Lipids in tissues of the eye

Biochimica f? Elsevier

et Biophysics Acta, Scientific Publishing

306 (1973) 329-339 Company, Amsterdam

- Printed

in The Netherlands

BBA 56257

LlPIDS VIII.

IN TISSUES

OF THE

SPHINGOMYELINASE

H. ROELFZEMA,

EYE OF THE

R. M. BROEKHUYSE

Institute of Ophthalmology (The Netherlands)

and Department

(Received

1972)

December

rjth,

LENS

and J. H. VEERKAMP

qf Biochemistry,

University

qf Nijmegen,

Nijmegerz

SUMMARY

T. Sphingomyelinase has been detected in the epithelium and the cortex of calf lens, using labeled lenticular sphingomyelins as substrates. 2. The enzyme preparations of both origins have a pH optimum of 4.6 and are activated by Triton X-100 like the enzyme found in rat liver and other tissues. 3. Sphingomyelin isolated from the epithelium, and sphingomyelin isolated from the rest of the lens, have considerable differences in their fatty acid composition, but are hydrolysed at the same rate by sphingomyelinase prepared from the epithelium. 4. The specific activity of sphingomyelinase in homogenates from different parts of the lens increases in the order: nucleus 4 cortex < equator < epithelium.

INTRODUCTION

A study of the sphingomyelin metabolism was started in our laboratory in view of the large increase in relative concentration of sphingomyelin in comparison to the other phospholipids during differentiation and aging of the lens cells’T2. The lens (Fig. I) is surrounded by a capsule and grows within it. There is a single layer of nucleated epithelial cells at the front surface. In the equatorial region the cells differentiate and elongate into fibres. New fibres grow from the equator towards the front and back, and are layered on top of previous fibres. Thus the oldest cells are situated in the lens nucleus and fibres containing subcellular organelles are largely confined to the equator3. As was shown previously, the hydrolysis of sphingomyelin to ceramide and phosphorylcholine is the most important pathway for the catabolism of sphingohas been studied in liver697, kidney’ and myelin4*“. The enzyme sphingomyelinase brain of rat’,“, human spleen”, aortaI and fibroblasts13 from skin and bone marrow. In this paper the presence, the distribution and some of the properties of sphingomyelinase of calf lens will be reported. Labeled lenticular sphingomyelins from epithelium and decapsulated lens were used as substrates.

H. ROELFZEMA lens

zonularfibers

of a cross-section

MATERIALS

AND

Preparations

of the lens parts

of the lens. The inner

equator

I

15.9 mm

I

Fig. I. Diagram are indicated.

eplthelwm

surface

et ul.

and outer

diameter

of the equatorial

zone

METHODS

Fresh lenses were obtained from eyes, which were collected immediately after death from 2-3-month-old calves. All subsequent procedures were performed at 4 “C. The capsule was stripped off the lens and the epithelial cells were removed with it. The decapsulated lens was then either stirred vigorously with 0.25 M sucroseI mM Tris-HCl (pH 7.4) for I min to disperse the outermost layers of the cortex, or frozen and the annular equator removed by cutting through with a cork-borer (diameter 14.1 mm) (Fig. I). Stirring for IO min in the same buffer yielded the rest of the cortex. The remainder, which was whitish in appearance, was considered to be the nucleus. The weight ratio, capsule plus epithelium, equator, cortex, nucleus was 0.3: 1.0: I 1.5:4.6. Preparation

of’ the enzyme suspension Homogenates of the different parts of the lens were prepared by dispersing the tissues in 0.25 M sucrose-Tris-HCl (pH 7.4) in a glass Potter-Elvehjem tube with a teflon pestle followed by ultrasonic treatment for I min. An epithelial preparation free from capsule was obtained from a homogenate of epithelium with capsule by standing till the capsules had settled down. To investigate the properties of sphingomyelinase from the epithelium and the cortex, parts of the lens were homogenized for 5 min with 3 vol. 0.25 M sucroseTris-HCl (pH 7.4) in a glass Potter-Elvehjem tube with a teflon pestle, rotating at 250 rev./min. The homogenate was centrifugedI in a 870 head rotor (I&,= 10.9 cm,

