ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

173,

l-10

(1976)

Sialyltransferase in Fetal Tissues: Incorporation Endogenous and Exogenous Glycoprotein MATHEW

Children’s

Hospital

Research

of Sialic Acid into Acceptors’

M. MADAPPALLY,2 JAMES R. WILSON,3 ERNEST F . ZIMMERMAN4 Foundation and Departments Cincinnati, Cincinnati, Received

December

of Pediatrics Ohio 45229

AND

and Pharmacology,

University

of

2, 1974

The properties of exogenous sialyltransferase activity in fetal mouse liver were compared with the endogenous enzyme activity. Nonionic detergents such as Tergitol inhibited endogenous activity but released the enzyme from the 30,OOOg particulate fraction and allowed it to react with the exogenous glycoprotein acceptors, partially desialylated a-fetoprotein and calf fetuin. Zinc ions partially transferred the enzyme activity from the particulate fraction to the supernatant, while incubation at 45°C preferentially inhibited endogenous enzyme activity. a-Fetoprotein, sialylated by the endogenous enzyme preparation, was determined to be 7% of the total incorporation of radioactivity by immunoprecipitation with mouse anti-a-fetoprotein and subsequent sodium dodecyl sulfat+polyacrylamide-gel electrophoresis. Sialyltransferase activity in fetal liver, yolk sac, placenta and embryo (minus liver) was measured during development. Exogenous enzyme activity of liver in early development (day 13.5) was twice as high as that in yolk-sac tissues that synthesize afetoprotein. Furthermore, enzyme activity increased 12-fold in fetal liver and 4-fold in yolk sac by day 18.5 of development. Activity in placenta increased 2.9-fold and then decreased, while embryo (minus liver) showed no increase. Endogenous enzyme activity increased temporally in these tissues, but the increase was less than that measured by using exogenous acceptors.

One of the secreted glycoproteins synthesized by mouse fetal tissues is ol-fetoprotein (1). This protein was shown to exhibit a developmental microheterogeneity due to differences in the number of sialic acid residues attached to the oligosaccharide chain (2, 3; Zimmerman et al., unpublished). Sialic acids occupy the terminal position of the oligosaccharide chains of glycoproteins, and it has been established ’ This work was supported by a grant from the American Cancer Society (No. NP86) and a Center Grant in Mental Retardation (No. HD-05221). * Present address: Coulter Diagnostics inc., Hialeah, Fla. 3 Sections of this paper represent part of the Ph.D. dissertation of J. R. Wilson to be submitted to the Graduate Program in Developmental Biology, University of Cincinnati. ’ To whom correspondence should be addressed.

Copyright All riphts

0 1976 by Academic Press, Inc. of rmroduction in anv form reserved

that these residues play an important role in their metabolism (4, 5). Polymorphonuclear leukocytes, when their membranes were stripped of sialic acid, were shown to lose their stimulation of phagocytosis by tuftsin (6). Sialic acid, which occupies a terminal position on the carbohydrate chains of many glycoproteins, may in fact mask or otherwise affect the cell-cell interactions and adhesion thought to be associated with the more internal sugars of glycoproteins (7, 8). Sialic acid is added to the growing oligosaccharide chain by sialyltransferase. It is possible that the developmental microheterogeneity of mouse cu-fetoprotein, due to differences in sialic acid content, results from different levels of sialyltransferase activity in tissues that synthesize the cyfetoproteins during embryonic develop-

2

MADAPPALLY,

WILSON

ment. The biological significance of the developmental microheterogeneity of mouse a-fetoprotein is not yet known. However, it would be interesting to know if there is a fluctuation of sialyltransferase levels concomitant with the increased sialylation of mouse a-fetoprotein during development. This paper deals with (a) the study of the changes in exogenous enzyme activity of CMP-sialic acid:glycoprotein sialyltransferase in mouse fetal liver, yolk sac, placenta and embryo (minus liver) during development; (b) a comparison with endogenous sialyltransferase activity since this would also reflect the endogenous acceptor levels during development and morphogenesis of different embryonic tissues; and (c) some of the properties of sialyltransferase found in the embryonic tissues. MATERIALS

