312

Biochimica et Biophysics Acta, 488 (1977) 312-321 @ Elsevier/~or~h-Holland Biomedical Press

BBA 57036

THE ISOLATION AND CHARACTERIZATION FROM HUMAN PLACENTAL TISSUE

OF SPHINGOM~ELINASE

PETER

E. GAL

G. PENTCHEV,

ROSCOE

0. BRADY,

ANDREW

Repartment ofHealth, Education and Welfare, National Institutes Bethesda, .&Id. 20014 (U.S.A.) (Received

February

17th,

and SUE R. HIBBERT

of Health,

1977)

Human placental sphingomyeli~ase activity was eluted as a single symmetrical peak from Sephadex G-200 with a molecular weight of 290 000; however, the enzyme behaved heterogeneously on ion exchange chromatography. A specific species of sphingomyelinase was purified approx. 10 OOO-fold to a constant specific activity of 274 000 nanomol of sphingomyelin hydrolyzed per mg protein per h. When the purified enzyme was examined on sodium dodecyl sulfate disc gel electrophoresis, two distinct protein bands in approximately equal proportions with molecular weights of 36 800 and 28 300 were found. The specificity of the enzyme is directed towards both the hydrophilic phosphocholine and the hydrophobic ceramide moieties of sphingomyelin. Possible interrelationships between the heterogenous forms of placental sphingomyelinases are discussed.

Introduction Sphingomyelin is the most abundant sphingolipid in tissues of higher animals. Its catabolism is initiated by the enzyme sphingomyelin~e, and the products of the reaction are phosphocholine and ceramide [l J. The substrate specificity of this phosphodiesterase is readily distinguished from other phospholipases in mammalian tissues. The heritable autosomal recessive disorder in humans called the Niemann-Pick disease is caused by an insufficiency of sphingomyelinase activity in the organs and tissues of the afflicted individuals [ 2 J . In order to begin a ~h~acte~zation of the molecular and catalytic properties of this enzyme and to initiate important studies towards the potential application of enzyme replacement therapy in Niemann-Pick disease it was necessary to obtain human sphingomyelinase acti&y in highly purified form. Although the partial purification of sphingomyelinase from various sources has been reported [ 1,3--6], none of these preparations are of sufficient purity

313

for either replacement studies or for precise characterization of the enzyme. In this communication, a procedure for the isolation of highly purified human placental sphingomyelinase is described and the properties of this enzyme are presented. Experimental

procedure

Materials. [Choline-Me-14C]sphingomyelin (0.37 mCi/mmol) was synthesized as described previously [ 71. Sphingosylphospho-[le-14C]choline [8] and p-nitrophenylphosphocholine [ 91 were prepared according to the published procedures. Cutscum (isoctylphenoxypolyoxyethanol) was obtained from Fisher Chemical Company, Silver Spring, Md. Dithiothreitol was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Sephadex products were obtained from Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey. Fresh human placentas were obtained from local hospitals over a period of 48 h and kept at 0°C until processed. Assay of enzyme activity. Sphingomyelinase activity was determined routinely by incubating the enzyme in 0.2 ml of 100 mM potassium acetate buffer, pH 5.0, containing 0.5 mg Cutscum, 0.5 mg human serum albumin and 12 nmol of [ 14C]sphingomyelin. The reaction was stopped by the addition of 1.0 ml of 1% (w/v) solution of human serum albumin and 0.1 ml of 100% (w/v) solution of trichloroacetic acid. Following centrifugation, the amount of [14C]phosphocholine in the supernatant solution was measured as described previously [ 11. One unit of enzymatic activity represents the hydrolysis of 1 nmol of sphingomyelin per h under these conditions. Protein was determined according to Lowry et al. [lo] using human serum albumin as standard. Purification of sphingomyelinase: extraction of enzyme. All procedures were carried out at 4°C including centrifugation, unless otherwise stated. Placentas were freed of their loose connective tissue and then passed through a meat grinder. Typically 7.0 Kg of ground tissue was suspended in 14 1 of distilled water and stirred for 1 h. The suspended material was homogenized in a Waring blendor for 1 min. The homogenate was centrifuged at 10 000 X g for 15 min and the sedimented material was taken up in 4 ~01s. of 25 mM citrate/phosphate buffer, pH 6.0, containing 2 mg of Cutscum per ml. The suspended material was homogenized in a Waring blendor for 1 min. The homogenate was centrifuged at 10 000 X g for 30 min and the supernatant solution was used for further fractionation. Ammonium sulfate fractionation. Solid ammonium sulfate (234 g/l) was added to the extracted enzyme and stirred until it was completely dissolved. The suspension was centrifuged at 10 000 X g for 30 min. Approx. 20% of the total protein could be removed in this first precipitate. An additional 96 g of ammonium sulfate were added to each liter of he supernatant solution. After centrifuging as before, the sedimented material was dissolved in sufficient 25 mM citrate/phosphate buffer pH 6.0 containing 2 mg of Cutscum per ml, to yield a final protein concentration of 40-50 mg/ml. Cutscum was added to this and all subsequent buffers to avoid aggregation of the enzyme. The solution was dialyzed 48 h against 25 mM citrate/phosphate buffer, pH 4.2, containing 2 mg of Cutscum per ml. The retentate was centrifuged at 48 000 X g for 30 min.