SPHINGOMYELINASE

OF THE LENS

331

Rmin= 6.5 cm) of a refrigerated IEC centrifuge (Model B20) at 990 x g,, for IO min. The sediment was rehomogenized in the same quantity of medium and centrifuged at 610 x g,, for IO min. After repeating the latter operation the supernatants were combined and made up to volume to form the I : IO cytoplasmic extract. To isolate an epithelial enzyme preparation this cytoplasmic extract was centrifuged for 30 min at 3 I 430 x g,, (S,i, = 8 IO). The supernatant was decanted and the sediment resuspended in 0.25 M sucrose-r mM Tris-HCl (pH 7.4). After ultrasonic treatment for 3 min this suspension was used as the epithelial enzyme preparation. In order to isolate a cortical enzyme preparation the I : IO cytoplasmic extract was centrifuged for IO min at 4420 x g,, (S,i, = I 7 300). The supernatant of this fraction was centrifuged for 20 min at 16700xgaV (Smin= 2300). The supernatant was decanted and the sediment resuspended in 0.25 M sucrose-t mM Tris-HCl (pH 7.4). This suspension (173002300 S) was treated ultrasonically for 3 min and used as the cortical enzyme preparation. The enzyme preparations were stored at - 25 “C and showed no decrease of sphingomyelinase activity for three weeks. Chemicals and reference compounds Triton X-100 was obtained from the British Drug Houses and [‘4C]nlethy1iodide from the Radiochemical Center, Amersham, England. Sphingosylphosphorylcholine was obtained from Supelco, Pa. U.S.A., silica gel HR from E. Merck A.G., Germany and phospholipase C (EC 3.1.4.3) (Clostridium welchii) from Sigma, St. Louis, MO., U.S.A. Reference sphingomyelin was prepared from blood p1asma15. Ceramide was prepared by splitting sphingomyelin with phospholipase Cl6 and sphingosine by alkaline hydrolysis of ceramide’ 6. [32P]phospholipids were isolated from rat liver”. Preparation ~j’[Me-‘~c] sphingomyelirl Sphingomyelin was prepared from epithelium and decapsulated lens after extraction with chloroform and methanol according to Folch et a118. After washing with o. I M KC1 and evaporation of the organic layer, the crude lipids were subjected to mild alkaline hydrolysis”. After neutralization with ethyl formate the solution was evaporated and a Folch partition was performed. The chloroform layer was subjected to silicic acid column chromatography’ ’ or preparative thin-layer chromatography in the solvent systems chloroform-methanol-7 M ammonia (90: 54: I I, by vol.) and chloroform-methanol-acetic acid-water (90: 40: I 2 : 2, by vol.)*‘. Lipid components were visualized by spraying with a 0.001 % aqueous Rhodamine 6G solution. Phospholipids were quantitatively eluted with chloroform-methanol-water (I : 3 :0.4), by vol.). The chromatographically pure sphingomyelin was 14C-labeled within thecholine moiety with a modified procedure according to Stoffel et al.2’. 0.5 mmole sphingomyelin from decapsulated lens was demethylated with 2.5 mmoles sodium thiophenolate in 20 ml dimethylsulfoxide. The mixture was heated with magnetic stirring under N, at 95-100 “C. After 6 h the mixture was cooled and 25 ml ice-cold 2 M HCI were added. The solution was extracted twice with 40 ml chloroform. The chloroform layer was dialysed for 24 h against water. After addition of methanol, a Folch partition was performed. The chloroform layer was subjected to preparative thin-layer chromatography using chloroform-methanol-5.6 M ammonia (60: 35 : 8, by vol.) as

332

H. ROELFZEMA er rrl.

a developer. Yield: o. I mmole ceramide-t-phosphoryl-N, N-dimethylethanolamine.55 ,umoles of this product were dissolved in chloroform-methanol (2:1, by vol.) and aqueous NaOH was added up to pH 10.5. The solution was taken to dryness and dried for 72 h over P,O,. The material was dissolved in 4 ml methanol and 1.4 ml 40 mM methanolic NaOH and 55 pmoles [r4C]methyliodide (9 Ci/mole) were added. After reacting for 3 h at room temperature, 110 Ltmoles nonradioactive methyliodide and 0.6 ml 40 mM methanolic NaOH were added. After 24 h, 4 ml 2 M HCl, 13.3 ml chloroform and 0.7 ml methanol were added and a Folch partition was performed. The chloroform phase was washed with synthetic upper phase as described by Folch was further purified by silicic acid column et al.“. The [Me-r4C]sphingomyelin chromatography. Yield: 20 pmoles with a specific activity of 5610 dpm/nmole. The same procedure was applied on microscale for 20 Llmoles epithelial sphingomyelin. In this case purification was performed by preparative thin-layer chromatography in chloroform-methanol-5.6 M ammonia (60:35:8, by vol.). Yield: 0.43 /{mole with a specific activity of 2330 dpm/nmole. Assajl of etizynle ac/ivitJ’

