Appearance of a proteoglycan in developing sea urchin embryos

385

Biochimica et Biophysica Acta, 541 (1978) 385--393

© Elsevier/North-Holland Biomedical Press

BBA 28553 APPEARANCE OF A PROTEOGLYCAN IN DEVELOPING SEA URCHIN EMBRYOS

KAYOKO OGURI * and TATSUYA YAMAGATA Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194 (Japan)

(Received December 9th, 1977)

Summary It was shown that a proteoglycan is synthesised by embryos of a Japanese sea urchin, H e m i c e n t r o t u s p u l c h e r r i m u s . This proteoglycan appears as a single peak on sucrose density gradient ultracentrifugation t h r o u g h o u t the development. A b o u t half of the mucopolysaccharide moiety in this proteoglycan was found to be dermatan sulphate and the rest to be chondroitinase-resistant mucopolysaccharides. Evidence is presented to show that both types of mucopolysaccharide do not exist in a free form but reside as an integral part of the proteoglycan. The linkage between mucopolysaccharide and protein moieties of the proteoglycan appeared n o t to be an O-glycosidic bond, which is c o m m o n among other proteoglycans such as proteochondroitin sulphate and proteodermatan sulphate.

Introduction Several histochemical studies have suggested the occurrence of mucopolysaccharides in sea urchin embryos. Possible roles of mucopolysaccharides in the development of sea urchin embryos have been discussed, based on their localization [1--4]. Some investigators consider that mucopolysaccharides play an important role when the primary mesenchymal cells fall into a blastocel and migrate along the inner surface of the blastular wall to form a characteristic arrangement of the cells, since they believe that mucopolysaccharides are found at the vegetal region of an early blastula and then in the surroundings of the primary mesenchymal cells [ 5--8]. Furthermore, embryos undergo abnormal development after the blastula stage when t h e y are cultured in sulphate-free artificial sea water [9]. As sul-

* Present address: D e p a r t m e n t 1 58, J a p a n .

o f B i o l o g y , Faculty of Science, T o k y o M e t r o p o l i t a n University, T o k y o

386 phate is an integral part of acid mucopolysaccharides, this observation may give further support to the postulate that mucopolysaccharides are indispensible for the early development of sea urchin embryos. Although many researchers have discussed the possible roles of mucopolysaccharides in the sea urchin development, studies on the nature of mucopolysaccharides of sea urchin embryos had not been performed until we identified a mucopolysaccharide from the embryos of Japanese sea urchins, Pseudocentrotus depressus [10] and Hemicentrotus pulcherrimus (Oguri, K. and Yanagisawa, T., unpublished), as oversulphated dermatan sulphate. The dermatan sulphate of the sea urchin larvae was reported to be composed of (1 -* 4)<~-Lidopyranuronosyl-(1 -~ 3)-2-acetamido-2-deoxy-fl-D-galactopyranosyl 4,6-disulphate units [10]. These studies showed for the first time the occurrence of dermatan sulphate in invertebrates. Since it is widely accepted that mucopolysaccharides appear as a part or' proteoglycan molecules in vertebrates, we u n d e r t o o k the study on the proteoglycan synthesised by sea urchin embryos during their early development. Materials and Methods Embryos of H. pulcherrimus were cultured in filtered sea water (5" 10 ~ embryos per ml) at 20°C with gentle stirring. At the stages desired, the embryos were transferred to sulphate-free artificial sea water [11] and labelled with [3SS]sulphate (carrier-free, 5 gCi per ml, Japan Radioisotope Association) for 1 h. They were then harvested, washed several times with acetone and dried. Chondroitinase ABC and AC were purchased from Seikagaku Kogyo Inc. Pronase-P was obtained from Kaken Kogyo Co. Guanidinium chloride (GnHC1) and cesium chloride (CsC1) were products of Nakarai Chem. and Merck, respectively, and other reagents were analytical grade commercial chemicals. We tried to find out the most effective method to extract the proteoglycan from sea urchin embryos. The [ aSS]sulphate-labelled materials in gastrulae were extracted by several methods. Gastrulae ( 5 . 1 0 6 embryos) were labelled at 20°C in 250 ml of sulphate-free artificial sea water containing 1 mCi of [3sS]sulphate. After 1 h of incubation, embryos were collected by centrifugation, and divided into a number of equal portions. Embryos were extracted with 15 vol. of several concentrations of GnHC1 in 0.05 M acetate buffer at pH 5.8 and GnHC1 in Tris • HC1, (pH 7.2) [12]/3 M MgC12 [13]/10% CaC12 (w/v) (pH 7.2) [14], at 4°C, with stirring, for 24 h. The extraction with 6 M urea at pH 7.2 was carried out at 60°C with a constant shaking for 16 h [15]. The extraction with 0.05 M sodium pyrophosphate was done exactly according to the m e t h o d described by Kinoshita [16]. All the extracts were centrifuged to obtain the supernatants, which were dialyzed against the extracting solution and assayed for radioactivity. Radioactivity was measured in a toluene-based scintillator by Beckman liquid scintillation spectrometer. The radioactivity thus obtained was expressed as a percentage of the radioactivity o-f the alkaline extract of the embryos. Alkaline extraction was performed in 0.5 N NaOH at 37°C for 13 h. All radioactivity incorporated to macromolecules was extracted under these conditions.

