©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Neutral Galactocerebroside Sulfate Sulfatidase from Mouse Brain (*)

Soma K. Sundaram , Jian-Hua Fan , Meir Lev (§)

From the (1) Department of Microbiology, CUNY Medical School/Sophie Davies School of Biomedical Education, New York, New York 10031

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have described an enzyme in brain that catabolizes galactocerebroside sulfatide with a pH optimum of 7.2. To our knowledge, this is the first description of a catabolic enzyme for sulfatide at a neutral pH. Activity at a neutral pH implies a non-lysosomal location for this sulfatidase. Galactocerebroside sulfate sulfatidase ( n-sulfatidase) activity was not apparent in crude microsomal extracts and was detected following partial purification of the enzyme. This enzyme, n-sulfatidase, differs from other arylsulfatases in its M, inability to bind to concanavalin A, and substrate specificity; n-sulfatidase was unable to hydrolyze p-nitrocatechol sulfate or estrone sulfate. The molecular mass of n-sulfatidase obtained by Sephacryl S-200 chromatography was 72 kDa, and the active fraction from this procedure was purified >600-fold by isoelectric focusing. Following SDS-polyacrylamide gel electrophoresis, two bands were obtained with apparent molecular masses of 58 and 66 kDa. Enzyme activity was regenerated from both of these bands, with the 66-kDa band showing greater activity. The Kof the sulfatidase was determined as 5.8 10 M. The pI of n-sulfatidase was 7.7 in contrast to the pI of 4.9 for the sulfotransferase. No requirement was found for Mgor ATP for sulfatidase activity; vitamin Kenhanced sulfatidase activity approximately 3.3-fold. Therefore, this enzyme may have a role in the pathogenesis of metachromatic leukodystrophy in which sulfatides accumulate in the nervous and other tissues and in myelination since sulfatides are an important component of myelin.


INTRODUCTION

We have studied the participation of vitamin K in sphingolipid biosynthesis initially in the vitamin K-dependent bacterium Porphyromonas (Bacteroides) levii (formerly Bacteroides melaninogenicus) (1) . When the bacterium was depleted of vitamin K, sphingolipid biosynthesis was inhibited, and further studies showed that vitamin K regulated the activity of the first enzyme of the sphingolipid pathway, serine palmitoyltransferase (3-ketodihydrosphingosine synthase) (EC 2.3.1.50) (2, 3) . The role of vitamin K in brain sphingolipid biosynthesis was then investigated by the administration of the vitamin K antagonist, Warfarin, to young mice. Warfarin administration resulted in a significant reduction in brain sulfatide levels, and this reduction was reversed by the administration of vitamin K (4) . Vitamin K was then shown to regulate the activity of galactocerebroside sulfotransferase, which catalyzes the biosynthesis of sulfatide, both in vivo (5) and in vitro. In vitro, the requirement of the enzyme for ATP was partially fulfilled by 5 mM orthophosphate plus vitamin K (6) . During experiments on the purification of galactocerebroside sulfotransferase from brain (7) , a new enzyme, galactocerebroside sulfate sulfatidase ( n-sulfatidase),() was found, which catabolizes sulfatide at a neutral but not at an acidic pH. This enzyme thus differs from the lysosomal catabolic enzyme, arylsulfatase A. n-Sulfatidase activity could not be detected in initial extracts of brain microsomes and only became apparent following partial purification of galactocerebroside sulfotransferase when catabolism of the product sulfatide was noted. Among other enzymes that break down sulfated compounds are arylsulfatase B, a lysosomal enzyme which catabolizes dermatan sulfate, chondroitin-4-sulfate, and arylsulfatase C, a microsomal enzyme which catabolizes sulfated steroid compounds at a neutral pH (8) .

The role of sphingolipids and their metabolic products have been studied extensively in cell signaling, including a role for sulfatides and galactocerebroside (9, 10) . A neutral sphingolipid-catabolizing enzyme, sphingomyelinase, has been shown to be involved in cell signaling (for reviews, see Refs. 11 and 12). A product of sulfatide catabolism, ceramide, was shown to be the active species in the sphingomyelin cycle and thus has a central role as a second messenger in this metabolic process (13) , although the role of ceramide in TNF- activation of NF-kB has recently been disputed (14) .

In this study, we report on the isolation, purification, and properties of the n-sulfatidase. A preliminary report of these results has been presented (15) .