The assay is based on the fact that sphingomyelin and the products of its hydrolysis are partitioned between a chloroform phase (sphingomyelin and ceramide) and an aqueous methanolic phase (phosphorylcholine)22. The standard assay system contains in a final volume of 0.5 ml: 55.9 nmoles [Me-‘4C]sphingomyelin (55000 dpm), 0.5 mg Triton X-I 00,20 pmoles sodium acetate buffer (pH 5.0) and an enzyme preparation of 138 pg epithelial protein or 95 /lg cortex protein. 4 ml chloroformmethanol (2: I, v/v) and 0.67 ml water were added after incubation with shaking for I h at 37 ‘C. After mixing and centrifugation, aliquots of I ml of the upper and the lower phase were taken for measurement of the radioactivity. The “zero time” control amounted to 0.3 :‘< of the total amount of the sphingomyelin. Ident$7catiotz i~f’the hydrolysis products

The products of enzymatic hydrolysis in the chloroform phase were investigated by thin-layer chromatography with chloroform-methanol-water (60: 35: 8, by ~01.)~ as a developer. The method of Schneider and Kennedy’ using chloroform-methanolacetic acid (90: 4: 4, by vol.) as developing system up to I 5 cm and chloroformmethanol-2 M ammonia (70:30:4, by vol.) up to 7 cm in the same direction was used as a control system. Sphingomyelin, ceramide, sphingosylphosphorylcholine and sphingosine were run concurrently as reference compounds. The products in the methanolic-aqueous phase were resolved by thin-layer chromatography in the system chloroform-methanol-cont. ammonia (70: 52: I 5, by vol.) with phosphorylcholine and sphingosylphosphorylcholine as reference compounds. Paper chromatography in the ascending direction with n-butanol-acetic acid-water (4: I : 5, by vol., upper phase)7 or with ethanol-20 mM acetic acid (6:4, v/v)” was used as a control system. Lipid components were detected in general with iodine vapour and ninhydrin. Cholinecontaining components were detected with Dragendorff’s reagensz3 and phosphoruscontaining components were visualized by using the spray according to Amelung and Biihmz4. Radioactivity was measured by counting the silicagel scraped off or by scanning with a Berthold thin-layer scanner (Type LB 2722).

SPHINGOMYELINASE

Analytical

OF THE

LENS

333

procedures

Sphingomyelin present in the enzyme preparation was determined after extraction according to Bligh and Dyer25. Lipid phosphorus was determined after quantitative two-dimensional thin-layer chromatography according to Broekhuyse2’ and visualization with I, vapour and ninhydrin. Protein was determined by the method of aqueous solutions was measured in IO ml of Bray of Lowry et al. 26. Radioactivity solution2’. Chloroform samples were counted after evaporation in scintillation vials, containing 0.4 “/, (w/v) PPO and 0.01 s/, (w/v) dimethyl-POPOP in toluene. Samples of silica gel were counted with 3.5 y0 Cab-0-Sil in the medium. Radioactivity was assayed in a Packard Tricarb liquid scintillation spectrometer Model 3380. Quenching corrections were made using the external standard method. Gas chromatography of fatty acids was performed as described earlier2’. RESULTS

AND

DISCUSSION

Some modifications proved to be necessary in the preparation procedure of [Me-14C]sphingomyelin according to Stoffel et al.” to get a pure microscale preparation. The time of methylation was extended to 24 h, because after 4 h the reaction appeared to be incomplete. Even after 24 h, not all of the demethylated sphingomyelin had reacted with the [‘4C]methyliodide. Moreover, the [Me-‘4C]sphingomyelin was extensively purified. The presence of sphingomyelinase in calf lens could be established by identification of the hydrolysis products after incubation of [Me-14C] sphingomyelin with the lens preparations. Ceramide proved to be present in the chloroform phase, sphingosine and sphingosylphosphorylcholine were absent. Phosphorylcholine was identified as hydrolysis product in the methanolic-water phase and no sphingosylphosphorylcholine was present (Table I). Stoffel and Assmann4, and Schneider and Kennedy5 concluded from their experiments that there was no breakdown of sphingomyelin to sphingosylphosphorylcholine in vivo or in vitro. Only Fujino et al. 29,30 detected sphingosylphosphorylcholine as an intermediate in the biosynthesis of sphingomyelin. The used enzyme preparations were isolated by differential centrifugation and contained the highest specific activity of sphingomyelinase compared to the other

TABLE

I

IDENTlFlCATlON

OF THE

[Me-“Clsphingomyelin Materials and Methods. plate or Whatman No.