387 The proteoglycan was prepared by a slightly modified technique of Hascall et al. [17], since it turned out to be the best extracting method. The acetonedried embryos were extracted with 4 M GnHC1/0.05 M acetate buffer (pH 5.8) containing 0.1 M 6-aminohexanoic acid/0.005 M benzamidine hydrochloride/ 0.01 M EDTA, at 4 ° C. After 48 h the extract was clarified by centrifugation. Solid CsC1 was added to the supernatant to give a density of 1.50 g/ml (0.59 g of CsC1 per g of solution). A density gradient was formed by centrifugation a t 43 000 rev./min for 38 h at 22°C in a Spinco 50 Ti rotor. A fraction, above a density of 1.50 g/ml, was concentrated and dialysed against the extracting solution using Diaflo apparatus (with UM-10 membrane). The fraction was put on a 5--20% (w/v) sucrose density gradient formed in the presence of 4 M GnHC1/0.05 M Tris • HC1 buffer, pH 7.2 and centrifuged at 24 000 rev./min for 38.5 h at 4°C in a Spinco SW 27.1 rotor [18]. Results

Comparison of various methods tested for the extraction of proteoglycans Fig. 1 shows that GnHC1 at pH 5.8 was the most effective extracting agent of [3SS]sulphate-labelled materials from sea urchin embryos. When the length of time needed for satisfactory extraction was checked with 4 M GnHC1 at pH 5.8 (not shown), the extraction for 48 h was found to be sufficient to extract almost 100% of the [3SS]sulphate-labelled material from embryos. 3 M MgC12 and 10% CaC12 extracted about 60% in 24 h. 6 M urea extracted about 40%. Extraction of proteoglycan was reported to be carried out with 0.05 M sodium pyrophosphate in 30 min at 0°C from embryos of the sea urchin, Clypeaster ]aponicus [16], but, in our experiment, 0.05 M sodium pyrophosphate was able to extract only 20% of labelled materials. Based on the above results, in the present study the extraction of proteoglycans was performed with 4 M GnHC1/ 0.05 M acetate buffer (pH 5.8) supplemented with protease inhibitors [17].

Some properties of proteoglycans extracted from gastrulae Proteoglycans were extracted and purified from embryos as mentioned in Materials and Methods. After centrifugation of the extract of the gastrulae in CsC1 density gradient, 70% of the [3SS]sulphate-labelled material was recovered in the b o t t o m fraction (p ~> 1.50). Only 10% of the protein applied appeared in this fraction (Fig. 2A). The sample from each fraction tube was treated with chondrointinases ABC and AC to study the nature of the [3SS]sulphate-labelled materials. None of the samples was susceptible to digestion by chondroitinase AC, whereas digestion with chondroitinase ABC resulted in the degradation of about 50% of the radioactive materials (Fig. 2B and Table I). The digested products were separated by paper chromatography and identified [10]. The major product appeared to be unsaturated disaccharide 4-sulphate, and the minor one to be disulphated derivative (Table I). We can conclude that about a half of [3SS]sulphate-labelled materials was dermatan sulphate. The fractions from 1 to 4 (p >t 1.50) were pooled, concentrated and centrifuged on a sucrose density gradient [18]. The [3SS]sulphate-labelled material appeared as a single peak between the two peaks of proteochondroitin sulphates (proteochondroitin sulphates H and L), which are the well-studied

388 $

m

.2 100

1.5

o

D E

o

o

1.

