Shaw, and G. F. White The mechanism of action of primary alkylsulphohydrolase and arylsulphohydrolase from a detergent-degrading microorganism. Biochem. J. 167: Delisle, G. J., and F. H. Milazzo The isolation of arylsulphatase isoenzymes from Pseudomonas aeruginosa. Biochim. Biophys. Acta 212: Delisle, G. J., and F. H. Milazzo Characterization of arylsulphatase isoenzymes from Pseudomonas aeruginosa. Can. J. Microbiol. 18: Dodgson, K. S Observations on the arylsulphatase of Proteus vulgaris. Enzymologia 20: Dodgson, K. S Determination of inorganic sulphate in studies on the enzymic and non-enzymic hydrolysis of carbohydrates and other sulphate esters. Biochem. J. 78: Dodgson, K. S., and G. M. Powell Arylsulphatase activity in the digestive juice and digestive gland of Helix pomatia. Biochem. J. 73: Dodgson, K. S., and F. A. Rose Sulfohydrolases, p In D. M. Greenberg (ed.), Metabolic pathways, vol. 7: Metabolism of sulfur compounds. Academic Press, Inc., New York. 13. Dodgson, K. S., and F. A. Rose Observations on the biological roles of sulphatases, p In Sulphur in biology. Ciba Foundation symposium 72. Excerpta Medica, Amsterdam. 14. Dodgson, K. S., F. A. Rose, and B. Spencer The isolation and characterization of biosynthetic arylsulphates. Biochem. J. 60: Farooqui, A. A Sulfatases, sulfate esters and their metabolic disorders. Clin. Chim. Acta 100: Fitzgerald, J. W Sulfate ester formation and hydrolysis: a potentially important yet often ignored aspect of the sulfur cycle of aerobic soils. Bacteriol. Rev. 40: Fitzgerald, J. W Naturally occurring organosulfur compounds in soil, p In J. 0. Nriagu (ed.), Sulfur in the environment, part II: ecological impacts. John Wiley & Sons, Inc., New York. 18. Fitzgerald, J. W., and M. E. Cline The occurrence of an inducible arylsulphatase in Comamonas terrigena. FEMS Lett. 2: Fitzgerald, J. W., and L. C. Kight Physiological control of alkylsulfatase synthesis in Pseudomonas J. BACTERIOL. aeruginosa: effects of glucose, glucose analogs, and sulfur. Can. J. Microbiol. 23: Fitzgerald, J. W., W. H. Maca, and F. A. Rose Physiological factors regulating tyrosine-sulphate sulphohydrolase activity in Comamonas terrigena: occurrence of constitutive and inducible enzymes. J. Gen. Microbiol. 111: Fitzgerald, J. W., and F. H. Milazzo Further studies on the heterogeneity of arylsulphatase from Proteus rettgeri and some properties of one chromatographic form of the enzyme. Int. J. Biochem. 6: Fowler, L R., and D. H. Rammler Sulfur metabolism of Aerobacter aerogenes: the purification and some properties of a sulfatase. Biochemistry 3: Freney, J. R Sulphur-containing organics, p In A. D. McLaren and G. H. Peterson (ed.), Soil biochemistry, vol. 1. Marcel Dekker, Inc., New York. 24. George, J. R., and J. W. Fitzgerald Tyrarninemediated enhancement of bacterial arylsulphatase activity. FEMS Lett. 3: George, J. R., and J. W. Fitzgerald Tyraminemediated enhancement of arylsulphatase purified from Pseudomonas C,2B. Biochem. Soc. Trans. 7: George, J. R., and J. W. Fitzgerald Arylsulfatase from Pseudomonas sp. strain C12B: purification to homogeneity, immunological analysis, and physical properties. J. Bacteriol. 145: Harada, T., and Y. Murooka Participation of tyramine oxidase in multiple control of bacterial arylsulfatase synthesis. Mem. Inst. Sci. Ind. Res. Osaka Univ. 37: Henderson, M. J., and F. H. Milazzo Arylsulfatase in Salmonella typhimurium: detection and influence of carbon source and tyramine on its synthesis. J. Bacteriol. 139: Kice, J. L., and J. M. Anderson Mechanism of the acid hydrolysis of sodium arylsulfates. J. Am. Chem. Soc. 88: Lineweaver, H., and D. Burk The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: Murooka, Y., T. Yamada, S. Tanabe, and T. Harada Immunological study of the regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes. J. Bacteriol. 132: Murooka, Y., M.-H. Yim, and T. Harada Formation and purification of Serratia marcescens arylsulfatase. Appl. Environ. Microbiol. 39: Oka, M., Y. Murooka, and T. Harada Genetic control of tyramine oxidase, which is involved in derepressed synthesis of arylsulfatase in Klebsiella aerogenes. J. Bacteriol. 143: Okamura, H., T. Yamada, Y. Murooka, and T. Harada Purification and properties of arylsulfatase of Klebsiella aerogenes: identity of the enzymes formed by non-repressed and derepressed synthesis. Agric. Biol. Chem. 40: Roy, A. B L-ascorbic acid 2-sulfate: a substrate for mammalian arylsulfatases. Biochim. Biophys. Acta 377: Roy, A. B Sulphatase A-an arylsulphatase and a glycosulphatase, p In Sulphur in biology. Ciba Foundation symposium 72. Excerpta Medica, Amsterdam. 37. Spencer, B Enzymic cleavage of aryl hydrogen sulfates in the presence of H2"8O. Biochem. J. 69: Steiner, R. F., R. W. Lippoldt, H. Edelhoch, and V. Frattali Ultraviolet fluorescence of proteins: influence of conformation and environment. Biopolymers 1: Tabatabai, M. A., and J. M. Bremner Forms of sulfur and carbon, nitrogen and sulfur relationships, in Iowa soils. Soil Sci. 114: