Regulation of sulfate transport in neurospora by transinhibition and by inositol depletion

Neurospora possesses two distinct sulfate transport systems, a low-affinity form (Permease I) which is the only type found in conidia, and a second species (Permease II) which predominates during the mycelial stage. Although methionine represses the synthesis of both of these permeases, inorganic sulfate only partially represses the mycelial form and does not affect the synthesis of Permease I. Both transport systems are also regulated by transinhibition. The transinhibition which occurs in mycelia is not due to an intracellular pool of inorganic sulfate, but is instead exerted by an early intermediate of the sulfate assimilatory pathway.The development of functional sulfate transport activity depends upon genetic and metabolic events which affect the cell membrane. The synthesis of sulfate permease activity in the inos mutant requires an exogenous supply of inositol. The effect of the cot mutant, which is thought to interfere with membrane synthesis, also prevents the development of sulfate permease at the restrictive temperature. The maintenance of pre-existing functional sulfate permease activity apparently also requires a continuous renewal of membrane components since withdrawal of inositol from inos mutants results in a rapid inactivation of transport activity.     

Regulation of sulfate metabolism in Neurospora crassa: Transport and accumulation of glucose 6-sulfate

Neurospora crassa can utilize glucose 6-sulfate as its sole sulfur source, although this compound cannot serve as a carbon source for this organism. Neurospora possesses a transport system capable of glucose 6-sulfate uptake; the system is energy dependent, is inhibited by extracellular sulfate, and is clearly distinct from the permeases responsible for the uptake of glucose and those for sulfate transport. The metabolism of glucose 6-sulfate apparently involves its transport as an intact molecule, followed by a slow intracellular hydrolysis. Methionine, which represses the synthesis of a number of enzymes of sulfur anabolism, also represses the synthesis of the transport system responsible for glucose 6-sulfate uptake. A regulatory gene, cys-3, which controls the synthesis of aryl sulfatase, choline sulfatase, choline-O-sulfate permease, and two distinct permease species, also regulates the permease for glucose 6-sulfate.     

Developmental regulation of choline sulfatase and aryl sulfatase in Neurospora crassa

Regulation of the synthesis of several enzymes of sulfur metabolism in Neurospora is a function of both metabolic regulation and the genetic control exerted by the cys-3 and scon regulatory genes. Additional control mechanisms appear to regulate the synthesis of choline sulfatase and aryl sulfatase in different developmental stages of the life cycle. The metabolic regulation of enzyme synthesis in conidia differs from that which occurs in the mycelial stage. During conidial germination and mycelial outgrowth, the synthesis of these enzymes is not coordinate but begins at different times and occurs at different rates. A rapid and early synthesis of choline sulfatase was observed during conidial germination under derepressing conditions; furthermore, synthesis of the enzyme also occurred for a brief period in germinating conidia even in the presence of repressing levels of sulfate. The results of this study suggest that several enzymes of sulfur metabolism are independently controlled by a developmental system which is superimposed upon the cys-3 regulatory mechanism. It was also found that choline sulfatase undergoes rapid turnover while aryl sulfatase is a stable species.     

Genetic and metabolic control of sulfate metabolism in Neurospora crassa: A specific permease for choline-O-sulfate

Neurospora crassa can use choline-O-sulfate as its sole sulfur source; the utilization of this compound involves its entry followed by intracellular hydrolysis. Neurospora possesses a transport system for the uptake of choline-O-sulfate which is specific for the sulfate ester and does not transport, nor is it inhibited by, either choline or inorganic sulfate. Mutant strains of Neurospora that are unable to transport or grow on inorganic sulfate can, nevertheless, utilize choline-O-sulfate for growth and transport the intact organic sulfate at a normal rate. Methionine, which represses a number of enzymes of sulfur anabolism, also represses the synthesis of the specific permease for choline-O-sulfate. A regulatory gene, cys-3, which controls the synthesis of choline sulfatase, aryl sulfatase, and several other related enzymes, also regulates the synthesis of the choline sulfate permease. Evidence is presented that the activity of choline sulfate permease is also regulated by a turnover process, the transport system having a functional half-life of approximately 3 hr.This investigation was supported by Public Health Service Grant 1 RO1 GM-18642 from the National Institute of General Medical Services.     

The structural basis of anomalous kinetics of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K_i values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of ³⁵S-labeled substrate at pH values near the pH optimum (pH 5–6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.     

