Cell Differentiation, 8 (1979) 173--186 173 © Elsevier/North-Holland Scientific Publishers Ltd.

T H E C A R B O H Y D R A T E C O N T E N T O F I S O L A T E D Y O L K P L A T E L E T S F R O M E A R L Y D E V E L O P M E N T A L S T A G E S OF X E N O P U S L A E V I S

NANCY ROBERTSON*

Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, CA 94720 (U.S.A.)

Accepted December 12th, 1978

Yolk platelets (YP) from blastulae and gastrulae of Xenopus laevis contain the neutral hexoses glucose and mannose at 12--42 t~g[mg protein and amino sugar at 2.6-- 3.8 ~g/mg protein. Carbohydrate is present in lipovitellin I and lipovitellin 2 and glycolipid was found. Little or no sialic acid, galactose, fucose, or glycosaminoglycan (GAG) was found. The results are discussed in terms of possible functions of these complex carbo- hydrates. The saccharide moiety of one or more of the glycoproteins may promote uptake of vitellogenin by the developing oocyte; glycolipids may be ready made compo- nents for membrane biogenesis during embryogenesis.

The 1973 findings o f Kos he r and Searls t ha t YP of Rana pipiens em- b r y o s i n c o r p o r a t e inorganic 3sSO42- at any stage of d e v e l o p m e n t up to S h u m w a y stage 18 i m p l y t h a t G A G are ac t ive ly syn thes ized on these organ- elles. G A G are k n o w n to be syn thes ized in the rough e n d o p l a s m i c r e t i cu lum and Golgi vesicles of d i f f e r en t i a t ed cells such as c h o n d r o c y t e and synovia l cells (S too lmi l l e r and D o r f m a n , 1969; Lev i t t and D o r f m a n , 1974) and the s y n a p t o s o m e f rac t ion o f e m b r y o n i c ch ick brain (Brand t e t al., 1975) . A l though G A G are p re sen t in p r e c h o n d r o g e n i c e m b r y o s o f Rana (Koshe r and Searls 1973) and sea urch in (Karp and Solursh, 1974) , the subcel lu lar loca l iza t ion of the i r synthes is is n o t known . Synthes is o f G A G on YP would cons t i t u t e a novel site for this func t ion .

T o date , the m o s t de ta i led i n f o r m a t i o n on the c a r b o h y d r a t e (CHO) c o n t e n t o f a m p h i b i a n YP is given by Ansari et al. (1971) . T h e y f o u n d t h a t v i te l logenin, the p recu r so r o f the t w o m a j o r YP pro te ins , l ipovitel l in (LV) and phosvi t in (PV)~ of X e n o p u s laevis o o c y t e s have sialic acid, na tu ra l hexose and h e x o s a m i n e . R e d s h a w and Fo l l e t t (1971) f o u n d neu t ra l hexose in the l ipovitel l in of X e n o p u s oocy te s , b u t n o t in phosvi t in .

The p u r p o s e of this s t udy is to def ine the CHO c o n t e n t o f YP isola ted

*Present address : Jules Stein Eye Institute, The Center for Health Sciences, University of California, Los Angeles, CA 90024, U.S.A. Abbreviations: CHO, carbohydrate; GAG, glycosaminoglycan(s); LV, lipovitellin; PAS, perodic acid Schiff; PV, phosvitin; PVP, polyvinylpyrollidone-10 (mol. wt 10 000); YP, yolk platelet(s)

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from blastulae and gastrulae of Xenopus laevis embryos using a new gradient for YP isolation.

MATERIALS AND METHODS

E m b ~ o s

Embryos from stages 7 and 8 (5 and 6 h), representing blastula, and from stages 10, 11, and 12 (8, 10.5, and 13 h), representing gastrula, were obtained as described previously (Robertson, 1978).

YP isolation

YP were isolated and purity checked by electron microscopy using pro- cedures already described (Robertson, 1978).

Protein determination

A modification of the Lowry method of protein determination was done on YP suspended in 0.5 M KC1 (Hartree, 1972).

