From the
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
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),
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-
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) .
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 CO
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
[
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
[
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 [
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
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
[
We thank Peter Cherry for helpful discussion on enzyme
kinetics.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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 K
of 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
Mg
or ATP for sulfatidase activity; vitamin K
enhanced 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.
(
)
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) .
activation of NF-kB has recently been disputed
(14) .
asphyxiation, 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 MgCl
and 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:H
O, 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.
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
K
in 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.
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) .
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
SO
production 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.
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 K
showed 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) .
; 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.
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.