AND

METHODS

Materials. CMP-[U-‘4C]sialic acid and CMP[3H]sialic acid were purchased from New England Nuclear Corporation, phospholipase A and Tergitol NPX from Sigma Chemical, neuraminidase (Vibrio cholerac) from Calbiochem, and trypsin from Boehringer-Mannheim Corporation. Breeding of mice. Appearance of a vaginal plug the morning after overnight mating of mice was used as a criterion for conception. Inbred strains C3H/An and A/J were obtained from Cumberland View Farms, Clinton, Tenn., and Jackson Laboratories, Bar Harbor, Me., respectively. Most experiments employed the C3H strain except for those described in Fig. 3 and Table IV (endogenous sialylated u-fetoprotein) which used the A/J strain. There were no differences between the two strains in either exogenous or endogenous sialyltransferase activity during development. Preparation of tissue homogenates. Pregnant mice from day 12.5 to 18.5 of gestation were killed by cervical dislocation of the neck, and the uteri were removed and kept on ice. After the fetuses were separated from the uterine sacs, the placentas, yolk sacs, livers and total embryos (minus livers) were removed and pooled. A 10% homogenate of each tissue was prepared in sucrose medium containing 0.02 M Tris (pH 7.0), 1 mM EDTA, 1 mM reduced glutathione and 0.25 M sucrose by using a PotterElvehjem homogenizer for 2 min. The homogenates were successively centrifuged at 600g for 10 min and 30,OOQg for 30 min at 4°C. The pellet fractions were suspended in appropriate volumes of the sucrose medium by two strokes in a Dounce homogenizer. Sialyltransferase activity was determined in both pellet and supernatant fractions to study develop-

AND

ZIMMERMAN

mental changes in enzyme activity in various embryonic tissues. However, since most of the enzyme activity was located in the 30,OOOg pellet, this fraction was used in all other experiments. Protein was estimated by method of Lowry et al. (9). Preparation of glycoprotein acceptors, Fetuin was purified from fetal calf serum by the method of Spiro (10). The purified calf fetuin was then partially desialylated by incubation with 3 units of V. chlolerae neuraminidase per mg of protein (11). After a 24-hr incubation, the proteins were applied to a Bio-Gel P30 column (1.4 x 6 cm), and the partially desialylated calf fetuin which elutes in the void volume was collected and precipitated with four volumes of 100% ethanol at -20°C. The precipitate was collected by centrifugation at SOOg for 10 min and dissolved in 0.05 M sodium acetate buffer (pH 5.6). The mouse (Ifetoprotein mixtures, purified by polyacrylamidediscontinuous (disc) gel electrophoresis, were obtained from the following sources: Fp l-3 from day12.5 amniotic fluid, Fp l-5 from day-15.5 amniotic fluid and Fp5 from day-18.5 fetal plasma. In addition, a FplN and FP~~ mixture was prepared by desialylating the Fp l-5 mixture with V. choler-w neuraminidase (50 wg of protein, 30 units of enzyme) as described for calf fetuin. Enzyme assay. Sialyltransferase activity was determined essentially as described by Kim et al. (12). The reaction system contained, unless otherwise indicated, 66 pmol of cacodylateacetate (pH 6.81, 200 pg of desialylated calf fetuin, 100 pmol of CMPlL4C]sialic acid (229 mCi/ mmol), 0.5 mg of Tergitol and 50 ~1 of enzyme preparation (approximately 290 pg of protein) in a final volume of 0.33 ml. The reaction mixture was incubated at 37°C for 2 h and the reaction was stopped by adding 5 ml of 1% phosphotungstic acid in 0.5 M HCl. The precipitate was collected by centrifugation, washed three times with 5 ml of phosphotungstic acid, and dissolved in 1 ml of Soluene, and the radioactivity was determined in a liquid scintillation spectrophotometer by using 10 ml of liquid scintillator (4 g of 2,5-diphenyloxazole and 100 mg of 1,4-bis(4-methyl-5-phenyloxazol-2-yl)benzene per liter of toluene). The values for endogenous sialyltransferase activity were determined by the same assay system but deleting Tergitol and desialylated calf fetuin. Under conditions for the assay of exogenous sialyltransferase activity, the amount of desialylated calf fetuin employed was saturating; the radioactivity incorporated was proportional to the concentration of enzyme protein at all times in development. Enzyme activity was also linear during the 2h incorporation. At the pH employed, enzyme activity was 93% of that which is optimal (pH 7.2). Under conditions of the endogenous assay, activity was proportional to enzyme protein. The pH of 6.8 was optimal for the endogenous activity. The reaction was linear at 37°C for only 10 min. Thereaf-