314

~h~~~at~~a~h~c ~~s~~~tio~. The pH values of the various buffers were determined by trial to provide the best resolution and yield. The supernatant solution obtained in the preceding step was adjusted to pH 5.0 with 1 M NaOH and aliquots of 350 ml of this solution were carefully layered on columns of Sephadex G-200 (90 X 10 cm) that had been previously equilibrated with 25 mM citrate/phosphate buffer, pH 5.0, containing 2 mg of Cutscum per ml. The samples were percolated through the columns with the equilibrating buffer solution and the fractions containing the greatest sphingomyelinase activity were pooled. The pH was adjusted to 6.5 with 1 M NaOH. The solution was then applied to a column of DEAE-Sephadex (70 X 10 cm) previously adjusted to pH 6.5 with 25 mM citrate/phosphate buffer containing 2 mg of Cutscum per ml. Sphingomyelinase did not bind under these conditions, nor at higher pH values where stability did not become a major problem, and all the enzymatic activity was eluted from the column with equilibrating buffer solution. The pH of the eluate was adjusted to 4.8 with citric acid and 20% glycerol (v/v); 5 mM EDTA, and 1 mM dithiothreitol were added. The solution was applied to a column of SP-Sephadex C-25 (50 X 5.0 cm) previously equilibrated with buffer, glycerol, EDTA, Cutscum and dithiothreitol as above. The enzyme was retained as a sharp band at the top of the column. Sphingomyelinase was eluted with 2 1 of a linear gradient of NaCl ranging from 0 to 400 mM, that contained 25 mM citrate/phosphate buffer pH 4.8,20% glycerol (v/v), 5 mM EDTA Cutscum and 1 mM dithiothreitol. Fractions between 60 to 90 mM NaCl contained the highest sphingomyelin~e activity. These fractions were pooled and dialyzed against 25 mM citra~/phosphate buffer, pH 4.5, that contained 2 mg of Cutscum per ml, 40% glycerol (v/v), 5 -mM EDTA and 1 mM dithiothreitol. The dialyzed sample was applied to a SP-Sephadex C-25 column (30 X 2.0 cm) equilibrated with the above solution at pH 4.5. The column was washed successively with 100 ml of the equilibrating buffer solution and 200 ml of the same solution containing 60 mM NaCl. Sphingomyelinase was eluted from the column with 200 ml of the buffer solution with a linear gradient of NaCl between 60 and 100 mM. Peak enzyme-containing fractions were pooled, dialyzed as before, and applied to a 10 ml SP-Sephadex C-25 column (15 X 0.9 cm) equilibrated with the dialyzing solution. The column was washed with 100 ml of the same buffer solution and subsequently with this buffer cont~n~ng 60 mM NaCl. The enzyme was again eluted with 200 ml of the glycerol-buffer solution with a linear gradient of NaCl between 60 and 100 mM. Following this third chromatography on SP Sephadex, the specific activity of the enzyme preparation remained constant when eluted from an additional SP column under identical conditions. The final product was stabilized by dialyzing the enzyme against 25 mM citrate/phosphte buffer, pH 5.0, containing 2 mg of Cutscum per ml, 60% glycerol (v/v), 5 mM EDTA and 1 mM dithiothreitol. The enzyme retained full catalytic activity over a S-month period when stored at -20°C under these conditions.