Sulfate Uptake in Saccharomyces cerevisiae: Biochemical and Genetic Study

Sulfate uptake is the first step of the sulfate assimilation pathway, which has been shown in our laboratory to be part of the methionine biosynthetic pathway. Kinetic study of sulfate uptake has shown a biphasic curve in a Lineweaver-Burk plot. The analysis of this plot indicates that two enzymes participate in sulfate uptake. One (permease I) has a high affinity for the substrate (K(m) = 0.005 mM); the other (permease II) shows a much lower affinity for sulfate (K(m) = 0.35 mM). Regulation of the synthesis of both permeases is under the control of exogenous methionine or S-adenosylmethionine. It was shown, moreover, that synthesis of sulfate permeases is coordinated with the synthesis of the other methionine biosynthetic enzymes thus far studied in our laboratory. An additional specific regulation of sulfate permeases by inhibition of their activity by endogenous sulfate and adenosyl phosphosulfate (an intermediate metabolite in sulfate assimilation) has been shown. A mutant unable to concentrate sulfate has been selected. This strain carried mutations in two independent genes. These two mutations, separated in two different strains, lead to modified kinetics of sulfate uptake. The study of these strains leads us to postulate that there is an interaction in situ between the products of these two genes.     

Evidence of an essential histidine residue in rabbit liver aryl sulfatase A.

A detailed study of the pH dependence of the Michaelis-Menten constants (V and K_m) of aryl sulfatase A (EC 3.1.6.1) from rabbit liver indicates that at least two functional groups (pK's ~4.3 and ~7 in the enzyme-substrate complex) participate in the enzymic degradation of substrate. Aryl sulfatase A is inactivated by diethyl pyrocarbonate (ethoxyformic anhydride). The enzyme that has been modified with this reagent can in turn be reactivated by treatment with hydroxylamine. The pH dependence of inactivation reveals a reactive group having a pK of 6.5–7.0. The results indicate that at least one histidine plays an important catalytic role in rabbit liver aryl sulfatase A, consistent with the results of earlier workers who employed diazotized sulfanilic acid. Phosphate ion, a competitive inhibitor, partially protects the enzyme from inactivation by diethyl pyrocarbonate whereas sulfate ion, also a competitive inhibitor, increases the rate of inactivation by diethyl pyrocarbonate. This result is of particular significance in view of the anomalous kinetics of aryl sulfatase A. The kinetic effects of even small amounts of sulfate ion impurities in many commercial sulfate ester substrate preparations is also discussed.     

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.     

Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.     

Purification and properties of a homogeneous aryl sulfatase A from rabbit liver.

Aryl sulfatase A (aryl sulfate sulfohydrolase EC 3.1.6.1) has been purified > 10,000-fold from rabbit liver; by disc gel electrophoresis the enzyme appears homogeneous. Various properties of the enzyme have been determined and comparisons are made with other aryl sulfatases. Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is made up of monomers of molecular weight ～ 70,000. At pH 7.4 the enzyme exists as a dimer whereas a tetrameric form predominates at pH 4.8.The enzyme exhibits the anomalous kinetics often observed with aryl sulfatase A from mammalian tissues (the enzyme is modified to an inactive form while degrading substrate and the inactive form can be reactivated by sulfate ion). The enzyme activity has been studied under a variety of reaction conditions. Two pH optima are observed and neither enzyme concentration or changes in ionic strength appear to have an effect on the relative magnitudes of the optima. Aryl sulfatase A is competitively inhibited by potassium sulfate, potassium phosphate, and sodium sulfite (K_i = 2.9 × 10^?3 M, 3.4 × 10^?5 M, and 1.1 × 10^?6 M, respectively). Kinetic constants for some substituted phenyl sulfate esters have been determined. The variation in V is not consistent with a reaction mechanism involving a rate-limiting breakdown of a common intermediate.The inactive (modified) form of the enzyme has been isolated from reaction mixtures containing aryl sulfatase A and substrate. A procedure is presented for determining the relative amount of modified and native enzyme in these preparations. In the presence of substrate, sulfate displaces the equilibrium between native and modified enzyme in favor of native enzyme. In the absence of substrate neither sulfate or phosphate have an effect on the equilibrium. A study is made of the temperature dependence of the process in which the modified enzyme is converted back to native enzyme. The relatively small entropy of activation for the conversion of the modified to the native form (ΔS3 = ?8 cal/mole deg) does not seem to be consistent with a major modification of protein conformation.     

The Saccharomyces cerevisiae YCC5 (YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine

The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.     