Carbohydrate determinations : Monosaccharide composition of YP

Solutions of YP in KC1 were hydrolysed in 4 N HC1 at 100°C for 7 h to release amino and neutral sugars from complex CHO. Hydrolysates were dried and made to small volumes with H20 for passage through small Dowex 50 cation exchange columns, 200--400, hydrogen form, to separate neutral and amino sugars (Spiro, 1966).

The H20 eluates were evaporated and made to small volumes with H20 and total neutral sugars were determined by the primary cysteine-H2SO4 reaction of Dische {1955) from measurements at A410 using D-Man as a standard.

For qualitative determination of neutral hexoses, 20 to 120 t~l of H20 eluate were spotted onto Whatman No. 1 paper along with 6 pg each of authentic D-Gal, D-Glc, and D-Man, and developed by descending chroma- tography in ethyl acetate/pyridine/H20 (8 : 2: 1). Chromatograms of the same standard sugars with 9 pg each of authentic L-Fuc, NANA, D-Xyl, and 15--20 pg each of D-Glucuronic acid (GlcUA) D-GlcNH2, D-GalNH2, D-GlcNAc, and D-GalNAc were run in the same solvent. Subsequently, samples of the H20 eluates were chromatographed in n-butanol/pyridine/ H20 (10 : 3 : 3) (Hough and Jones, 1962). Sugar spots were visualized with the silver nitrate method of Trevelyan et al. (1950).

The HC1 eluates were evaporated and redissolved in small volumes of H20. Total hexosamine was determined by the Elson-Morgan reaction (Davidson, 1966) using D-GlyNH2 as a standard.

Sialic acid was assayed on solutions of YP with the resorcinol method

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(Spiro, 1966). This method was chosen in addition to the thiobarbituric acid method (Spiro, 1966) since the latter detects sugars in DNA, and the presence of nucleic acids in YP remains an unsettled question (see Wallace, 1963; Bruce and Emanuelsson, 1975; Kelley et al., 1971; Opresko et al., 1977). It was desirable not to have interference from deoxy sugars, if present. For the resorcinol test, absorbances were measured at 580 and 450 rim, the latter to correct for contribution by neutral sugars, and NANA and D-Man were used as standards. Absorbances were read at 549 nm for the thiobarbi- toric acid test with NANA as a standard.

Methyl pentose was determined with the Dische-Shettles reaction (Dische, 1955) using L-Fuc and D-Man as standards. The amount of methyl pentose present was calculated from the difference between A396 and A4zT. This test was done on samples of the H20 eluates from hydrolysed YP and also on solutions of native YP in 0.5 M KC1 (Spiro, 1966, 1972). In addition, a variation of this reaction in which addition of H20 obliterates the formation of chromogens in neutral sugars but not in methyl pentose was done on un- hydrolysed YP (Dische, 1955). The quanti ty of methyl pentose was calcu- lated on the basis of A 396 with L-Fuc as a standard.

Carbohydrate determinations: glycosaminoglycans

Any GAG present on YP were released for assay by digestion with papain (Antonopoulos et al., 1964), or with pronase (Nameroff and Holtzer, 1967).

Ten, 20 and 40 pl of pronase and papain digested YP were put on a 20 × 20 cm plain silica gel thin layer plates (Brinkman Chemical Co.) and cochromatographed with 10 pg each of authentic chondroitin sulfates 4 and 6, hyaluronic acid (Miles Laboratories), and heparin (Nutritional Biochemical Co.). The plates were developed by ascending chromatography and stained according to the methods of Marzullo and Lash, 1967.

One to three pl of pronase and papain digests of YP were applied to 3 $ 18.5 or 1 × 15 cm cellulose acetate strips, and cochromatographed with 1 or 2 pg each of authentic chondroitin sulfates 4 and 6, hyaluronic acid, and heparin. The strips were electrophoresed in 0.1 M barium acetate buffer, (pH 8.3) at 80 V for 5 h. The strips were dried and stained with 0.5% tolui- dine blue in 3% acetic acid and destained in 1% acetic acid and HzO (Seno et al., 1970; Wessler, 1971).