Results In contrast with previous experiments on the isolation of glucocer~brosid~e from human pIacental tissue [II], the detergent Cutscum was needed to maxi-

315

mize the extraction of sphingomyelin~e (Table I). The initial steps in the purification of the solubilized enzyme represent standard techniques. The DEAE exclusion column showed more variation than the other steps yielding enzyme fractions 2 to 4-fold purification (Table II). As the extract washed through the column, stationary and welldefined yellow bands could be seen forming throughout the gel bed. The elution of the enzyme from cation exchange resins presented a major obstacle in the isolation of sphingomyelinase. Irrespective of the pH (4.0-6.0), type of resin (CM-Sephadex, SF-Sephadex, CM-cellulose, SP-cellulose), detergent (Cutscum, Triton, sodium taurocholate) or variation in the concentration of detergent (2.0-20 mg/ml) physical treatment (sonication, freezing, warming), the enzyme eluted with a sharp front and extended tailing (Fig. 1).Part A illustrates a typical elution profile of sphingomyelinase activity and protein from SP-Sephadex columns with a linear gradient of NaCl. Two significant observations were made when the eluted sphingomyelinase was pooled according to the four fractions indicated in Fig. 1 and rechromatographed on four separate identical columns. Sphingomyelinase from Fraction I eluted as a sharp symmetrical peak which was distinguishable from the other three enzyme fractions. The activities of the remaining three fractions appeared essentially indistinguishable as broad peaks on rechromatography. Although the activity of Fraction I represented only a portion of the total sphingomyelinase eluted from the column, the specific activity of this particular fraction was considerably higher than that of the remaining peaks. In addition, this activity, in contrast to other sphingomyelin~e fractions, eluted as a sharp peak. Further purification procedures were developed for this species of sphingomyelinase. Two additional chromatographic steps were needed to obtain enzyme with maximum specific activity (Table III). The question of the purity of the final enzyme preparation cannot be answered unambiguously at the present time. When various sphingomyelinase preparations with widely different degrees of purity were applied to gel electro-

TABLE

I

EXTRACTION

OF SPHINGOMYELINASE

FROM

HUMAN

PLACENTAL

TISSUE

Fresh placental tissue was passed through a meat grinder. A portion of the ground tissue was homogenized directly with 3 ~01s. of the indicated buffers in a Waring blendor and subseqnently centrifuged at 48 000 X g for 30 min. Another portion of tissue was first suspended with stirring in 2 ~01s. of distilled water for 15 min and subsequently centrifuged at 10 000 X g for 10 min. A homogenate of the washed tissue was then prepared as above. The conditions for measuring enzymatic activity are defied in the text. Homogenizing

medium

Water wash

Enzymatic

activity

Units per g of tissue

Phosphate, 20 mM Buffer. pH 6.0 Phosphate,

recovered Units per mg of protein

+

161 61

4.4 a.7

+

251 182

6.3 25.0

20 mM

Buffer, pH 6.0, + 2.0 mg of Cutscum per ml --___

316 TABLE

II

INITIAL

STEPS IN THE PURIFICATION

OF HUMAN

PLACENTAL

SPHINGOMYELINASE

The values represent the average of five preparations. The initial extract was prepared from 7 kg of fresh human placental tissue. Sphingomyelinase assays were made at a suboptimal concentration of [14ClsphingomyeIin (60 PM) in order to conserve the labeled substrate. __~~ Fraction

or step

Activity (units)

Protein

Volume

(mg)

(mu

Initial extract

2.5

106

1.3

10s

2.2

103

Ammonium sulfate precipitation

2.4

106

2.2

104

3.8

10’

Specific activity (unitsimg protein)

Recovery (%o)

Purihcation (-fold)

19 109

96

5.7

10.8

Acid dialysis

1.8

106

8.8

103

4.3

102

205

72

G-200

filtration

8.2

105

7.4

102

5.5

102

1100

33

58.4

DEAE exclusion

7.0

105

2.9

102

1.4.