EXPERIMENTAL PROCEDURES

Male Swiss mice (ICR) weighing 8-12 g were obtained from Harlan-Sprague-Dawley, Inc. and were used at 16 days old postweaned. Animals were sacrificed by COasphyxiation, and the brains were removed. Brain microsomes were prepared as previously described (5) . The brains were homogenized in 0.32 M sucrose to give 40-60 mg/ml protein. After a 30-min centrifugation at 50,000 g, the supernatant was centrifuged at 100,000 g for 90 min. The microsomal pellet was stored suspended in 0.32 M sucrose at 80 °C at a concentration of 5 mg/ml protein. The microsomal preparation was extracted with an equal volume of 100 mM imidazole buffer (pH 7.2) containing 2.5 mM MgCland 0.5% Triton X-100 (buffer 1) with a protein to detergent ratio of 5:2. The Triton-extracted material was centrifuged (100,000 g, 30 min), and the supernatant was used for further purification.

Preparation of S Substrate from Mouse Brain

S-Labeled sulfatide was prepared by intracranial injection of 16-day-old mice with 1 µCi of carrier-free Na-S-O/g of body weight. We have shown previously that vitamin K stimulates brain sulfatide turnover (5) , and we have used this property of vitamin K to increase the specific activity of the [S]sulfatide. Mice were injected intraperitoneally with 1 mg of Aquamephyton (vitamin K) on each day 2 days previous to injection with Na-[S]O, and after 24 h, the mice were sacrificed. Sulfatides were extracted and purified from brain tissue as previously described (5) . The vitamin K procedure results in a 60% increase in sulfatide specific activity over that obtained from mice that were not vitamin K-treated.() [S]Lysosulfatide was prepared by the following procedure.() [S]Sulfatide was heated in 0.8 M KOH in methanol for 8 h in a pressure flask. The methanol was removed under N, and the residue was dissolved in chloroform:methanol, 2:1 and applied to a silica gel plate. The plate was developed in chloroform:methanol:HO, 65:25:4; the lysosulfatides were detected following radioautography.

Enzyme Purification

Cerebroside sulfotransferase was purified and assayed as described using Sephacryl S-200 and PLP-ligated Sepharose column chromatography (7) . This partially purified sulfotransferase preparation from the PLP-ligated Sepharose column also contained the sulfatide-degrading enzyme. In the present studies, sulfatidase was purified starting from the Sephacryl S-200 fractions containing sulfatidase activity. These fractions were pooled, concentrated by ultrafiltration through a membrane with 10,000 molecular mass cutoff, and analyzed further by preparative scale IEF in the Rotofor (Bio-Rad) isoelectric focusing cell.

To minimize the time required for focusing and to avoid pH extremes, the Rotofor cell was prefocused with approximately 48 ml of 3 M urea containing 0.5% Triton X-100, 5% glycerol, and 2% ampholytes, pH 5-8 (Bio-Rad) (solution A), at 15 watts constant power, 3 °C for 1 h to establish the pH gradient. The sample containing concentrated sulfatidase fraction (1-2 ml, 5 mg of protein) was mixed with an equal volume of solution A and was injected near the middle of the chamber. Focusing was continued for 3 h. The initial conditions were 1400 V, 7 mA, and at equilibrium the conditions were approximately 2200 V, 4 mA. 20 fractions were harvested, and the pH, protein concentration, and sulfatidase activity were determined. Fractions containing activity (at pH 7.6) were pooled and rerun in the small Rotofor cell at 12 watts without additional ampholytes. Fractions with sulfatidase activity (pI = 7.6) were pooled, desalted, and used for determination of sulfatidase properties. The pI of n-sulfatidase was then determined as 7.7 using a tube gel and a surface electrode. A similar procedure was used to determine the pI of the sulfotransferase using 2% ampholytes, pH 4-6.5, in the Rotofor cell.