Aliquots of the protease digests were treated as follows before electro- phoresis : Solid residue was pelleted at 15 000 rev./min for 30 min at 4°C. Enzymes were precipitated with 10% trichloracetic acid at 4°C, and pelleted at 10 000 rev./min for 45 min. Digests were dialysed at 4°C against H20 for 47 h. One and two pl of standards and dialysed digests were applied to the cellulose acetate strips.

Sequential extraction of the major types of GAG using cetylpyridinium chloride and graded concentrations of NaC1 was done on other aliquots of papain or pronase digests of YP according to the methods of Schiller et al.

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(1961). One to three pl of such extracts were put onto strips for electro- phoresis.

SDS gel electrophoresis

To get rid of KC1, YP solutions were precipitated in H20, and the preci- pitate was consolidated by centrifugation at 1100 g for 15 min at room temperature. This was repeated once. The opalescent YP material in the second supernatant was pelleted at 10 000 rev./min at 4°C for 10 min.YP pellets were suspended in a mixture containing 0.5% SDS, 0.1 M sodium phosphate (pH 7), 1.25% mercaptoethanol, and 0.005% bromphenol blue, the tracking dye. The mixture was boiled for 15 min, a time sufficient to dissolve all samples and to prevent proteolysis (Bergink and Wallace, 1974).

Gels (16 cm × 1.3 cm) of 5% acrylamide were prepared according to the method of Shapiro et al. (1967) and were run in 0.1 M sodium phosphate buffer (pH 7.0), with 2.5 mM mercaptoethanol. 0.9 mg of denatured YP were loaded onto the gels which were run at 25 V, 100 mA for 8 h.

Gels were stained overnight in Coomassie brilliant blue in a 45% methanol, 9% acetic acid solution for proteins, and destained in the solvent and washed in H20. Other gels, run concurrently, were stained for CHO using the perio- dic acid-Schiff (PAS) basic fuchsin procedure of Zacharius et al. (1969).

Gels (8 cm × 0.4 cm) were prepared as above, and 46 ug YP material and 26 pg aspartic transcarbamylase, in which neither CHO nor lipid have been found (Jacobson and Stark, 1973; Benisek, 1971), were loaded onto the gels which were run in the solutions described above at 100 mA for 4 h. Gels were stained for CHO, protein, or lipid. The last is a modification of a stain developed for paraffin embedded tissue sections (Weesner, 1970). A satura- ted solution of Sudan IV was prepared (0.4% per 100 ml of 50% ethanol) and filtered once through Whatman No. 1 paper. Gels were stained overnight. Lipids appear as brown bands on a yellow background. Gels are placed in H:O to return them to their original sizes for comparison with the other gels.

RESULTS

Electron microscopy

As was true for Wallace and Karasaki (1963), the unit membrane was generally absent. Only a fraction of the YP circumference (5%) exhibited membranes on YP that had them. Thirty-five percent of the platelets in a test sample (148 out of 517 YP) displayed small vesicular fragments of mem- brane.

Of 22 YP batches, 10 display detectable contamination, which was unusu- ally present on 2 of the 4 grids examined per batch. In 5 cases, the contam- inant was a piece of vitelline membrane. In the remaining instances, contam- inants were lipid, membranes and, rarely, pigment granules. Generally, each

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piece of contaminant was considerably smaller than virtually all YP, and at most represented 6.5% of the total population of cellular components by number of pieces, the rest being YP. No glycogen particle was ever seen. The contaminant that is of greatest concern is the vitelline membrane which is about 8 -11% CHO (Wolf et al., 1976). Vitelline membrane interference proved to be below the limits of detection because YP appear to lack the fucose, galactose and sialic acid present in this contaminant.