10’

2410

28

126.8

IA

-1

mM

Nacl

IN ELUTION

BUFFER

Fig. 1. Elution profile of partially purified sphingomyelinase from two successive SP-Sephadex columns. A sample of partially purified sphingomyelinase (7.0 X lo5 units and 250 mg protein) in 100 ml of 25 mM citrate/phosphate buffer, PH 4.8. containing 2.0 mg of Cutscum per ml, 20% glycerol, 5 mM EDTA and 1 mM di;hiothreitoI were added to a SP-Sephadex column (25 X 2.5 cm) previously adjusted and equilibrated with the same buffer. The column was eluted with 1 1 of this buffer with a linear gradient of NaCl from a concentration of 0400 mg. (A), the effluent was pooled into the four indicated fractions and dialyzed against the initial buffer to remove N&l. The total sphingomyelinase activity and protein content of the four pools were: Fraction I, 5.8 X lo4 units and 3.9 mg protein; Fraction II, 2.0 X lo5 units and 50 mg protein; Fraction III, 1.2 X lo5 units and 120 mg protein; Fraction IV, 3.0 X lo4 units and 60 mg protein. (B) following dialysis. the fractions were rechromatographed on four identical columns (25 X 2.5 cm) as before. The protein elution profiles of B II and B III were essentially indisguisbable from B IV. Solid line (-_) indicates enzyme activity. Broken line (- - - - - -) indicates protein.

317

TABLE

III

FINAL

STEPS IN THE ISOLATION

step

OF HUMAN

Activity (units)

PLACENTAL

Protein

Volume

(mg)

(ml)

SPHINGOMYELINASE Specific activity (units . (units protein

1st SP-Sephadex

column

3.5.104

2nd SP-Sephadex

column

1.7

3rd SP-Sephadex

column

8.6.103

. 104

. lOI 10’

2.33

8.7

0.24

2.8

0.044

1.1 . 101 I---_r____-

Purifi-

(o/o)

(-fold)

. mm’ . h-l )

15,000

1.40

791

71.000

0.68

3700

195,000

* Based on the values of the initial extract in Table II. ** The specific activity at this step is 275 000 unitslmg of protein

Recovery*

**

at substrate

0.34

10,300

saturation.

phoresis systems containing no denaturing agents, the protein migrated very slowly into the gel as a single band indicating major aggregation of protein during the stacking procedure. The enzyme was inactivated under conditions required for isoelectric focusing. The only means available for qualitative anal-

-

1

“““‘l’i .1 2

3

.4

5

.6

.7

.8

9

1.0

I?

Fig. 2. Protein patterns obtained on SDS gel electrophoresis at various stages gomyelinase. The discontinuous buffer system of Neville [121 was employed Ph 9.81. The protein samples in 0.1 M NaZC03, 1% SDS (w/v), 8 M urea and were heated at 1OO’C for 5 min followed by dialysis against upper gel buffer (w/v). 0.05% dithiotbreitol and 2.0% sucrose (w/v). Aliquots of 0.5 ml were glass tubes containing 0.5 ml of stacking gel and 2.5 ml of separating gel. The right contained sphingomyelinase fractions with specific activities of 100, 195 000. The gels were stained with 0.1% Coomassie Blue.

in the purification of sphinwith a lower gel buffer of 10% mercaptoethanol (v/v) cl21 containing 0.1% SIX applied to 5-mm diameter alternate tubes from left to 1100, 7000. 70 000 and

Fig. 3. Estimation of molecular weight of sphingomyelinase subunits by SDS gel electrophoresis. The RF values of standard subunit markers were compared to that of highIy purified human sphingomyeiinase as described in Fig. 2. The standard proteins were: (1) P-galactosidase Wr = 130 000). (2) phospborylase A (Mr = 94 000). (3) bovine serum albumin f&f, = 68 000). (4) catalase (Mr = 60 000). f5) ovalbumin @fr = 43 000, (6) DNAaw (iVr = 31000), (7) chymotrypsinogen @fr = 25 7001, (8) hemoglobin @fr = 15 500) and (9) cytochrome c (MT = 11 700). The open circles (0) represent the sphingomyelinase bands.