A PEST-Like Sequence Mediates Phosphorylation and Efficient Ubiquitination of Yeast Uracil Permease

Uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. Uracil permease located at the cell surface is subject to two covalent modifications: phosphorylation and ubiquitination. The ubiquitination step is necessary prior to permease endocytosis and subsequent vacuolar degradation. Here, we demonstrate that a PEST-like sequence located within the cytoplasmic N terminus of the protein is essential for uracil permease turnover. Internalization of the transporter was reduced when some of the serines within the region were converted to alanines and severely impaired when all five serines within the region were mutated or when this region was absent. The phosphorylation and degree of ubiquitination of variant permeases were inversely correlated with the number of serines replaced by alanines. A serine-free version of this sequence was very poorly phosphorylated, and elimination of this sequence prevented ubiquitination. Thus, it appears that the serine residues in the PEST-like sequence are required for phosphorylation and ubiquitination of uracil permease. A PEST-like sequence in which the serines were replaced by glutamic acids allowed efficient permease turnover, suggesting that the PEST serines are phosphoacceptors.     

Developmental regulation of protein synthesis during Polysphondylium pallidum cyst germination: A two-dimensional gel electrophoresis study

Microcyst germination in Polysphondylium pallidum can be used as a model for studying gene expression because temporally regulated modulations in protein synthesis occur in this developmental pathway. Germinating cysts were labeled with [³⁵S]methionine for half-hourly periods during the synchronous germination sequence, and the proteins labeled in each period were resolved by two-dimensional polyacrylamide gel electrophoresis. Three major classes of proteins observed were distinguished by the time of onset and duration of their synthesis: (a) proteins made throughout germination; (b) proteins synthesized only during a portion of the germination pathway; and (c) polypeptides whose synthesis started at 1 or 1.5 h and then continued throughout germination.     

The monomer -- dimer association of rabbit lver aryl sulfatase A and its relationship to the anomalous kinetics.

The polymerization of aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) has been studied by frontal gel chromatography on Sephadex G-200 and Bio-Gel A-5m under various conditions of pH, ionic strength, and temperature. The aryl sulfatase A molecule exists as a monomer and as a dimer at pH 7.5 and pH 4.5, respectively. The extent of dissociation is markedly pH-, protein concentration-, and ionic strength-dependent. Only a small effect of temperature was observed. The enthalpy change (ΔH^o) for the dissociation was ?2.5 ± 1 kcal/mol at pH 5.5–5.6, and the entropy change for dissociation of the enzyme dimer to two monomeric units was ?47 cal mol^?1 deg^?1. Sulfate ion has little effect on the extent of dissociation of the enzyme at pH 5.6. The present studies suggest that the dissociation of rabbit liver aryl sulfatase A is regulated by the ionization of amino acid residues whose apparent pK is between pH 5 and 6. The driving force for the association of the subunits of the enzyme is primarily ionic and/or ionic/hydrogen bond formation. The small enthalpy change and the fact that dissociation is strongly favored by an increase in the ionic strength suggest that hydrophobic interactions play only a minor role in stabilizing the dimeric quaternary structure relative to the monomeric state. The monomeric form of the enzyme exhibits the anomalous kinetics often observed with sulfatase A but the dimer does not show anomalous kinetics. Since aryl sulfatase A is probably in the dimeric form in the lysosome, the anomalous kinetics of the enzyme are unlikely to be of physiological importance in the intact lysosome.     

STEROID SULFATASE IN BRAIN: COMPARISON OF SULFOHYDROLASE ACTIVITIES FOR VARIOUS STEROID SULFATES IN NORMAL AND PATHOLOGICAL BRAINS, INCLUDING THE VARIOUS FORMS OF METACHROMATIC LEUKODYSTROPHY

Abstract– The enzymatic hydrolysis by brain homogenate of the sulfate esters of estrone, pregnenolone, dehydroepiandrosterone, testosterone, cholesterol and p-nitrophenol was studied. With homogenate of young rat brain, the pH optima of estrone sulfatase ⁴ 4 The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout.
and arysulfatase C (p-nitrophenyl sulfate as substrate) were 8.2 and all other steroid sulfatases had pH optima at 6.6. Apparent K_ms for these steroid sulfates were widely different. The highest K_m value was 32.2 μm for estrone sulfate and the lowest was 0.66 μm for testosterone sulfate; the K_m for p-nitrophenyl sulfate was 30 fold higher than for estrone sulfate. Specific activity was also highest with estrone sulfatase and lowest with testosterone sulfatase; specific activity with aryl sulfatase C was over 3 fold higher than with estrone sulfatase. Estrone sulfatase activity was inhibited noncompetitively by sulfate esters of dehydroepiandrosterone, pregnenolone, and cholesterol; on the other hand, other steroid sulfatases were inhibited by these latter three sulfates competitively. Developmental changes of these sulfohydrolase activities in rat brain were almost identical with the exception of testosterone sulfatase activity; the latter sulfatase had a peak activity at 30 days old, while all other sulfatase had a peak at 20 days old. Thermal stability of all these activities was identical. Testosterone sulfatase activity in neurological mouse mutants, jimpy, msd, and quaking mice, was less than one half of littermate controls, while other steroid sulfatase levels in these mutants' brain were normal. All sulfatase activities were diminished in the brain of a metachromatic leukodystrophy patient with multiple sulfatase deficiency. The brains of classical metachromatic leukodystrophy patients contained normal levels of all steroid sulfatases and arylsulfatase C, with the single exception of testosterone sulfatase which level was less than 50% of control.     