WATER-SOLUBLE

HYDROLYSIS

PRODUCT

was incubated for 3 h with an epithelial enzyme The methanolic water phase was concentrated I paper. RF values are given for all compounds.

Sq’stetn

Thin-layer chromatography with chloroformmethanol-cont. ammonia. (70 : 52 : 15, by vol.) Paperchromatography with n-butanol-acetic acid-water (4 : I : 5. by vol., upper phase) Paperchromatography with ethanol-20 mM acetic acid (6 : 4, by vol.)

preparation and applied

as described in to a thin-layer

Sphingosylphosphwylcholine

Phosphorylcholine

Hydrul.vsis prodrrcr

0.27

0.05

0.05

0.57

0.07

0.07

0.85

0.43

0.43

H. ROELFZEMA

334

et nl.

fractions. The properties of these enzyme preparations from epithelium and cortex were studied on [Me-14C]sphingomyelin from decapsulated lens. A correction was always made for the endogenous sphingomyelin concentration, which was 36_+ 2 (n = 4) nmole per mg protein for the epithelial preparation and 65 4_ 8 (n = 2) nmole per mg protein for the preparation of the cortex. Hydrolysis of sphingomyelin by the enzyme preparation from the epithelium was linear with time for at least 90 min at a substrate concentration of 121 nmoles per ml (Fig. 2). After this time 12.6 % of the sphingomyelin had been hydrolyzed. The optimal substrate concentration was determined by measuring the velocity of the reaction at several substrate concentrations (Fig. 3). To obtain the maximal sensitivity for the hydrolysis of radioactive sphingomyelin a substrate concentration of about 120 nmoles sphingomyelin per ml was chosen as standard condition for a rate of hydrolysis of 5 nmoles/h. An apparent K,,, of 8.rom5M was calculated for sphingomyelin. For the rat liver enzyme a Km between 1.8.10-~-9.0. tom4 was reported6.7, whereas a K,,, of 3.8. IO-~ was reported for the enzyme from the human

1

V (n

moles/ h I~~-

10 1

0

60

120 time

180 (min )

0

50

100

150

200

S (nmoles/ml

I

Fig. 2. Time dependence of the hydrolysis of sphingomyetin by epithelial sphingomyelinase. Assay conditions are described in Materials and Methods. Total substrate concentration was 121 nmoles/ml. Fig. 3. Rate of hydrolysis of sphingomyelin in dependence of the substrate concentration. Assay conditions are described in Materials and Methods except that the pH was 4.6. The total substrate concentration is a sum of the added and the internal sphingomyelin, nmoles of phosphorylcholine are plotted along the ordinate.

spleen’ ‘. Maximal sphingomyelinase activity was obtained at pH 4.6 in acetate buffer for the enzyme preparation from both epithelium and cortex (Fig. 4). The optimum pH is in the range of values found in other tissues6.7.9.‘1%‘2 for this enzyme. The epithelial enzyme as well as the enzyme from the cortex were activated by Triton X-100 (Fig. 5). A remarkable shift of the activation curve was obtained at increasing protein concentrations. The rate of hydrolysis was linear with the enzyme

SPHINGOMYELINASE

OF THE

LENS

335

Fig. 4. Dependence of the rate of hydrolysis of sphingomyelin on the pH. 1‘--‘~, epithelial enzyme preparation; A----& cortical enzyme preparation. Assay conditions are described in Materials and Methods. In case of the epithelial enzyme the total substrate concentration was IZI nmoles/ml. The cortical enzyme was incubated for 2h at a total substrate concentration of I 52 nmoles/ml.

v(n

v(nmoles/h) optimal 1.0,

triton

X-100

(mg/ml P

moles/h)

)

I I Ll.5

Fig. 5. Dependence ofthe rate of hydrolysis of sphingomyelin on the Trition X-100 concentration and the protein concentration. Epithelial enzyme preparation: O---C), O. I 38 mg protein per ml; O----O, 0.276 mg protein per ml; a--& 0.552 mg protein per ml; COrtiCd enzyme preparation: A----& o.t90 mg protein per ml. Assay conditions are described in Materials and Methods, except that the rate of hydrolysis at a protein concentration of 0.552 mg protein per ml was measured for 0.5 h.