SIALYTRANSFERASE

IN

ter, little additional activity could be shown. The instability of the endogenous acceptor system can also be observed in the experiment presented in Fig. 2. Polyacrylamide-gel electrophoresis of sialylated proteins. Analytical separation of sialic acid-labeled exogenous glycoproteins was carried out as described by Ornstein (13) and Davis (14). The proteins were separated in 6 x 90-mm separating gels for about 2 h at 3.5 mA per gel until the tracking dye was approximately 1 cm from the end of the gel. The gels were extruded and stained in 1% aniline blueblack in 7% acetic acid, and the absorbance patterns were recorded by scanning at 600 nm in a Gilford spectrophotometer equipped with a densitometer. The gels were sliced into 2 mm pieces and each slice was placed in a separate scintillation vial. To each vial 0.1 ml of 30% H,O, was added, the vials were capped and incubated overnight at 65°C. The radioactivity was determined in 5 ml of a detergent liquid scintillator (16.5 g of 2,5-diphenyloxazole in a mixture of 2 liters of toluene and 1 liter of Triton X-100). Immunotitration of a-fetoprotein present in the endogenous preparation. Ten replicate assays for endogenous enzyme activity were carried out by employing 150 pg of protein in the 30,OOOg pellet fraction from day-17.5 fetal liver in each tube. The composition of each reaction mixture was as previously described except that 85 pmoles of CMP-[3Hlsialic (2.33 Ci/mmol) was used. After a 2-h incorporation at 37”C, duplicates were assayed for total incorporation of radioactivity in the following manner. The reaction was stopped by addition of 3 ml of phosphotungstic acid, and the precipitate was collected on a 13-mm glass filter (Gelman A-E), washed three times with 3 ml of phosphotungstic acid, and dissolved in 0.3 ml of Soluene. Three milliliters of liquid scintillator were added, and radioactivity was counted. The remaining reaction mixtures were chilled and recombined, and an equal volume of a detergent mixture (2% Triton X-100 and 2% sodium deoxycholate) was added. Final volume was 5 ml. The preparation was sonicated for 30 s and centrifuged at 30,OOOg for 30 min, and the supernatant fluid was centrifuged at 105,OOOg for 90 min. Duplicate aliquots (50 ~1) of the supernatant solution were incubated for 2 h at 37°C and then 1 day at 4°C with 25, 50, 100, 150 and 200 ~1 of mouse anti-cufetoprotein. Day-15.5 mouse amniotic fluid (5 ~1) containing 6 wg of cY-fetoprotein was added as carrier; 30 ~1 of detergent mixture was also added, and phosphate-buffered saline was added to make the final volume 300 ~1. The antibody was a purified IgG preparation (71 mg/ml) and is monospecific for mouse a-fetoprotein (2). The immunoprecipitates were washed by the method of Rhoads et al. (15). 5 Abbreviations sodium dodecyl

used: sulfate.

IG,

immunoglobulin;

SDS,

FETAL

3

TISSUES

Each immunoprecipitate was layered over 100 ~1 of 1 M sucrose and detergent mixture in a 0.4-ml conical polyethylene centrifuge tube, centrifuged at 15,OOOg for 15 min, the solution frozen and the tip of centrifuge tube cut off. The immunoprecipitate in the tip was dissolved in 0.3 ml of Soluene and counted as before. RESULTS

