SHORT COMMUNICATIONS 48I

I I R. AXEN AND J. PORATH, Acta Chem. Scan&, 18 (1964) 2193. 12 R. AXEN, J. PORATH AND S. ERNBACK, Nature, 214 (I967) 13o2. 13 J. PORATH, R. AXEN AND S. ERNBACK, Nature, 215 (1967) 1491.

Received Ju ly 25th, 1969

Biochim. Biophys. Acta, 191 (1969) 478-481

BBA 63421

Lysosomal localization of sphingomyelinase in rat liver

Several recent studies have shown tha t rat- l iver lysosomes conta in an acid hydrolase tha t splits sphingomyel in to ceramide and phosphorylcholine 1-4. However, it is no t clear from the data given whether the enzyme is restricted solely to lysosomes or is present also in other hepatic cell organelles. The results of HELLER AND SHAPIRO 1 suggest a p redominan t lysosomal localization of sphingomyelinase, bu t these authors have found no detectable ac t iv i ty in the post-mitochondrial supe rna tan t fraction, even though this fraction conta ined 30% of the tota l acid phosphatase activity. The da ta of WEINREB et al. 3 are even more puzzling. The dis t r ibut ion pa t t e rn reported by these authors for the enzyme in normal rat liver allows for an association with

Acicl phosphotose SphincJomyelinase

8 L L

I S N P $

ii Cylochr orne oxldose Esterose

P S i

; , , , ; o ; , ,

S

' ,;o Per cent of totoL protein

Fig. I. Intracellular distribution of enzymes in livers of normal rats. Fractions are represented separately in the ordinate scale by their relative specific activity (percentage of total recovered activity/percentage of total recovered protein). In the abscissa scale, each fraction is represented (cumulatively from left to right) by its protein content, expressed as percentage of total re- covered protein.

Biochim. Biophys. Acta, 191 (1969) 481-484

482 S H O R T C O M M U N I C A T I O N S

0_.

Acid phospholose

4

i p l s , /

Sphingomyelinose

ML

P I =

Cytochrorne oxidose Eslerose

P , s f ~ l ML

; ' ' ' ,;o ; ' ' Per cem of totol protein

S

Fig . 2. I n t r a c e l l u l a r d i s t r i b u t i o n o f e n z y m e s in l i v e r s o f r a t s t r e a t e d w i t h T r i t o n W R - 1 3 3 9 - R e s u l t s a r e r e p r e s e n t e d as in F ig . i .

lysosomes of only a minor fraction of the total sphingomyelinase activity of the liver. Nevertheless, they obtained a 97-fold purification of sphingomyelinase in lysosomes purified by floatation from the livers of rats treated with Triton WR-I339, as opposed to a 3I-fold purification of the lysosomal marker acid phosphatase. These discrepan- cies suggest that some unknown factors may have complicated the assay of sphingo- myelinase in these experiments.

The subcellular distribution of sphingomyelinase in rat liver has been reinvesti- gated in the present work. The following experimental conditions were used for assay of the enzyme. The reaction mixture, in a total volume of o.25 ml, contained 7-5 mM sphingomyelin (dispersed by sonication in the presence of 2% Triton X-ioo), 0.2 M acetate buffer (pH 5.o), 0.8% Triton X-Ioo, and enzyme. After incubation at 37 ° for I h, the reaction was stopped, and the remaining substrate removed by extraction into chloroform as described in a previous paper4; the aqueous phase was then assayed for total phosphate. The excess of water-soluble phosphorus found in the test system over that recovered from the enzyme and from the substrate incubated separately was taken as a measure of enzyme activity. Under these conditions, good proportionality with amount of enzyme was obtained if less than 15% of the added

T A B L E I

DISTRIBUTION OF ENZYMES BETWEEN SUBFRACTIONS FROM LIVERS OF RATS TREATED WITH TRITON W R - I 3 3 9

T h e M L f r a c t i o n o f F ig . 2 w a s f u r t h e r s u b f r a c t i o n a t e d b y f l o t a t i o n t h r o u g h a d i s c o n t i n u o u s s u c r o s e g r a d i e n t n. R e s u l t s a r e g i v e n a s p e r c e n t a g e o f t h e c o n t e n t o f t h e M L f r a c t i o n .

Fraction Density Protein Acid Sphingo- Esterase Cytochrome of (%) phospha- myelinase (%) oxidase sucrose tase (%) (%)

(%)

I i . o 6 2. 5 46 .3 60 .9 4 .9 o .o I I 1.15 i . I 13.9 11.3 3 .8 o .7

I I I 1.21 86 .6 40 .5 38 .6 85 .o 88 .0

Biochim. Biophys. dcta, 191 (1969) 4 8 1 - 4 8 4

SHORT COMMUNICATIONS

T A B L E I I

C O M P L E M E N T A R Y D A T A ON E X P E R I M E N T S OF F I G S . 1 A N D 2

483

Enzyme Untreated rats Rats treated with Triton W R - I 3 3 9

Act iv i ty Recovery* Act ivi ty Recovery (units]g (%) ** (units/g (%) ** liver) * liver) *

Acid p h o s p h a t a s e 9.77 lO4-3 9.00 96.6 Sph ingomye l inase o.o 37 i o i . 2 o.o 36 i o2.9 Es te rase ~ 2 i . o 99.9 124 . 4 94. o Cy tochrome oxidase 14.8 90.9 17.5 89.2 Pro te in 217. 5 98. 7 200.o 98.2

* One u n i t of cy toch rome oxidase a c t i v i t y is the a m o u n t of enzyme oxid iz ing 90 % of the reduced cy tocb rome c p resen t in ioo ml of i ncuba t i on m i x t u r e per min ; one un i t of hydro la se a c t i v i t y is the a m o u n t of enzyme sp l i t t i ng I / , m o l e of subs t r a t e per min (ref. 6). P ro te ins are g iven in nlg/g of l iver .