Assay of n-sulfatidase was performed using [S]sulfatide of known specific activity prepared as described above and purified by TLC. Sulfatide (10,000 cpm, about 10 nmol) was dissolved in 25 µl of chloroform:methanol (2:1, v/v) containing Triton X-100 (4%), evaporated under N, and resuspended in buffer 1 (0.2 ml) containing 30 µg of vitamin K(Aquamephyton), 0.3 ml of enzyme in total volume of 0.5 ml. After incubation for 30 min at 37 °C, the reaction was terminated by the addition of 5 ml chloroform:methanol, and the mixture was partitioned into 0.2 volume aqueous 0.88% KC1. Sulfatides were then assayed by TLC (7) , or the aqueous fraction was counted. In some experiments, sulfatide was tritiated (16) and used where indicated. Labeled sulfatide showed a negligible (3%) loss in activity following a 30-min incubation period at 37 °C in a reaction mixture containing inactive enzyme.

Arylsulfatase A Assay

Arylsulfatase A activity was assayed as previously described (5) in purified sulfatidase fractions. Assays were performed in 0.2 ml containing 10 mM p-nitrocatechol sulfate, 0.2 M sodium acetate buffer, pH 5.0, and enzyme. The reaction mixture was incubated (37 °C, 30 min), and the reaction was stopped by the addition of 5 ml of 1 N NaOH and read at 500 nm. In other experiments, 10 nmol of [S]sulfatide was used as substrate, and following incubation, catabolism of sulfatide was determined as described above.

Regeneration of n-Sulfatidase Activity Following SDS-PAGE

SDS-PAGE was performed as shown (Fig. 4), and enzyme activity was regenerated (17) . The SDS was removed by washing twice in 100 ml of 20% 2-propanol in Tris-HCl, pH 8.0, for 1 h followed by 250 ml of Tris buffer containing 5 mM 2-mercaptoethanol for 1 h at room temperature. The enzyme was then denatured by treating with two changes of 6 M gaunidine HCl for 1 h and then renatured in five changes of 250 ml of Tris buffer containing 0.04% Triton X-100 at 4 °C. A portion of the gel was silver stained; areas corresponding to the two bands, shown in Fig. 4 , lane D, were cut out, and the protein was eluted by electroelution. n-Sulfatidase activity was then assayed as described above.


Figure 4: SDS-PAGE analysis of n-sulfatidase at stages in purification. Microsomal extracts were partially purified by Sephacryl S-200 chromatography; the active fractions were concentrated and run in the Rotofor. The active fraction from this procedure (pI = 7.6) was collected, concentrated, and rerun in the Rotofor. Lane A, standards (-galactosidase, 116 kDa; phosphorylase B, 97.4 kDa; serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa; aprotinin, 6.5 kDa); lane B, Sephacryl S-200 active fraction (5 mg of protein); lane C, IEF first run active fraction about 2 mg of protein; lane D, IEF second run active fraction about 1 mg of protein. Two major bands are present (molecular masses of 58 and 66 kDa).



Materials

[S]PAPS (1.6 Ci/mmol), Na-[S]O(carrier free), and [H]estrone sulfate (49 mCi/mmol) were obtained from Dupont NEN, Aquamephyton (colloidal suspension of vitamin Kin detergent/glucose/benzyl alcohol) was obtained from Merck Sharp and Dohme, and EAH-Sepharose 4B and PD-10 columns were obtained from Pharmacia Biotech Inc. Galactocerebroside was obtained from Matreya, Inc. (Pleasant Gap, PA) and was a mixture of hydroxy and non-hydroxy fatty acids. Other chemicals were obtained from Sigma. All of the chemicals were reagent grade or the best quality available.


RESULTS

Detection of Neutral Sulfatidase (n-Sulfatidase) Activity

Brain microsomes were prepared and extracted as described under ``Experimental Procedures.'' Assay of sulfotransferase in brain microsomes results in a linear increase in sulfatide formation up to 90 min in our experiments, after which the level remains constant on further incubation as previously reported (18, 19) .

However, when the extracts were passed through a PLP-ligated column and fractions were assayed for sulfotransferase activity, an increase in sulfatide formation occurred, which was followed by a decrease in lipid [S]sulfate (Fig. 1) in contrast to results obtained using whole microsomal extracts. This decrease in sulfatide level following biosynthesis indicated the presence, in the partially purified preparation, of an enzyme-catabolizing sulfatide. In an experiment using a highly purified preparation of sulfotransferase (Fig. 1), the level of sulfatide did not decrease, supporting the presence of a catabolic enzyme in the partially purified material.