Properties

of Embryonic

Sialyltransferase

Sialyltransferase catalyzes the transfer of sialic acid from CMP-sialic acid into glycoproteins and therefore the measure of incorporation of sialic acid into a glycoprotein acceptor is the method most widely used for the determination of the enzyme activity. Since our goal was to assay the sialyltransferase that incorporated sialic acid into mouse a-fetoprotein, it would be better to use desialylated a-fetoprotein as the glycoprotein acceptor in our assay system. However, availability of purified CYfetoprotein is very limited, and therefore we have examined whether desialylated calf fetuin would function as a suitable sialic acid acceptor. Table I shows the incorporation of [14Clsialic acid into various exogenous acceptors in the presence and absence of Tergitol. It is known that in many instances nonionic detergents like Tergitol stimulate membrane-bound enzymes, and therefore it was included in the assay system (14). The total incorporation of radioactivity in the absence of Tergitol remained approximately the same whether or not the exogenous acceptors were added to the incubation mixture. These results suggest that there was no V4Clsialic acid incorporation into the exogenous acceptors in the absence of Tergitol. When Tergitol was added in the absence of exogenous acceptors there was only a negligible incorporation into the endogenous acceptors (208 cpm). Addition of exogenous acceptors in the presence of Tergitol resulted in appreciable incorporation. The radioactivity incorporated into each exogenous acceptor was calculated by subtracting the radioactivity in the absence of exogenous acceptors from the total radioactivity incorporated. There was a large incorporation of radioactivity into both the FplN and Fp3N mixture (2165 cpm) and desialylated calf fetuin (10,235 cpm). Less

MADAPPALLY,

WILSON

AND

TABLE INCORPORATION

-

Acceptor

Protein (CLg)

OF

[‘CISIALIC

ZIMMERMAN

I

ACID

INTO EXOGENOUS

ACCEPTORS”

-Tergitol Total incorporation kpm)

+Tergitol Incorporated into acceptor (cpm)

Total incorporation (cpm)

Incorporated into acceptor (cpm)

Counts per minute per microgram of acceptor protein

None 4,398 208 FplN and FP~~ 5 4,243 2,373 2,165 433 Fpl-3 25 4,438 40 724 516 21 Fpl-5 38 4,604 206 280 2 72 29 4,465 67 228 20 1 Fp5 Desialylated calf 100 3,640 10,443 10,235 100 fetuin DFetal livers from day 18.5 of development were homogenized, successively centrifuged at 600 and 30,OOOg and the pellet fraction (30,OOOg) was suspended in sucrose medium. An aliquot of 50 ~1 (290 pg of protein) was added to each incubation mixture and incubated for 2 h at 37”C, and the radioactivity incorporated was determined as described in the text.

radioactivity was incorporated into the Fpl-3 mixture (516 cpm), and only negligible activity was observed with the Fpl-5 mixture and with Fp5. These results indicate that there was a progressive decrease in incorporation of labeled sialic acid into a-fetoprotein mixtures as the four available sites on the glycoprotein were progressively occupied with sialic acid residues (Zimmerman et al., unpublished results). However, incorporation into these cY-fetoprotein components from amniotic fluid or plasma was much less than would be expected for a four-sialic acid model: Fp5, 4 sialic acid residues; Fp4, 3; Fp3, 2; Fp2, 1; Fpl, 0. A possible explanation might be that these a-fetoprotein components show microheterogeneity with respect to the penultimate galactose; oligosaccharide chains lacking galactose would not accept sialic acid. Although the FplN and Fp3N mixture (80% Fpl and 20% Fp3) was a very efficient acceptor (433 cpmlpg of acceptor protein) relative to desialylated calf fetuin (100 cpm/pg>, the radioactivity incorporated into desialylated calf fetuin was appreciable. In fact, since calf fetuin was present at near-saturating conditions and the FplN and Fp3N mixture was limited in the reaction mixture, a true comparison between the exogenous acceptors cannot be made. In any case, desialylated calf fetuin appeared to be a useful glycoprotein

acceptor and was employed to study sialyltransferase activity in embryonic tissues. The results in Table I show that endogenous activity is almost totally inhibited by Tergitol and, further, as stated earlier, Tergitol appears to be an obligatory requirement for incorporation of sialic acid into exogenous glycoprotein acceptors. In order to confirm this conclusion, desialylated calf fetuin and an a-fetoprotein mixture (FplN and Fp3N) were incubated for 2 h with CMP-[14C]sialic acid and fetal liver sialyltransferase in the presence and absence of Tergitol. The incubation mixture was then centrifuged at 6OOg, and the supernatant solution was subjected to polyacrylamide-disc gel electrophoresis. When the incubation mixture contained Tergitol most of the radioactivity incorporated was found in the supernatant proteins. However when Tergitol was deleted, almost all activity was associated with the pellet fractions (data not presented). Figure 1 shows the protein profile obtained by scanning the gels at 600 nm and shows the amount of radioactivity incorporated into each of these proteins. When Tergitol was present in the incubation mixture, the radioactivity peaks (closed circles) corresponded with the absorbance peaks of the acceptor proteins: The labeled sialylated a-fetoprotein was slightly faster moving than Fpl, which contains no sialic acid