concentration up to a concentration of 276 pg protein per ml, provided an optimal Triton X-I oo concentration was present. Sodium taurocholate has also an activating effect on the hydrolysis of sphingomyelin (Fig. 6), although the activation was less than with Triton X-loo. Here two maxima were observed instead of one maximum when Triton X-loo was used. Tween 80 showed little activation and cetyltrimethylammonium bromide exerted no effect. The surface-active agents are necessary for the solubilization of the enzyme, which is otherwise precipitated at the pH of the reaction and for the proper dispersion of sphingomyelin. The activating effect of detergents on the hydrolysis of sphingomyelin was also found with rat liver’ and brain’ preparations. In these studies the amount of protein added in the assay was not taken into account when Triton X-loo was used as detergent, leading to different optimal Triton X- IOO

concentrations. Our finding that the optimal Triton X-too concentration was dependent on the protein concentration is stlpported by studies of Pradal et d3’ and Wat~~au~ and de Duve”‘. An effect of cetyltrim~thyIanlmonium bromide at a concentration of 0.2 mg per ml as was found by Keller and Shapira’ in rat liver could not be detected in our system. Barnholz el ~1.~ found in rat brain with cetyltrimethyia~~monium bromide an inhibition of 50 7; at a concentration of 0.25 mg/ml and complete inhibition at I mg~mi. The activating effect of detergents and the acid pH optimum points to the fact that the enzyme is possibly located in lysosomes, as has been proved for rat liver3”. Tentative results obtained from d~fferentja~ centrifuga~~on of ~o~u~e~a~~s of lens parts and dct~rmination of the activity of marker enzymes also points in this direction. Further investigations on this aspect are in preparation. The enzyme from epithelium was shown to retain its activity in the presence of substrate and detergent during I h in the range from 27-47 “C (Fig. 7) An Arrhenius plot showed a decrease in the activation energy at temperatures above 37 “C

Fig. 6. Influence of sodium laurocholate. Tween 80 und c~tyI&~metbylammonium bromide on the hydrolysis of sph~ngomyeliil by epithelial s~hi~~omy~li~as~. *t. --.-?. sodium ta~rocbolate~ *----8, Tween 80; : L--G . cetyltrimethylammonium bromide, Assay coil~it~ons are described in Materials

Fig. 7. The effect of temperature on the hydrolysis of sphi~gomyeli~l by epithelial sph~n~omyclinase, Assay conditions are described j, ~;~ter~a~sand klethods. A---A*, 27 ‘C; :. -. P,. 32 “C; +--e. 37 “C; x__ -_x, 42 ;c: ,:__.-“.~i* 47 ;c. Fig. 8. Arrhenius plot ofthe rate of hydrolysis of sphingomyelin by epithelial spbil~go~~yc~i~~ise.

SPHINGOMYELTNASE

OF THE

LENS

337

(Fig. 8). The cause of this decrease is still obscure. Schneider and Kennedy” found for the enzyme of the human spleen after I h a temperature optimum of 60 “C. Preliminary experiments with j2P-labeled phospholipids showed that the lens preparations exhibit no phospholipase C action on phosphatidylethanolamine and phosphatidylcholine like rat liver7 and brain” preparations. Because the sphingomyelinase activity might depend on the composition of the sphingomyelin, the activity of the epithelial enzyme was tested on epithelial sphingomyelin and sphingomyelin from the decapsulated lens. Both of the substrates were hydrolyzed at the same rate (Table II), in spite of the marked differences in fatty acid composition (Table II 1). From epithelium to nucleus there is a sharp increase in 24: I, 16: o and 14: o, while 24: o and 20: I show a sharp decline, altogether resulting in a decrease of the mean effective chain length. Barnholz et ~1.~used bovine brain sphingomyelin and sphingomyelin from spleen of patients with Niemann-Pick’s disease as substrates. They observed similar rates of hydrolysis with an enzyme preparation from rat brain, in spite of the differences in fatty acid composition of the used substrates34*35. The sphingomyelinase activity was determined in homogenates of different TABLE

II

ACTION OF EPITHELIUM Assay

EPITHELIAL SPHINGOMYELINASE AND THE DECAPSULATED LENS

conditions

were described

in Materials

Substrate ____ ~~__. ~~ Epithelial sphingomyelin Decapsulated lens sphingomyelin

TABLE

ON

and Methods

SPHINGOMYELINS

Total substrate present (nmoles)

that the pH was 4.6 ~____ Substrate hydrolyzed (nmoles/h)

52.5 53.3

3.4 + 0.2 3.1 IO.1

_~

except

FROM

-.