Carbohydrate determinations

Neutral sugars. The total neutral hexose content of YP ranges from 11.5 to 27.8 pg CHO per mg YP (dry wt) or 12 and 31 pg CHO per mg protein for blastula and 15.4 pg per mg YP (dry wt) or 42 pg per mg protein for gastrula (Table I). Apparent differences in the neutral hexose content of blastula and gastrula cannot be considered significant on the basis of the amount of data available.

At most, only 2 sugar spots were seen on the chromatographs of the H20 eluates, and these comigrated with D-Glc and D-Man in n-butanol/pyridine/ H20 (10 : 3 : 3 :) (Fig. 1, Table II). YP neutral sugars do not cochromato- graph with D-Gal in ethyl acetate/pyridine/H20 (8 : 2 : 1), so this sugar is absent above the 1.8 pg per mg YP (dry weight) level. It can also be seen in the table that D-Glc and D-Man do not in turn cochromatograph with L-Fuc, NANA, xylose D-GlcNH2, D-GalNH2, or D-GlcUA. Since GlcNAc and GalNac are deacetylated under the hydrolysis conditions used here to GlcNH2 and GalNH2, respectively (Carlson, 1972), these N-acetylated sugars are likewise not present in the H20 eluates.

Amino sugars. Amino sugars in blastula YP ranged from 2.6 to 3.4 ~g/mg YP (dry wt) or 2.6--3.9 pg/mg protein. Corresponding values for gastrula were 4.4 and 4.0 t~g/mg YP and 3.7 and 3.8 pg/mg protein (Table I). Again, insufficient data are present to permit a comparison of overall blastula and gastrula values to see if changes occurred in the amount of hexosamine present,

Methyl pentoses. Determinations on the H20 eluates of HC1 hydrolysed YP with the Dische-Shettles test reveals 0.01--0.05 pg methyl pentose/mg YP, dry wt. For unhydrolysed YP solutions, methyl pentose values ranged from 0 to 0.06 t~g/mg YP, wet wt. There are insignificant amounts of methyl pentose in YP from blastula and gastrula, and the results are identical for purified samples and whole YP (Table I).

The H20 induced color obliteration variation of the Dische-Shettles test shows that there is 0 t~g methyl pentose per mg YP (wet wt).

Sialic acid. The quanti ty of sialic acid in YP ranges from 0 to 6 × 10 -3 t~g/mg YP (wet wt) for the resorcinol method (Table I), and 0 pg/mg YP (wet wt)

TABLE I

CHO CONTENT OF YP

YP batches are indicated in parentheses

Blastula (Stages 7- 9)

ug CHO/mg protein CHO

ug CHO/mg YP (dry wt)

Neu tral hexose 12 (No. 8 ) (Experiment 2 ) 31 ( No. 12 ) H 2 O eluate of hydrolysed YP

Hexosamine 2.6 (No. 8) (Experiment 2) 3.9 (No. 2) HC1 eluate of hydrolysed YP

Sialic acid 0 (No. 12) (Experiment 1) 6 x 10 3 (No, 6) Unhydrolyzed YP (Wet wt)

Methyl-pentose (Experiment 1 )a (Dische-Shettles) 0.01 (No. 12)

o.o6 (No. 6) 0 (No. 6) 0 (No. 12) 0 (No. 6)

(Dische-Shettles with color obli teration)

0.067 0.172

0.015 0.022

11.5 (No. 8) 24.5 (No. 2) 27.8 (No. 1) 20.0 (No. 7) 21.6 (No. 10)

2.6 (No. 8) 3.0 (No. 2) 3.4 (No. 10) 2.9 (No. 12)

(Experiment 2) b 0.03 (No. 2) 0.05 (No. 12) 0.01 (No. 1 0 )

Gastrula (Stages 10- 12)

ug CHO/mg protein

uM CHO

ug CHO/mg YP (dry wt)

Neutral hexose 42 (No. 4) (Experiment 2) 42 (No. 5) H 2 O eluate of hydrolysed YP

Hexosamine 3.8 (No. 4 ) (Experiment 2) 3.7 (No. 5) HCI eluate of hydrolysed YP

Sialic acid 3 × 10 -4 (No. 5) (Experiment 1 ) Unhydrolyzed YP (Wet wt)