Figure 1: Detection of n-sulfatidase activity in partially purified sulfotransferase and its absence in a highly purified preparation of sulfotransferase. Partially purified sulfotransferase (about 10 µg of protein) prepared by elution from a PLP-ligated column, an assay for sulfotransferase activity, resulted in an initial increase in sulfatide formation followed by a decrease in sulfatide level. Details are described under ``Experimental Procedures.'' This fall in activity ( open circles) indicated the presence of a sulfatidase. With a highly purified preparation of sulfotransferase prepared by Sephacryl S-200, PLP, and ATP-ligated column chromatography and assayed as described (7), no fall occurred in sulfatide level.



Sulfatidase activity in the partially purified brain microsomal preparation was shown directly following the addition of S-labeled sulfatide to a reaction mixture used for sulfotransferase assay. A decrease in sulfatide level was found after 30 and 60 min of incubation, and this decrease was accompanied by an increase in aqueous [S]sulfate (Fig. 2). The aqueous S fraction (96%) was precipitated by barium chloride, indicating the generation of free sulfate.


Figure 2: Catabolism of labeled sulfatide. The reaction mixture contained partially purified enzyme (10 µg of protein) imidazole buffer (100 mM, pH 7.2), 2.5 mM MgCl, 0.5% Triton X-100, and S-labeled sulfatide (10,000 cpm, about 10 nmol) in a total volume of 0.5 ml. The enzyme produced a decrease in sulfatide level, which was linear for 30 min, and the decrease in sulfatide was accompanied by an increase in SOproduction from sulfatide for the 30-min period.



Purification of n-Sulfatidase

n-Sulfatidase was purified by column chromatography on Sephacryl S-200; the peak n-sulfatidase activity corresponded to a molecular mass of 72,000 for the enzyme. The fractions containing sulfatidase activity were subjected to IEF on a Rotofor using ampholytes from pH 5-8. The active fraction was recovered from the Rotofor chamber at pI 7.6 (Fig. 3 A), and sulfotransferase activity was recovered at pI 4.9. This procedure was repeated and resulted in a 600-fold purification over the fraction obtained from the Sephacryl column (). An IEF tube gel was run, and the pI was determined as 7.7 (Fig. 3 B) using a surface electrode. The 600-fold purified sulfatidase fraction was subjected to PAGE on a native gel; a single band was obtained, which was cut out and eluted. This band possessed n-sulfatidase activity and, when subjected to SDS-PAGE, formed two bands corresponding to molecular masses of 58 and 66 kDa (Fig. 4). No sulfotransferase activity was detected in the 600-fold IEF purified fraction following incubation with galactocerebroside and PAPS.


Figure 3: A, purification of n-sulfatidase by IEF. Details are described under ``Experimental Procedures.'' This figure illustrates the pH profile and n-sulfatidase activity in Rotofor fractions of Sephacryl S-200 preparation of n-sulfatidase. B, IEF tube gel of n-sulfatidase. Purified n-sulfatidase (about 0.5 µg of protein was applied to a capillary tube gel containing 1% 7-9 ampholyte and focused at 750 V for 3.5 h. Activity was detected at arrow corresponding to a pI of 7.7 determined with a surface micro pH electrode. Gel shown was analyzed as above and silver stained. A gel was run with the following standards and silver stained: from positive 1, human hemoglobin C (pI 7.5); positive 2-4, lentil lectin (3 bands) (pI 7.8, 8.0, 8.2); positive 5, cytochrome C (pI 9.6).



Regeneration of n-Sulfatidase Activity Following SDS-PAGE

The gel was treated as described under ``Experimental Procedures,'' and the proteins were assayed for enzyme activity. n-Sulfatidase activity was present in both bands; the 66-kDa protein possessed 1.6-fold the enzyme activity of the smaller, 56-kDa protein (290 versus 178 nmol of sulfatide hydrolyzed/mg of protein/30 min).

Differentiation of n-Sulfatidase from Sulfotransferase and Arylsulfatases

As noted above, highly purified preparations of the sulfotransferase did not show a reduction in sulfatide level on prolonged incubation (Fig. 1). Further evidence that the two enzymes were distinct was obtained following chromatography on Sephacryl S-200 and IEF. In addition, IEF of the active fraction from Sephacryl S-200 chromatography gave a pI value of 7.7 for the sulfatidase and a pI of 4.9 for the sulfotransferase, indicating the differing characteristics of the two enzymes.