THE

~_

111

PROPORTIONAL FATTY ACID COMPOSITION LIUM AND DECAPSULATED LENS

OF SPHINGOMYELINS

FROM

EPITHE-

Sphingomyelins were isolated after alkaline hydrolysis of total lipids by repeated thin-layer chromatography. The fatty acid composition was determined by gas-liquid chromatography and is given in g per too g. The fatty acid methyl esters are designated by the number of carbon atoms, followed by the number of double bonds. Fatty acid

Epithelium

1410 1610

2.3 8.9

18:o 18:1 19:o

2.9 3.1 3.5

20:o

’

20:

I

22:o 22:

23:o 23: I 24~0 24: 1 > 24

11.2

2.7 2.7 0.2

1.9

.9

0.6

I I.,

3.4 3.7 0.8

I.4 2.3

0.7 38.6 5.3 14.1

lens ~~ -

26.5

2.1 I

Decapsulated

~_

I.3 5.5 3t.7 7.3 ~-~~

-

H. ROELFZEMA

338 TABLE

et al.

IV

SPHINGOMYELINASE

ACTIVITY

IN DIFFERENT

PARTS

OF THE

LENS

Sphingomyelinase activity was assayed in crude homogenates of the different lens parts as described in Materials and Methods at pH 4.6. ~~~~ ~~~~~ .._ ~~~ Protein content Sphingomyelin content Specific activity fnmoles/h) Tissue (g per g tissue) Ipmoleslg) Per mg proteitl Per g tissue Per nmole sphingomyelin Epithelium Equator Cortex Nucleus

0.14

I.523

0.22

0.352 0.477 0.323

0.29 0.43

5.2 I~ 0.2 0.58 l 0.03 o. I 6 + 0.04 0. I I i 0.02

749 z 30 128 4~6 51 z8 55 ! 6

0.49 i 0.02 0.37 + 0.01 0.1 I ) 0.02 0.17 0.02

lens parts and calculated per mg protein, per g tissue and per nmole sphingomyelin (Table IV). During the differentiation process in the equatorial zone the concentrations of all phospholipids decrease. During the aging process of the lens fibres, however, the sphingomyelin concentration is rather stable in comparison to the concentrations of the other phospholipids. This results in a higher relative concentration in the inner lens parts (nucleus 23.4 %) as compared to the outer layers activity of preparations (epithelium 9. I %, equator I 2.8 %)’ *‘. The sphingomyelinase from lens parts is low (87-1.8 punits per mg protein) compared to the activity in liver, kidney and spleen of man and rat (I 300-200 punits per mg protein)“. In the metabolically most active parts of the lens, the epithelium, the highest sphingomyelinase activity was found. The equator contains a much lower activity than the epithelium. In the former part the epithelial cell differentiates to a lens fibre, a process which is characterized by an increase in the area of the plasma membrane, an increase of mainly nonstructural protein (crystallins) and a fall in the number of cell organelles3. The sphingomyelinase activity of the equator is low compared with the epithelium, but is considerably higher than in the cortex, where the lens fibre is fully grown. The sphingomyelinase activity in the nucleus is comparable to the activity in the cortex. The decrease of sphingomyelinase activity from epitheliun to cortex corresponds in general with a decline in activity of most of the enzymes, which have been investigated in the lens36. Part of the sphingomyelinase activity in cortex and nucleus may be used to bring about the changes in fatty acid composition of the sphingomyelins in both lens parts. Besides the possibility that these changes are caused by chain shortening or desaturation in the intact sphingomyelin molecule, a hydrolysis of sphingomyelin via ceramide to sphingosine might occur. After this process specific fatty acids could be incorporated for the resynthesis of sphingomyelin37. The possibility of a deacylating-reacylating mechanism of sphingomyelin has not been found by Stoffel and Assmann” in rat liver. Studies on the biosynthesis of sphingomyelin in different parts of the lens, planned in our laboratory, may yield more information about this point. ACKNOWLEDGEMENTS

The authors are indebted to Mr A. L. M. de Leeuw for excellent technical assistance. The present investigations have been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).

SPHINGOMYELINASE

OF THE

LENS

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