** Sum of ac t iv i t i e s of f rac t ions expressed as pe rcen tage of a c t i v i t y of h o m o g e n a t e (nuclear f rac t ion plus cy top l a s m ic ext rac t ) .

substrate was consumed during the reaction. Assay methods for reference enzymes were those described previously 5 except for the determination of esterase, which was done by an automated method developed in our laboratory by Dr. Marco Baggiolini, using a-naphthyl acetate as substrate.

In Fig. I are shown the results obtained in a fractionation experiment performed according to DE DuvE et al. 6 on normal rat liver. An abbreviated version of the same fractionation scheme, in which the heavy (M) and light (L) mitochondrial fractions were separated as a single fraction, was followed in another experiment performed on the livers of rats killed 3.5 days after an intraperitoneal injection of Triton WR-I339 (0.85 g/kg body weight). As shown by WATTIAUX et al. 7, this t reatment causes the lysosomes to be filled with the low-density detergent, allowing their further purifi- cation by floatation in a density gradient 5. Fig. 2 and Table I show the results ob- tained on the animals treated with Triton WR-I339. Enzyme recoveries in the fractionation experiments are listed in Table II . Esterase was chosen as a microsomal marker and cytochrome oxidase as a mitochondrial marker.

There is a striking similarity in the distribution patterns of sphingomyelinase and acid phosphatase. The only difference is a somewhat higher level of acid phos- phatase in the microsomal fraction and a correspondingly lower level of this enzyme in the final supernatant. This difference is more pronounced after t reatment of the animals with Triton WR-I339, when a greater breakage of lysosomes upon homo- genization is likely to occur. The known ability of acid phosphatase to adsorb onto microsomes s is probably responsible for it, I t should be noted further that all the enzyme recoveries were quite satisfactory in these experiments, thus providing no indication of unknown interferences with the enzyme assays.

This conclusion has some bearing on the pathogeny of Niemann-Pick disease, which is characterized by the accumulation of sphingolipids in a number of tissues. I t has recently been found that sphingomyelinase activity is very low or absent in the tissues of patients afflicted with this disease 9-n. Morphological observations

Biochim. Biophys. Acta, 191 (1969) 481-484

484 SHORT COMMUNICATIONS

i n d i c a t e t h a t t h e a b n o r m a l l ip id d e p o s i t s occu r w i t h i n m e m b r a n e - b o u n d e d c y t o -

p l a smic vacuo le s 1~-14. These r e su l t s sugges t t h a t N i e m a n n - P i c k d isease is a t y p i c a l

i n b o r n l y s o s o m a l d isease , as de f ined b y HERS 15.

This r e s ea r ch was s u p p o r t e d b y G r a n t GB-5796 X f rom t h e N a t i o n a l Science

F o u n d a t i o n . I wish to t h a n k Mrs. M a r y a n n F o w l e r for he r exce l l en t t e chn i ca l ass is t -

ance .

The Rockefeller University,

New York, N . Y . ( U . S . A . )

STANLEY FOWLER*

i N. HELLER AND B. SHAPIRO, Biochem. J., 98 (1966) 763 . 2 J. N. KANFER, O. M. YOUNG, D. SHAPIRO AND R. O. BRADY, .]. Biol. Chem., 241 (1966) lO81 3 N. J. WE1NREB, a . O. BRADY AND A. L. TAPPEL, Biochim. Biophys. Acta, 159 (1968) 141. 4 S. FOWLER AND C. DE DUVE, d r. Biol. Chem., 244 (1969) 471. 5 V. LEIGHTON, B. POOLE, H. BEAUFAY, P. BAUDHUIN, J. W. COFFEY, S. FOWLER AND C. DE DUVE, J. Cell Biol., 37 (1968) 482.

6 C. DE DUVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPELMANS , Biochem. J., 60 (1955) 604

7 R. WATTIAUX, M. WIBO AND P. BAUDHUIN, in A. V. S. DE Reuck AND M. P. CAMERON, Ciba Foundation Symp. Lysosomes, Little, Brown, Boston, 1963, 176.

8 J. BERTHET, L. BERTHET, F. APPELMANS AND C. DE DUVE, Biochem. J., 50 (1951) 182. 9 ]~. O. BRADY, J. N. KANFER, M. B. 3/~OKC AND D. S. FREDRICKSON, Proc. Natl. Acad. Sci.

U.S., 55 (1966) 366. io P. B. SCHNEIDER AND E. P. KErCNEDY, J. Lipid Res., 8 (1967) 202. I I H. R. SLOAN, g. W. UHLENDORF, J. N. KANFER, R. O. BRADY AND D. S. FREDRICKSON, Bio-

chem. Biophys. Res. Commun., 34 (1969) 582. 12 B. W. VOLK AND B. J. WALLACE, 3m..]. Pathol., 49 (1966) 203. 13 S. S. LAZARUS, V. G. VETHAMANY, L. SCFINECK AND B. W. VOLK, Lab. Invest., 17 (1967) 155. 14 S. LusE, in S. M. ARONSON AND B. W. VOL~:, Inborn Disorders of Sphingolipid Metabolism,

Pergamon Press, London, 1967, p. 93- 15 H. G. HERS, Gastroenterology, 48 (1965) 625.

R e c e i v e d J u n e 26th, 1969

Present address : Laboratoire de Chimie Physiologique, Universitd de Louvain, 6, Deken- straat, Louvain, Belgium.

Biochim. Biophys. Acta, 191 (1969) 481-484