O

J_l -~

1.7

o

5o I

"o .2 >.

12345678 4

6

Bottom

8

Concentration of GnHCI

(M)

Top

Fraction number

12345678 Top

Bottom

Fraction number

Fig. 1. E x t r a c t i o n o f [ 3 5 S ] s u l p h a t e _ l a b e l l e d m a t e r i a l s b y v a r i o u s m e t h o d s . G n H C I / 0 . 0 5 M a c e t a t e b u f f e r at p H 5.8 (o), G n H C I / 0 . 0 5 M Tris - HCI b u f f e r at p H 7.2 (o), 3 M MgC12 (•), 10% CaCl 2 ( a ) , 6 M u r e a (~), 0 . 0 5 M s o d i u m p y r o p h o s p h a t e (A). R e f e r t o Materials a n d M e t h o d s for details. Fig. 2. T h e profile in t h e CsCl d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f the e x t r a c t o f gastrulae. A. A f t e r centrif u g a t i o n , f r a c t i o n s w e r e c o l l e c t e d f r o m t h e b o t t o m of t h e c e n t r i f u g a l t u b e s , a n d w e r e m e a s u r e d f o r t h e i r d e n s i t y (o). A l i q u o t s o f t h e f r a c t i o n s w e r e d i a l y s e d a g a i n s t distilled w a t e r a n d a s s a y e d f o r the r a d i o a c t i v i t y (o). P r o t e i n was r e v e a l e d b y t h e m e t h o d o f L o w r y et al. [ 2 3 ] using b o v i n e s e r u m a l b u m i n ( A r m o u r , F r a c t i o n V ) as a s t a n d a r d (D). B. A l i q u o t s o f t h e d i a l y s e d s a m p l e s w e r e t r e a t e d w i t h c h o n d r o i t i n a s e s ABC a n d AC a c c o r d i n g to t h e m e t h o d d e s c r i b e d b y Saito et al. [ 2 4 ] . T h e d i g e s t e d p r o d u c t s w e r e s e p a r a t e d b y p a p e r c h r o m a t o g r a p h y using a s o l v e n t o f n - b u t y r i c a c i d / 2 N N H 4 O H (5 : 3) [ 1 0 ] , a n d a s s a y e d for t h e radioactivity. The radioactivity of d e r m a t a n sulphate (shadowed) and chondroitinase-resistant m u c o p o l y saccharides (dotted).

proteoglycan standards, obtained from 13-day-old chick embryonic cartilage (Fig. 3A) [18]. In any fraction examined, a b o u t a half of [3SS]sulphate-labelled materials was found again to be dermatan sulphate, and the rest to be chondroitinase-resistant mucopolysaccharides (Fig. 3B). This [3SS]sulphate-labelled material was eluted at the void volume of a column of Sepharose 6B (Fig. 4A). After the digestion with Pronase-P, however, the labelled materials became smaller and retarded in the gel (Fig. 4B). Therefore, the [3SS]sulphate-labelled macromolecule was concluded to be a proteoglycan, which was composed of [3SS]sulphate-labelled mucopolysaccharide chains and a core protein. The size of the mucopolysaccharide chains of the proteoglycan resembled that of proteochondroitin sulphate H in their elution profiles on Sepharose 6B column [18]. A linkage between protein and chondroitin sulphate of the proteochondroitin sulphate of bovine nasal septum was reported to be an O-glycosidic bond b e t w e e n xylosyl and seryl residues [20] and undergoes fl-elimination in an alkaline solution. The proteoglycan extracted from the gastrulae was treated with 0.5 N NaOH at 4°C for 72 h or at 37°C for 3 h and applied to the column of Sepharose 6B. The radioactive material after the alkaline treatment was eluted at the void volume (Fig. 4C), giving the pattern identical to that of the i n t a c t proteoglycan (Fig. 4A). Since under the same or milder conditions (at 4°C for 16 h), the proteochondroitin sulphates of the chick embryonic cartilage underwent fl-elimination [18], it is unlikely that the conditions used may have