Aryl sulfatase of unusual specificity from the liver of marine mollusk Littorina kurila

An aryl sulfatase of unusual specificity has been isolated from the liver of marine mollusk Littorina kurila. It hydrolyzes p-nitrophenyl sulfate, does not affect the natural fucoidan, and catalyzes splitting off the sulfate group in position C4 of xylose residues within the carbohydrate chains of holostane triterpene glycosides from sea cucumbers. The properties of the enzyme were studied at pH 5.4. The protein is homogeneous according to electrophoresis and has M 45 ± 1 kDa. The semiinactivation time of the enzyme at 60°C is 20 min, and its K _m value for the hydrolysis of p-nitrophenyl sulfate is 8.7 ± 1 mM. It was shown that natural sulfated polyhydroxysteroids inhibit activity of the sulfatase; their I ₅₀ values depend on their structures and are within the range from 10^?3 to 10^?5 M.     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.     

Physiology of F-Pilin Synthesis and Utilization

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of Escherichia coli K-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [³⁵S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other tra products in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and traT product synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of traT product and membrane F-pilin, as assessed in an Flac tra mutant strain which is incapable of elaborating pili, was not rapid. Both traT product and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of traT product did not decrease even after the culture was incubated for 24 h. When wild-type Flac piliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the Flac tra mutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the Flac strain compared with the Flac tra strain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces.     

Subunit affinity chromatography and its application to the isolation of aryl sulfatase A enzymes

The monomeric form of rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) was covalently coupled to CNBr-activated Sepharose and the catalytic properties of the covalently coupled monomer subunit were examined. The immobilized subunit showed one pH optimum near pH 5.6 which appears to be the characteristic pH optimum of the monomer. The enzyme-Sepharose complex exhibited the characteristic anomalous kinetic behavior at pH 5.5 but there was no turnover-induced inactivation of the immobilized enzyme at pH 4.5. The covalently coupled subunit column was examined for its ability to act as a subunit affinity chromatography medium. It was found that dissolved aryl sulfatase A was removed from solution at pH 4.5 and pH 5.0, I = 0.2, and became associated with the affinity column of Sepharose-aryl sulfatase A. The retained subunit of the enzyme could subsequently be quantitatively eluted with 0.2 m Tris-HCl, pH 7.5. Extraneous protein such as bovine serum albumin did not measureably affect the rate or equilibrium for association of the enzyme to the covalently bound subunit. The extent of binding of the enzyme to the affinity column was found to be strongly dependent on the time of equilibration and on the pH. About 90% of the enzyme was retained after 24 h at pH 5.0, I = 0.2. Under otherwise comparable conditions, use of Sepharose-6MB resulted in slightly faster association than did Sepharose-4B. Under the experimental conditions employed, the total capacity of the affinity column was approx 50% of the total aryl sulfatase A coupled to the Sepharose. The rabbit liver subunit column also permits the purification of several other aryl sulfatase A enzymes. Thus, the subunit affinity column provides a simple, convenient, and rapid procedure for the isolation of most mammalian aryl sulfatase A enzymes as well as for studying inter- and intraspecific subunit association interactions.     

Covalent modification as the cause of the anomalous kinetics of aryl sulfatase A.

Mammalian aryl sulfatase A enzymes are known to exhibit an anomalous kinetic behavior in which the enzyme becomes inactivated as it catalyzes the hydrolysis of substrate. Part of the activity of this inactive, turnover-modified form of the enzyme can apparently be restored by the simultaneous presence of substrate and sulfate ion. The present experiments, conducted with 2-hydroxy-5-nitrophenyl [³⁵S]sulfate (nitrocatechol sulfate), establish that the turnover-modified enzyme is covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of ³⁵S per mole of enzyme monomer (each monomer of rabbit liver aryl sulfatase consists of two equivalent subunits). It is also shown that isolated, turnover-modified enzyme has lost 80% of its secondary structure when compared to the native enzyme. A commonly used sulfating agent, pyridine-sulfur trioxide complex brings about a similar loss of activity and of secondary structure.