318

ysis was sodium dodecyl sulfate (SDS) gel electrophoresis. Two distinct bands of protein with molecular masses of 36 800 and 28 300 were present in the final enzyme preparation that had a specific activity of 195 000 units~mg of protein (Figs. 2 and 3). An enzyme fraction of lesser specific activity (70 000 units/mg of protein) contained 7 protein bands including the two bands seen in the highly purified preparation. It appears that the ratio of the degree of staining of the two bands in the last two purification steps does not notably vary, suggesting the possibility of a single protein entity composed f two different sized subunits, The specific activity of the purified sphingomyelinase represented a 10 300 fold enrichment over the initial extract. Since a major portion of subcellular sphingomyelinase activity has been shown to be associated with lysosomes [ 131, it was of interest to learn whether the purified sphingomyelinase preparation contained any other lysosomal enzyme activity. The purified prep~ation was free of the following 13 lysosomal hydrolases the substrates of which were available for examination: cu-D-glucosidase, O-D-glucosidase, P-D-galactosidase, /3-L-fucosidase, P-D-fucosidase, a-D-mannosidase, fl-D-mannosidase, ol-D-glucosaminidase, cu-D-galactosaminidase, N-acetyl-fl-D-glucosaminidase, fl-D-glucuronidase, ai-L-arabinosidase and @-D-xylosidase. The purified enzyme is very unstable in the absence of high concentrations of glycerol. Consequently, the molecular weight of the isolated enzyme was not determined. However, less purified preparations of sphingomyelinase were not dependent on glycerol for stability, and the molecular weight of the partially purified enzyme could be readily determined by chromatography on Sephadex G-200. Sphingomyelinase eluted as a single symmetrical peak with a molecular mass of 290 000 i 20 000 (Fig. 4). In an extensive examination of possible substrates of the purified enzyme, only two compounds containing a phosphodiester linkage were hydrolyzed:

EFFLXNl

VOLUME

Fig. 4. Molecular weight dete~inat~on of crude sp~ingomye~nase on Sephadex G-200. Gel filtration was run in a column (84 X 1.6 cm) of Sephadex G-200 equilibrated with 50 mM citrate~phospbat~ buffer, pH 5.5, containing 2.0 mg of Cutscum per ml. Protein standards employed to calibrate the column were: (1) aldolase (Mr = 158 000). (2) ovalbumin (A!?, = 45 000). (3) chymotrypsinogen (Mr = 25 000) and (4) ribonuclease (M, 7 13 700). Sphingomyelinase activity represented a fresh extract of human placental tissue prepared as described in Methods. V,, elution volume of a particular protein: V,, void volume of the column. Solid circles (0) represent the standard protein markers and the open circles (0) sphingomyelinase activity.

319 TABLE

IV

SUBSTRATE

SPECIFICITY

Sphingomyelin

was assayed

OF PURIFIED as described

HUMAN

in section

SPHINGOMYELINASE on Methods

over a range of 5-500

ftM. The chromo-

genic substrate, 2-hexadecanoyiamino-4~nitrophenylphospbocholine. was assayed in 0.200 ~1 of 50 mM acetate buffer, pH 5.5, over a substrate range of 50-1000 IJM and albumin (0.5 mg) was added to enhance stability. The reaction was stopped and the chromogenic product measured as described 1161. [14CISphingosylphosphocholine was prepared from [14Clsphingomyelin according to the method of Kaller [S] and its hydrolysis was assayed under the conditions used for 114CIsphingomyelin. The hydrolysis of choline-methyl-[l’Clphosphatidylcholiine was assayed using the conditions employed for sphingomyelin or in 50 mM Tris buffer, PH 7.2, and 5 mM CalF as described for phospholipase C from C. perfrininges1171. The other chromogenic substrates were assayed at concentrations of 1 mM with 50 mM acetate buffer, pH 5.0. fncubations were carried out with 100 units of highly purified human placental sphingomyehnase (0.50 fig of protein). Compound

b’ (nmol

. mg-’

Sphingomyelin Sphingosylphosphocholine Phosphatidylcholine O-Hexadecanoylamino-

274 000 0 0

4-nitrophenylphosphocholine p-Nitrophenylphosphocholine Bis-p-nitrophenylphosphate p-nitrophenylphosphate _~

289 000 0 0

protein.

h-l)