389 TABLE I I D E N T I F I C A T I O N OF T H E [ 3 5 S ] S U L P H A T E - L A B E L L E D METHOD

M A T E R I A L BY T H E C H O N D R O I T I N A S E

T h e labelled m a t e r i a l o b t a i n e d f r o m g a s t r u l a e w a s f r a c t i o n a t e d i n t o eight f r a c t i o n s a f t e r c e n t r i f u g a t i o n , as s h o w n in Fig. 2. A l i q u o t s o f t h e f r a c t i o n s w e r e d i a l y s e d against w a t e r a n d t r e a t e d w i t h c h o n d r o i t i n a s e s ABC a n d AC a c c o r d i n g t o t h e m e t h o d of Saito et al. [ 2 4 ] . T h e d i g e s t e d p r o d u c t s w e r e s e p a r a t e d b y p a p e r c h r o m a t o g r a p h y [ 1 0 ] . A f t e r p a p e r w a s d r i e d , t h e r a d i o a c t i v i t y c o r r e s p o n d i n g to t h e origin, u n s a t u r a t e d d i s a c c h a r i d e 4 - s u l p h a t e a n d d i s u l p h a t e w a s m e a s u r e d . P e r c e n t d i s t r i b u t i o n is p r e s e n t e d in p a r e n t h e s e s . T h e a b b r e v i a t i o n s u s e d are: ADi-4S, 3 - O - A - g l u c u r o n o s y l - N - a c e t y l g a l a c t o s a m i n e 4-sulfate; ADi-diS, 3-O-Aglu cu r o n o syl-N-ac e t y l g a l a c t o s a m i n e 4,6-disulfate. Fraction number

C h o n d r o i t i n a s e ABC

C h o n d r o i t i n a s e AC

Total cpm

c p m at origin

c p m of ADi-diS

cpm of ADi-4S

Total cpm

c p m at origin

cpm of LxDi-diS

c p m of ADi-4S

1

1736 1963

3

2727

4

1181

5

1273

6

343

7

199

112 (6.5) 109 (5.6) 96 (3.5) 46 (3.9) 45 (3.5) 15 (4.4) --

22 (1.2) 22 (1.0) 6 (0.2) 39 (3.1) 25 (1.9) 7 (2.3) --

22

1790 (96.9) 2134 (96.9) 2988 (97.8) 1147 (91.4) 1226 (90.9) 285 (94.4) 132 . (100) 63 (100)

36 (1.9) 47 (2.1) 62 (2.0) 69 (5.5) 97 (7.2) 10 (3.3) --

8

433 (24.9) 940 (47.9) 1291 (47.3) 514 (43.5) 496 (39.0) 107 (31.2) 21 (10.6) --

1848

2

1192 (68.7) 914 (46.6) 1340 (49.1) 621 (52.6) 732 (57.5) 221 (64.4) 178 (89.4) 22 (100)

--

--

--

A

2203 3056 1255 1348 302 132 63

B 1©

o x

E eL

o

w

>" 4-

5

II _o

j

o

;::~::::;:::, 5 Bottom Fraction

10 number

15 Top

5

10

Bottom Fraction

15 Top

number

Fig. 3. A. S e d i m e n t a t i o n p r o f i l e s in t h e s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f [ 3 5 S ] s u l p h a t e - l a b e l l e d m a c r o m o l e c u l e s o f g a s t r u l a e ( e ) a n d p r o t e o c h o n d r o i t i n s u l p h a t e o b t a i n e d f r o m cartilage o f 1 3 - d a y - o l d c h i c k e m b r y o s (o) [ 1 8 ] . B. A f t e r d e s a l t i n g , e a c h f r a c t i o n w a s t r e a t e d w i t h c h o n d r o i t i n a s e s ABC a n d AC. T h e d i g e s t e d p r o d u c t s w e r e s e p a r a t e d a n d a s s a y e d f o r r a d i o a c t i v i t y as d e s c r i b e d in t h e l e g e n d f o r Fig. 2. The radioactivity of dermatan sulphate (shadowed) and chondroitinase-resistant mucopolysaecharides (dotted).