Methyl-pentose (Experiment 1 )a (Dische-Shettles) 0 (No. 5)

(Dische-Shettles 0 (No. 5) with color obli teration)

0.230 0.230

0.021 0.021

48.6 (No. 4) 45.5 (No. 5) 15.4 (No. 3) 57.7 (No. 6)

4.4 (No. 4) 4.0 (No. 5)

(Experiment 2 )b O.03 (No. 5) 0.05 (No. 4)

adata from unhydrolysed YP samples bdata from hydrolysed YP, H20 eluate

B I D - m a n ~ D-glc I

Or ig in I [ I I I J

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Front

Fig. 1. Paper c h r o m a t o g r a m of neu t ra l hexoses released by acid hydro lys i s o f ether ex t rac t s of YP, 80 ul of YP and 6 ~g each of D-Glc and D-Man were appl ied to W h a t m a n No. 1 paper . So lven t is n-butanol/pyridine/H:O (10 : 3 : 3). Visua l iza t ion wi th AgNO 3 .

for the thiobarbituric acid assay. Sialic acid is virtually absent from either blastula or gastrula YP.

Hexosamine varies from 3 to 20% of the neutral sugar values on a molar basis for blastula and 9% for gastrula.

GAG. The RF'S of authentic chondroi tm sulfates 4 and 6 and heparin on the thin layers, plates are 0.72, 0.72, and 0.42. Both hyaluronic acid and YP material remain at the origin and stain orthochromatically with toluidine blue. The other authentic GAG stain metachromatically. The YP material represents less than 2.3 pg sulfated GAG per mg YP (wet wt).

All YP material prepared for cellulose acetate electrophoresis behaved identically by remaining at the origin and staining orthochromatically with toluidine blue (Fig. 2). Heparin displays two magenta spots, one at the origin, and the other 2.5 cm therefrom. Chondroitin sufates 4 and 6 give purple spots 6.5 and 7 cm, respectively, from the origin. Hyaluronic acid gives an orthochromatic spot comigrating with the mobile portion of heparin. Thus, none of the standard GAG are present in YP at the 0.5 ug level of d_eteetion

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TABLE II Paper chromatography RFs of hexoses from HC1 hydrolysates of YP. HzO eluates Whatman No. 1 paper. AgNO2 visualisation. Each numbered column represents samples done on same sheet.

Ethyl acetate/pyridine/H20 (8 : 2 : ~)

n-Butanol/pyridine/H2 0 (10 : 3 : 3)

1 2 3 4 5 6 7 8

Glc Gal Man L-Fuc NANA Xyl Glc NH~ Gal NH 2 Glc-NAc Gal-NAc Glc UA

0.33 0.17 0.33 0.29 0.14 0.29 0.37 0.20 0.36 0.43 O.29 0.08 0.29

0.09 0.06 0.07 0.07 0.05 0.05

0.21 0.21 0.20 0.19 0.20

origin origin

0 .23 0.21 0.24 0.27 0.27 0.28

YP stage 8 11-12 8 8 20 ~l 0.34 0.22 0.24

0.26 40 ul 0.32 0.22 0.21

0.27 0.26 80 ~l 0.22

0.28

for 840 t~g of wet YP in the ce ty lpyr id in ium chloride/NaC1 ex t rac ted protease digests, dialysates of the lat ter only, or for 2.1 mg of protease digested YP (wet wt).

The crystall ine main b o d y of YP does no t con ta in appreciable quanti t ies o f GAG.