To distinguish the activity of this sulfatidase from that of the lysosomal catabolic enzyme, arylsulfatase A, enzyme preparations were incubated at pH 5.5. No sulfatidase activity was evident at pH 4.0, 4.5, and 5.5, although sulfatide catabolism occurred at pH 7.2. The pH curve for n-sulfatidase over the range 5.5-8.0 is shown in Fig. 5 ; the activity of the enzyme falls rapidly at pH values less than 6.8. In addition, arylsulfatase A activity was not detected in purified sulfatidase preparations when assayed at pH 5.0 using p-nitrocatechol sulfate or [S]sulfatide as substrate, and p-nitrocatechol sulfate was not hydrolyzed by n-sulfatidase at pH 7.2 (data not shown).


Figure 5: pH curve for n-sulfatidase activity. Buffers were prepared at the pH values indicated. The reaction mixture contained 16.7 mM [H]sulfatide and enzyme (active fraction from Sephacryl S-200 column chromatography). Following incubation (30 min, 37 °C), H-cerebrosides were separated by DEAE column chromatography and counted.



Properties of n-Sulfatidase

A time course for sulfatide hydrolysis showed that the reaction reached a maximum at 30 min incubation and then leveled off. The Kwas determined as 5.8 10 M. The hydrolysis of sulfatide was dependent on the amount of sulfatide added over the range 5-50 µg/ml (Fig. 6). Arylsulfatases are known to bind to concanavalin A, and attempts were made to determine whether the n-sulfatidase possessed this property; n-sulfatidase did not bind to a concanavalin A column. Vitamin K, which we have shown to enhance the activity of brain galactocerebroside sulfotransferase (5, 6) , was examined for its ability to enhance the activity of n-sulfatidase. An assay of n-sulfatidase with and without vitamin Kshowed that vitamin K enhanced n-sulfatidase activity approximately 3.3-fold over the preparation without vitamin K (Fig. 7).


Figure 6: Lineweaver-Burk plot for n-sulfatidase. The conditions are those described in Fig. 2. 1/v are reciprocal values of micrograms of sulfatide hydrolyzed per 30 min per microgram of protein.




Figure 7: Enhancement of n-sulfatidase activity by vitamin K. A sulfatidase assay was performed using [S]sulfatide (6.5 nmol) plus 50 µg of vitamin K( open circles) and without vitamin K ( closed circles). After incubation at 37 °C for the times indicated, the reaction was stopped by the addition of chloroform:methanol (2:1), and the sulfatides were extracted and counted.



Minimal Requirements for n-Sulfatidase Activity

There was no requirement for Mg, ATP, or dithiothreitol; phosphatidylserine, which had been shown to stimulate neutral sphingomyelinase activity (20) , had no effect on n-sulfatidase. PAPS, adenosine 3`-5`-diphosphate, lysosulfatide, galactocerebroside, and ceramide were not inhibitory at a level of 0.5 µM.

Substrate Specificity

The n-sulfatidase possesses a higher degree of specificity than arylsulfatase A since, as noted above, the artificial substrate p-nitrocatechol sulfate, which is used to assay arylsulfatase A, was not hydrolyzed by n-sulfatidase at pH 7.2. [S]Lysosulfatide was not hydrolyzed by n-sulfatidase, and [H]estrone sulfate, a substrate for arylsulfatase C, was not catabolized by n-sulfatidase when assayed as described (21) .


DISCUSSION

n-Sulfatidase was detected following studies on the purification of brain galactocerebroside sulfotransferase. Assays of the sulfotransferase with partially purified preparations showed an initial formation of the product sulfatide, which after 90 min was degraded. The degradation after 90 min suggested the presence of a sulfatidase acting at this time. The apparent lack of sulfatidase activity in the first 90 min is due to the excess sulfotransferase activity, and sulfatidase activity becomes apparent following depletion of PAPS and the resulting loss of sulfotransferase activity after 90 min. The lability of PAPS in sulfotransferase assays has been previously noted (18, 22, 23) . With a highly purified preparation of sulfotransferase, degradation of sulfatide does not occur.