390

A

Va

Vo

B

Vo

1 1-

Vt

Vt

Vt

IJ

.e

g

._o

50

100 "/

50

\

1(10"

50

100

Fraction number Fig. 4. E l u t i o n p r o f i l e s o n a c o l u m n o f S e p h a r o s e 6B (1 × 6 5 c m ) o f t h e p r o t e o g l y c a n o b t a i n e d f r o m g a s t r u l a e . T h e c o l u m n w a s e l u t e d w i t h 2 M G n H C 1 / 0 . 0 5 M T r i s • HCl b u f f e r at p H 7.2. A, I n t a c t p r o t e o g l y c a n ; B, a f t e r P r o n a s e - P t r e a t m e n t ; a n d C, a f t e r a l k a l i n e t r e a t m e n t . A. A f t e r t h e s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , s a m p l e s f r o m f r a c t i o n n u m b e r 1 1 - - 1 6 in Fig. 3 w e r e p o o l e d a n d c o n c e n t r a t e d . A p a r t o f t h e s a m p l e w a s d e s a l t e d a n d a p p l i e d t o t h e c o l u m n . B. A l i q u o t s o f t h e d e s a l t e d s a m p l e w e r e d i g e s t e d w i t h P r o n a s e - P in 0 . 5 M Tris • HC1 ( p H 8.0) at 4 0 ° C f o r 48 h, a n d a p p l i e d to t h e c o l u m n . C. A l i q u o t s o f t h e d e s a l t e d s a m p l e w e r e t r e a t e d w i t h 0 . 5 N N a O H at 4 ° C f o r 72 h or at 3 7 ° C f o r 3 h, a n d a p p l i e d to t h e c o l u m n a f t e r n e u t r a l i z a t i o n . A r r o w s i n d i c a t e v o i d v o l u m e (V•) a n d t o t a l v o l u m e ( V t ) .

been insufficient to cause H-elimination. It is also unlikely that the occurrence of an alkali-susceptible bond in the compound could be undetected by using the Sepharose 6B column, because the digested products with Pronase-P retarded in Sepharose 6B gel. The data suggested that the linkage between

! w

-*

5

I

o o,jS 5

10 Fraction

15

20

number

Fig. 5. S e d i m e n t a t i o n p r o f i l e s in t h e s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f t h e i n t a c t p r o t e o g l y c a n (o), a n d t h e c h o n d r o i t i n a s e - t r e a t e d p r o t e o g l y e a n ( o ) b e a r i n g chondroitinase-resistant [35S]sulphatelabelled m u c o p o l y s a c c h a r i d e s . The p r o t e o g l y c a n e x t r a c t e d f r o m gastrulae was digested with chondroitinase A B C . 2 vol. o f e t h a n o l w e r e a d d e d to t h e r e a c t i o n m i x t u r e in t h e p r e s e n c e o f 1% p o t a s s i u m a c e t a t e t o remove disscharides [19]. The ethanol precipitate was dissolved and analysed by centrifugation.

391 a

bc

d

e

f

g

>.

•

•

•

...........

E %

..J......

E

o. w

•:

..~,'"

:'"°'"i"..:...~...

".....'"

"'~'t"".

g

>.

: .....

....

i.

..e...;..

0 "D 0 c¢ ,

,

,

,

i

121

i

J

,

i

i

214

i

,

Time after fertilization

i

i

,

361

i

,

i

i

i

4/8

( h )

Fig. 6. T h e rate o f i n c o r p o r a t i o n o f [ 3 S S ] s u l p h a t e i n t o 10% t r i c h l o r o a c e t i c a c i d - i n s o l u b l e f r a c t i o n o f t h e e m b r y o . Up to p l u t e u s stage f r o m f e r t i l i z a t i o n o n w a r d s , e m b r y o s w e r e labelled e v e r y 2 h w i t h [ 3 5 S ] s u l p h a t e in s u l p h a t e - f r e e artificial sea w a t e r f o r 30 rain. T h e e m b r y o s w e r e w a s h e d w i t h 10% t r i c h l o r o a c e t i c acid t h r e e t i m e s a n d w i t h 50% e t h a n o l t w i c e . T h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o 10% t r i c h l o r o a c e t i c acid insoluble f r a c t i o n was m e a s u r e d . Stages are r e p r e s e n t e d b y t h e f o l l o w i n g letters; a, 4-cells; b, h a t c h i n g b l a s t u l a e ; c, s w i m m i n g b l a s t u l a e ; d, m e s e n c h y m a l b l a s t u l a e ; e, early g a s t r u l a e ; f, late g a s t r u l a e ; g, plutei.