Km (iiM) 24.5

204

---._

--~

sphingomyeli~, the natural substrate, and 2-hexadec~oyl~ino-~-n~~ophenylphosphocholine (Table IV). The latter compound has been shown to be a specific substrate analogue and it is used for the detection of homozygotes and heterozygous carriers of Niemann-Pick disease [16]. In support of this, the ratio of activities of the natural to chromogenic substrate did not vary when measured with either crude placental extracts or highly purified sphingomyelinase. The pH hydrolysis profiles with these substrates are illustrated in Fig. 5. When the effects of some potential inhibitors of the hydrolysis of sphingomyelin were examined, only inorganic phosphate was found to affect the reaction (Table V). Albumin was included in the assay mixture when sphingomye-

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

PH Fig. 5. PH profiles of natural and synthetic substrate hydrolysis by purified human sphingomyelinase. Ail (60 PM) and 2-hexaassays were carried out in 50 mM sodium acetate buffer. [ 14C]Spingomyelin decanoyl~~o~-~trophenylphosphoehol~e (200 pM) were incubated with aiiouots containing 0.05 fig denotes natural substrate and broken of enzyme and assayed as previoudy described. Solid line ( -_) line (- - - - ” -) synthetic substrate.

320

TABLE EFFECT

V OF

The

subsrate

tion

with

VARIOUS in this

purified

Additions

SUBSTANCES study

was

sphingomyelinase ._-.-__

incubated

as described

Enzyme (Q

activity

of control)

100

-

50 50

mM mM

sec-

.._.___

KI

_ .._ -_-____ ~~~~~ -

100

50

Mg2+

Methods

_ ~ ~~~~__~~~_.

mM

ca2+

in the

per incubation.

mM

mM

3.6

&M)

-__ _-.._..._._

-

50

9944

(60

gig of protein

ACTIVITY

100

EDT.4

PO&

using 0.05 ~~

_

I_...-.-_.~.-

SPHINGOMYELINASE

Clsphingomyelin

___

Concentration --..

AY

[I4

ON

12

2.0

_. ._-

__.__

mM

100

lin was used as the substrate, since it was discovered dies that the reaction was stabilized by this reagent.

early in the isolation

stu-

Discussion A specific sphingomyelinase fraction of human placental tissue was purified extensively and was shown to be composed of two different sized proteins when examined in SDS gel electrophoresis. It is unlikely that the two protein bands represent multimers of a smaller subunit, since the denaturing and reducing conditions employed during the preparation of the protein sample for electrophoresis were rigorous and extensive. The question of whether the final purified enzyme contains only sphingomyelinase protein components cannot be satisfactorily answered until considerably more of the highly purified protein becomes available for examination. However, the observations of Callahan and coworkers [ 18,191 and the present findings indicate that sphingomyelinase activity of crude tissue extracts is composed of a number of catalytic species. These observations are consistent with a heterogenous quaternary protein structure. Based on a molecular weight of 290 000 for the native enzyme and molecular weights in the range of 28 000-37 000 for subunits, the catalytically active complex can be estimated to be composed of 8-10 monomers. The catalytic specificity of the enzyme is directed towards both phosphocholine and the ceramide moiety (or its close structural analogue Z-hexadec~oylamine~-nitrophenol) since phosphatidylcholine, sphingosylphosphocholine, and p-nitrophenylphosphocholine were not hydrolyzed by the purified sphingomyelinase. Furthermore, there was no detectable hydrolysis of simple mono- or diphosphate esters such as p-nitrophenylphosphate or bis-p-nitrophenylphosphate. It was of particular interest to examine the effect of AY-9944 (tram+1, 4-bis(2chIorobenzylaminomethyl)-cyclohexane dihydrochloride) on purified sphingomyelinase. Although this compound has been known for some time to inhibit cholesterol synthesis f20], it has recently been shown that injection of this agent into young rats causes the formation of cytoplasmic inclusion bodies in the lens and retina that resemble those seen in ~iem~n-Pick disease [21], Examination of lysosomal hydrolase activities in the tissues of the treated animals revealed a specific decrease of sphingomyelinase activity 1221. Since it has been