SDS gel electrophoresis

Figure 3 shows PAS- and Coomassie blue-stained 16 cm gels and 8 cm gels stained for p ro te in only. The major pro te in bands were ident if ied as lipo- vitellins 1 and 2 on the basis o f compar i son with the migrat ion proper t ies o f these two subuni ts o f lipovitellin as de te rmined by Bergink and Wallace (1974) . No t all p ro te in f rom the unex t r ac t ed YP entered the gel. Figure 3A shows tha t one o f the two large PAS bands comigra tes with lipovitellin 2 and the small band with lipovitellin 2. The o ther large PAS band is ahead of the dye f ron t and does no t comigra te with any prote in , so it is identif ied as

181

I I D

I I

I II

I I +

Origin

/ Heporin

I ts6

]cs4

IHA

I YP pronose

I YP papain

Fig. 2. Cellulose acetate electrophoresis of protease digested YP and standard GAG. CS 6, Chondroitin 6-fulfate; CS4, chondroitin 4-sulfate; HA, hyaluronic acid.

A 0 ~ ~ B 0 i

LVl

LV2

LVl

LV2

TD :<

!Jii!iil

TD

GL

i;iiii!!iiii !!ii!!!iji j!

I 2

J:ii!!

::i!!

I 2 3 Fig. 3. 5% SDS-polyacrylamide gels. (A) 16 cm gels; 1-Coomassie blue, 2-PAS; (B) 8 cm gels; 1-Whole YP, 2-Ether extracted YP aqueous layer, 3-Aspartic transcarbamylase 0; loading end; LVI and LV2, Lipovitellins 1 and 2; GL, glycolipid; TD, tracking dye.

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TABLE III

Comparison of bands on.gels of YP 11

Also asparatic transcarbamylase controls (ATCase). Distance (,f band from loading end (ram). Bromphenol blue marker = 45 mm.

Sample Gel stain Lipovitellin YP bands Lipovitellin Glycolipid ATCase 1 2 bands

1 2

YP Coomassie 16 20 23 28 32 36 none blue PAS 17 66--72 Sudan IV 18 39 64

ATCase Coomassie 35 43 blue PAS no bands Sudan IV no bands

g lycol ip id on the basis o f these fea tures (B lumenfe ld and Zvi l ichovsky, 1972; Bikel and Knight , 1972) .

Tab le I I I gives the migra t ion dis tances o f the CHO, lipid, and ma jo r pro- te ins of YP and aspar t ic t r an s ca rbam y l a s e in the 8 cm gels (Fig. 3B). The la t te r p ro t e in is be r e f t o f CHO and lipid bands , a t tes t ing to the spec i f ic i ty of the stains used fo r th ree substances . Lipovi te l l in I had lipid and CHO. Sugar was n o t d e t ec t ed at the 46 pg level for l ipovitel l in 2 a l though lipid was present .

This m a y be re la ted to the fac t t ha t the PAS band fo r l ipovitel l in 2 was bare ly visible fo r 0.9 mg of loading mate r ia l on the 16 cm gel. Bands t ha t were ahead of the dye f r o n t s ta ined posi t ively fo r lipid and sugar, bu t no t p ro te in . These were ident i f ied as glycol ipid.

DISCUSSION

In s u m m a r y , the crys ta l l ine main b o d y of YP o f blas tulae and gastrulae have two g lycop ro t e in s and glycol ipid. T w o neut ra l sugars, m a n n o s e and glucose, and an a m i n o sugar m a k e up these c o m p l e x ca rbohyd ra t e s . In c o m p a r i s o n , Ansari e t al. (1971) s howed tha t c i rcula t ing Xenopus vitello- genin has h e x o s a m i n e , neu t ra l sugar, and sialic acid. T h e y do no t spec i fy which m e m b e r s of the f irst m e n t i o n e d classes of sugars are presen t , bu t the presence of sialic acid implies the p resence of e i ther galactose or galacto- samine as the p e n u l t i m a t e sugar to which it is a t t ached .