protein and mucopolysaccharides of the proteoglycan cff sea urchin embryos, was n o t the O-glycosidic bond of the xylosyl-serine type. The chondroitinase ABC
392 A

B

C

D

E

F

4.

§

o .£ >, 4o. >

J ..g

o

og

5

15

5'

Fraction

1'0

15 '

5

10

15

number

Fig. 7. S e d i m e n t a t i o n profiles in t h e s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of t h e p r o t e o g l y c a n s o b t a i n e d f r o m ( A ) m o r u l a e , (B) early b l a s t u l a e , (C) s w i m m i n g b l a s t u l a e , (D) e a r l y gastrulae, (E) late gastrulae a n d (F) plutei. E a c h p r o t e o g l y c a n c e n t r i f u g e d was p u r i f i e d as d e s c r i b e d for the p r o t e o g l y c a n o b t a i n e d f r o m gastrulae. T h e d i s t r i b u t i o n o f t h e p r o t e o d e r m a t a n s u l p h a t e was a n a l y s e d as d e s c r i b e d in t h e l e g e n d for Fig. 2. T h e r a d i o a c t i v i t y o f d e r m a t a n s u l p h a t e ( s h a d o w e d ) a n d c h o n d r o i t i n a s e - r e s i s t a n t m u c o p o l y s a c c h a rides ( d o t t e d ) .

The profiles of the sucrose density gradient centrifugation of the proteoglycans from these six stages are shown in Fig. 7. The distribution of dermatan sulphate among the [3SS]sulphate-labelled proteoglycan were examined as mentioned above. Dermatan sulphate was found to be distributed in every fraction belonging to the peak of the [3SS]sulphate-labelled proteoglycan t h r o u g h o u t development.

Discussion The appearance of a proteoglycan in sea urchin embryos has been suggested by some investigators [6,16]. By means of autoradiography, for instance, a parallel incorporation was observed of 14C-labelled amino acids and of [3sS]sulphate into mesenchyme and endomesoderm of the gastrulae of Paracentrotus lividus. The mesenchymal cells were supposed to be embedded in a matrix o f sulphated proteoglycan [6]. But actual occurrence of the complex was n o t shown in this experiment. It was reported that a proteoglycan was extracted from the embryos of C. japonicus with 0.05 M sodium pyrophosphate and it was claimed that the extraction with 0.05 M sodium pyrophosphate was more effective than that with 1.0 M GnHC1 from the embryos of Clypeaster [16], contrary to our data shown in Fig. 1. The molecular size of the proteoglycan isolated from Clypeaster [16] was much less than that of the proteoglycan of Hemicentrotus in our study. Moreover, the mucopolysaccharide moiety of Clypeaster was reported to be resistant to chondroitinase ABC [16]. It was found, however, in the embryos of C. ]aponicus that 18% of [3sS]-

393 sulphate labelled macromolecules was dermatan sulphate (Yamagata, T., unpublished). Admitting the species differences, we are still not certain if the intact proteoglycan was extracted and analysed in the preceding work [16]. We show clearly in the present study the occurrence of dermatan sulphate and chondroitinase-resistant mucopolysaccharides as the proteoglycan in the embryos of H. pulcherrimus. It is known that some proteoglycans have different types of mucopolysaccharide attached on a core protein in vertebrate [21]. The structure of protein-mucopolysaccharide linkage region of this proteoglycan was not determined. We only suggested that the O-glycosidic bond did not take part in the linkage because H-elimination was not observed by the alkaline treatment. As the linkage region of proteodermatan sulphates is k n o w n to be an O-glycosidic bond of the xyrosyl-serine t y p e [22], the proteoglycan in sea urchin embryos appeared unique in the linkage structure.