321

shown in the present experiments that there is no direct inhibition of sphingomyelinase by AY-9944 (Table VI), more involved modes of in vivo inhibition of sphingomyelinase by this compound must be considered. The increasing interest and encouraging results obtained in enzyme replacement in Fabry’s disease [23] and Gaucher’s disease [24] provide a strong stimulus for similar considerations for fi.iemann-Pick disease. This possibility seems particularly reasonable for patients with the Type B form of this disorder where the central nervous system is spared. The availability of highly purified human sphingomyelinase will now provide important opportunities for testing the potential application of sphingomyelinase for this purpose. It will be of critical importance in this respect to test the potential hemolytic activity of the enzyme in animafs and in vitro, since such an effect has been reported for sphingomyelinase activity of microbial origin 1251. Infusion of the enzyme in animals will also provide a model for studying the organ distribution and turnover of the exogenous enzyme (Pentchev, P.G., Kusiak, J.W. and Brady, R.O., unpublished results). Finally, the presently reported isolation and characterization of sphingomyelinase will provide the guidelines for developing the means for the effective large scale preparation of the enzyme. Acknowledgment We thank the National Lipid Diseases Foundation Sue R. Hibbert during these investigations.

for generous

support

to

References 1 Kanfer. J.N., Young, O., Shapiro, D. and Brady, R.O. (1966) J. Biol. Chem. 241,1081-1084 2 Brady, R.O., Kanfer, J.N., Mock, M.B. and Fredrickson, D.S. (1966) Proc. Natl. Acad. Sci. 55.366-369 3 Barnholz, Y., Roitman, A. and Gatt, S. (1966) J. Biol. Chem. 241. 3731-3737 4 Heller, M. and Shapiro, B. (1966) Biochem. J. 98.763-769 5 Schneider, P.B. and Kennedy, E.P. (1967) J. Lipid REs. 8, 202-209 6 Sloan, R.H. (1972) Methods Enzymol. 28, 874-879 7 Shapiro, D. and Flowers, H.M. (1962) J. Am. Chem. Sot. 84.1047-1050 8 Kaller, H. (1961) Biochem. Z. 334.451456 9 Kurioka, S. (1968) J. Biochem. 63,678--680 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193.265-275 11 Pentchev, P.G., Brady, R.O.. Hibbert, S.R., Gal, A.E. and Shapiro, D. (1973) J. Biol. Chem. 5256-5261 12 NevUe, Jr., D.M. (1971) J. Biol. Chem. 246,6328-6334 13 Weinreb. N.J., Brady, R.O. and Tappel, A.L. (1968) Biochim. Biophys. Acta 159.141-146 14 Ho, M.W., O’Brien, J.R., Radin, N.S. and Erickson, J.S. (1973) Biochem. J. 131,173--176 15 Pentchev, P.G. and Brady. R.O. (1973) Biochim. Biophys. Acta 297,491496 16 Gal, A.E. Brady, R.O., Hibbert, S.R. and Pentchev. P.G. (1975) N. Engl. J. Med. 293. 632-636 17 MacFarlane, M.G. and Knight, B.C.J.G. (1941) Biochem. J. 35, 884-902 18 Callahan, J.W., Khalil, M. and Gerrie, J. (1974) Biochem. Biophys. Res. Commun. 58,384-390 19 Callahan, J.W., Khalil, M. and Philippart, M. (1975) Pediatr. Res. 9.908-913 20 Kraml, M., Bagli, J.F. and Dvomik, D. (1964) Biochem. Biophys. Res. Commun. 15.455-457 21 Sakuragawa, M. (1976) Invest. Ophtai. l&1022-1028 22 Sakuragawa, N., Sakuragawa. EM., Kurabara, T., Pentchev, P.G., Barranger, J.A. and Brady, 11977) Science 196,317-319 23 Brady, R.O.. Tallman, J.F.. Johnson, W.G., Gal, A.E., Leahy, W.R.. Quirk. J.M. and Dekaban. (1973) N. Enpl. J. Med. 289, S-14 24 Brady, R.O., Pentchev, P.G., Gal, AX., Hibbert, S.R. and Dekaban, AS. (1974) N. Engl. J. 291.989-993 25 Bemheimer, A.W., Avigad, L.S. and Kim, K.S. (1974) Ann. N.Y. Acad. Sci. 236, 292-306

U.S.

248.

R.O. A.S. Med.