Putative role o f complex carbohydrates in oogenesis

The presence of sialic acid in v i te l logenin and its absence in YP suggest

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a function for some YP CHO analogous to that of the oligosaccharides in ceruloplasmin in mammals (Ashwell and Morrell, 1974). Perhaps vitellogenin remains in circulation in the parent frog as long as sialic acid is present. Removal of this sugar may result in the uptake of vitellogenin by the develop- ing oocyte. The uptake of ceroloplasmin by the liver requires the presence of galactose or N-acetylgalactosamine at the terminus following desialation. In another system, Sly {1977) presented data indicating that uptake of ~-glucuronidase by fibroblasts depends on the presence of terminal mannose or mannose phosphate, subsequent to removal of the distal saccharides. The paucity of Gal in YP may indicate that terminal Man is the uptake facilitating sugar for vitellogenin, in homology to Sly's system. This means that vitello- genin must be both desialated and degalactosylated in order to expose mannose which allows entry into the oocyte.

Relationship of YP to membrane biogenesis during embryogenesis

That YP components may be important in membrane biogenesis in sugges- ted by the electron micrographs of YP of Rana undergoing demolition during development (Karasaki, 1963). Unravelling lamellae of the typical membrane triiaminar structure, 50--100 h thick, the spacing of the crystalline structure of YP, are commonly seen peeling off the crystalline main body. This pattern of unravelling is also seen in the YP of the salamander, Ambystoma (L. Lemanski, pers. comm.) during cardiac development.

YP have many of the kinds of molecules found in plasma membranes. There are 5 fat ty acids, saturated and unsaturated (Ansari et al., 1971) (11 g neutral lipid/100 g protein, Redshaw and Follett, 1971) YP have 2 glycopro- teins, but it is not known whether or not both are broken down entirely to their consti tuent amino acids for utilization as food. Perhaps intact parts of these glycoproteins are utilized for direct incorporation into membranes. Clearly, data are not needed to define the exact mode of molecular utiliza- tion of amphibian YP components.

Most important from the standpoint of membrane biogenesis is the presence of glycolipids in YP. These molecules are uniquely constituents of membranes, whereas glycoproteins are often not associated with mem- branes. YP could thus serve as a warehouse for ready made glycolipids destined for incorporation into nascent plasma membranes.

Dain (1972) reported that sialic acid-containing gangliosides do not appear in the embryos of Rana until late gastrula. It is conceivable that shorter gang- liosides of the structures

Gal -~ Glc -~ ceramide Glc -~ ceramide

are present prior to that stage, especially the latter since little UDP-Gal transferase is present before late gastrula. If Xenopus YP glycolipids contain gangliosides, they are probably primarily the simplest ones containing only glucose because of the paucity of galactose and the absence of sialic acid.

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The putat ive presence o f the asialo gangliosides in YP until mid gastrula of Xenopus would coincide with the presence o f the same in Rana at this stage.

ACKNOWLEDGEMENTS

This work is a partial fulfillment of the requirements for the Ph.D. degree. I thank Doctors Robley Williams, C. Arthur Knight and John Gerhart for use of their facilities and for helpful discussions. I also thank John Gerhart for reading the manuscript and Mr. Michael Wu for help in learning the gel techniques used herein. I am also grateful to Mr. Nick Story for the drawings. This work was supported in part by NIH research Grant No. GM 19363 from the National Institute of General Medical Sciences and National Science Foundation Grant No. PCM 76-01549.

Note added in proof

Recent ly , sugar de te rmina t ions were done by gas-liquid c h r o m a t o g r a p h y on large and small YP f rom oocy te s (Spiro, 1972 ; Porter , 1975). The follow- ing quanti t ies o f monosacchar ides , as alditol acetates, were found :

Sugar Protein {determined by absorbance at 280 nm) (pg/mg)

Fuc 2.19 Glc NH2 0.57 Gal NH2 1.50 Man 5.14 Gal 0 .15 Glc 4.86

As in the case of blastula and gastrula YP, Glc and Man are the mos t a b u n d a n t neutra l sugars and Gal is the least abundant . The Dische react ions for m e t h y l p e n t o s e gave ambiguous results for e m b r y o n i c YP, bu t the presence of L-Fuc in oocy te s was conf i rmed by gas c h r o m a t o g r a p h y .

Sialic acid, as de te rmined by the th iobarbi tur ic acid test, was absent in o0cy tes as well as in embryos .

REFERENCES

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