Acknowledgements The authors thank the staffs of the Misaki Marine Biological Station for the use of the Station's research facilities. The authors' thanks are also due to Drs. Y. Kato and T. Yanagisawa for their interest in this work.

References 1 M o a n 6 , L. a n d S l a u t t e r b a e k , D.B. ( 1 9 5 0 ) Exp. Cell Res. 1 , 4 7 7 - - 4 9 1 2 Monn6, L. a n d S l a u t t e r b a c k , D.B. ( 1 9 5 2 ) Ark. Zool. 3, 3 4 9 - - 3 5 6 3 Immers, J. ( 1 9 5 6 ) Ark. Zool. 9 , 3 6 7 - - 3 7 5 4 0 k a z a k i , K. a n d Niijima, L. ( 1 9 6 4 ) E m b r y o l o g i a 8, 8 9 - - 1 0 0 5 M o t o m u r a , I. ( 1 9 6 0 ) Bull. Mar. Biol. A s a m u s h i 1 0 , 1 6 5 - - 1 6 9 6 I m m e r s , J. ( 1 9 6 1 ) E x p . Cell Res. 24, 3 5 6 - - 3 7 8 7 S u g i y a m a , K. ( 1 9 7 2 ) Dev. G r o w t h Differ• 14, 6 3 - - 7 3 8 K a r p , G.C. a n d Solursh, M. ( 1 9 7 4 ) Develop. Biol. 4 1 , 1 1 0 - - 1 2 3 9 Herbst, C. ( 1 8 9 7 ) A r c h . E n t w i e k l u n g s m e c h . Org. 5, 6 4 9 - - 7 9 3 1 0 Y a m a g a t a , T. a n d O k a z a k i , K. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 372, 4 6 9 - - 4 7 3 11 Okazaki, K. ( 1 9 6 2 ) E m b r y o l o g i a 7, 2 1 - - 3 8 12 Hascall, V.C. a n d Sajdera, S.W. ( 1 9 6 9 ) J. Biol• C h e m . 2 4 4 , 2 3 8 4 - - 2 3 9 6 13 SaJdera, S.W. a n d Hascall, V.C. ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 7 7 - - 8 7 14 Muir, H. a n d J a c o b s , S. ( 1 9 6 7 ) B i o c h e m . J. 1 0 3 , 3 6 7 - - 3 7 4 15 Toole, B.P. a n d L o w t h e r , D.A. ( 1 9 6 8 ) A r c h . B i o c h e m . Biophys. 1 2 8 , 5 6 7 - - 5 7 8 16 K i n o s h i t a , S. ( 1 9 7 4 ) Exp, Cell Res. 85, 3 1 - - 4 0 17 Haseall, V.C., O e g e m a , T.R. a n d B r o w n , M. ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 3 5 1 1 - - 3 5 1 9 18 K i t a m u r a , K. a n d Y a m a g a t a , T. ( 1 9 7 6 ) FEBS Lett. 71, 3 3 7 - - 3 4 0 19 Y a m a g a t a , T., Saito, H., H a b u c h i , O. a n d Suzuki, S. ( 1 9 6 8 ) J. Biol. C h e m . 243, 1 5 2 3 - - 1 5 3 5 20 R o d 6 n , L. a n d S m i t h , R. ( 1 9 6 6 ) J. Biol. C h e m . 2 4 1 , 5 9 4 9 - - 5 9 5 4 21 Hascall, V.C. a n d H e m e g a r d , D. ( 1 9 7 5 ) in E x t r a c e l l u l a r M a t r i x I n f l u e n c e s o n Gene E x p r e s s i o n (Slavkin, H.C. a n d Greulieh, R.C., eds.), p p 4 2 3 - - 4 3 3 , A c a d e m i c Press, New Y o r k 22 Stern, E.L., L i n d a h l , B. a n d R o d 6 n , L. ( 1 9 7 1 ) J. Biol. Chem. 2 4 6 , 5 7 0 7 - - 5 7 1 5 23 L o w r y , O.H., R o s e b r o u g h , N.J., F a r r , A.L. a n d R a n d a l l , R.J. ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 24 Saito, H., Y a m a g a t a , T. a n d Suzuki, S. ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 1 5 3 6 - - 1 5 4 2 •

o