The n-sulfatidase has been purified 600-fold over a Sephacryl S-200 fraction using IEF. Since we could not detect activity in the original microsomal fraction, the degree of purification is probably severalfold higher since we do not account for any purification obtained as a result of the Sephacryl chromatography. The 66-kDa band seen following SDS-PAGE, which contains the major activity following regeneration, may correspond to the 72-kDa activity peak from Sephacryl S-200 chromatography, indicating that the enzyme is monomeric. Since regeneration of enzymatic activity was found for both 66- and 58-kDa bands, the relationship if any between the two proteins remains to be determined.

The n-sulfatidase differs from the biosynthetic enzyme sulfotransferase in several ways. n-Sulfatidase has no requirement for ATP or Mg; the pI for n-sulfatidase is 7.7, whereas that for sulfotransferase is 4.9; the sulfotransferase is retained by an ATP-ligated column, whereas the n-sulfatidase is not. Because of the wide difference in pI values of sulfotransferase and n-sulfatidase, there is little chance of contamination of sulfatidase with the sulfotransferase. In addition, purified sulfotransferase had no sulfatidase activity, and n-sulfatidase preparations possessed no detectable sulfotransferase activity. These facts would rule out any involvement of the sulfotransferase in sulfatide degradation. It is interesting to note that sulfotransferase activity can be recovered following the IEF procedure at a low pH.

It was important to determine that the neutral sulfatidase we have described differs from known sulfatases. The major difference was in its activity at a neutral but not at an acidic pH. It also differs from arylsulfatase C, which is active at neutral pH and which catabolizes cholesteryl and estrone sulfate since the n-sulfatidase does not catabolize estrone sulfate. Moreover, arylsulfatase C and other arylsulfatases bind to concanavalin A-ligated columns (24, 25) , whereas n-sulfatidase does not. The inability to bind to concanavalin A would suggest that the enzyme is not glycosylated or does not contain the required mannose residues. Our results indicate, therefore, that this n-sulfatidase is a distinct enzyme. Based on its activity at a neutral pH and the fact that lysosomes have been removed during the initial preparation of the microsomes, the probable location of this enzyme is the cell membrane, although the exact location has yet to be determined. A further distinguishing feature was the high degree of substrate specificity of the enzyme; as noted above, p-nitrocatechol sulfate, the artificial substrate for arylsulfatase A, lysosulfatide, and estrone sulfate were not degraded.

The highly restricted substrate specificity and a neutral pH optimum may suggest a role for n-sulfatidase in a specific mechanism such as cell signaling rather than a purely degradative role. A role in cell signaling has been demonstrated for another neutral enzyme of sphingolipid catabolism, sphingomyelinase, where a sphingomyelinase cycle has been extensively studied (see Refs. 11 and 12 for reviews). Sulfatides and the products of sulfatide catabolism, galactocerebroside and ceramide (9, 10, 26, 27, 28, 29) , have also been shown to participate in cell signaling. Sulfatides bind to a number of compounds, including thrombospondin, p-selectin, and gp120 (see Ref. 30 for review; see also Refs. 24 and 31), and participate in cell signaling via p-selectin (32) in macrophage-endothelial cell interaction.

In addition to our previous studies demonstrating a role for vitamin K in brain sulfatide biosynthesis and turnover (4, 5, 6) , we now have used this property to prepare [S]sulfatide of a higher specific activity than previously obtained. This result confirms a regulatory role for vitamin K in brain sulfatide metabolism. We have shown previously that vitamin K enhances the activity of brain galactocerebroside sulfotransferase in vivo and in vitro (6, 7) . We now show that vitamin K enhances the activity of n-sulfatidase; the mechanism of the vi-tamin K activation of n-sulfatidase will be the subject of future studies.

  
Table: Partial purification of n-sulfatidase by Sephacryl S-200 chromatography and IEF



FOOTNOTES

*
This work was supported by Research Center for Minority Institutions Grant RR03060, a PSC-CUNY award, and a grant from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Microbiology, CUNY Medical School, 138th St. and Convent Ave., New York, NY 10031. Tel.: 212-650-7788; Fax: 212-650-6696.

The abbreviations used are: PAPS, 3`-phosphoadenosine-5`-phosphosulfate; n-sulfatidase, galactocerebroside sulfate sulfatidase; PLP, pyridoxal-5`-phosphate; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing.

S. K. Sundaram, J.-H. Fan, and M. Lev, unpublished observations.

M. C. Seidel, personal communication.


ACKNOWLEDGEMENTS

We thank Peter Cherry for helpful discussion on enzyme kinetics.


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