SIALYTRANSFERASE

GEL SLICE NUMBER

FIG. 1. The sialylated proteins were identified by polyacrylamide-gel electrophoresis. The FplN and FP~~ mixture or desialylated calf fetuin were incubated for 2 h with CMP-[%lsialic acid in the presence of sialyltransferase prepared from the 30,OOOg pellet of day-18.5 fetal liver. The reactions were stopped by chilling the tubes on ice. The proteins in the supernatant fraction obtained after centrifugation at SOOg for 10 min at 4°C were separated by polyacrylamide-gel electrophoresis as described in Materials and Methods. (A), FplN and FP~~ mixture; (B), desialylated calf fetuin. Radioactivity: (01, presence of Tergitol; (O), absence of Tergitol. Absorbance patterns ( - 1 of gels from extracts containing Tergitol are presented.

(Fig. lA), due to the increased anodic charge of the carboxyl group on the sialic acid. Thus one or two negatively charged sialic acid residues were incorporated into the glycoprotein which resulted in the shift of the radioactivity peak from the absorbance profile of Fpl. In contrast, the radioactivity peak corn&rated with the absorbance peak of calf fetuin in Fig. 1B. This correspondence of peaks might be due to the fact that the calf fetuin was only partially desialylated, and the addition of labeled sialic acid residues were small in relation to the preexisting ones and hence did not markedly increase the negative charge of the protein. When the enzyme

IN

FETAL

TISSUES

5

preparations were incubated in the absence of Tergitol (open circles), negligible incorporation of sialic acid into both exogenous glycoproteins and supernatant proteins was observed (Figs. 1A and B). It was possible that Tergitol treatment not only inhibited the endogenous sialyltransferase activity but also released the enzyme from the particulate fraction and thus allowed the enzyme from the particulate fraction to incorporate sialic acid into the exogenous acceptor. An experiment was therefore conducted to examine this possibility, and the result is shown in Table II. Endogenous activity was almost exclusively in the pellet fraction when no exogenous glycoprotein acceptor and Tergitol were added (control). This activity was almost totally inhibited by the addition of Tergitol, as found earlier. When desialylated calf fetuin was added alone to the incubation mixture, sialyltransferase activity remained the same, suggesting that desialylated calf fetuin did not inhibit the endogenous activity nor react with the enzyme. Exogenous sialyltransferase activity was 30% greater as indicated by the radioactivity incorporated in the presence of desialylated calf fetuin and Tergitol. The endogenous as well as exogenous activities in the control supernatant solutions were negligible (430-719 cpm) compared to their respective activities in the pellet fractions (12,475-17,206 cpm). Next the low speed supernatant fraction was preincubated with Tergitol and subsequently fractionated at 30,OOOgto ascertain if enzyme activity was released to the high speed supernatant. This Tergitol pretreatment prevented endogenous activity in both the pellet and the supernatant fractions as expected. Upon addition of desialylated calf fetuin there was a significant incorporation of radioactivity in both the pellet (9620 cpm) and the supernatant fractions (14,650 cpm). Thus Tergitol pretreatment released sialyltransferase activity from the 30,OOOgpellet to the 30,OOOg supernatant fraction (comparing addition of calf fetuin under control and experimental conditions). However, when the extract was pretreated with Tergitol, the combined incorporation in the pellet and su-

6

MADAPPALLY,

RELEASE

-

WILSON

OF MEMBRANE-BOUND

Additions

Control

(not

-

None Tergitolb Desialylated calf Desialylated calf and TergitoP

fetuin fetuin

ZIMMERMAN

TABLE II SIALYLTRANSFERASE preincubated Tergitol)

BY TERGITOL~

with

Experimental

(preincubated Tergitolb)

with

Pellet

-

AND

13,080 923 12,475 17,206

Supernatant

Pellet

647 0 719 430

Supernatant

92

273

9,620

14,650

(1 After removing unbroken cells and debris by centrifugation of the 10% homogenate of day-16.5 fetal liver at 12lg for 10 min, the supernatant fraction was divided into two equal parts and to one Tergitol (1%) was added. After a 15-min incubation at room temperature, the enzyme preparations (Tergitol-treated and untreated) were centrifuged at 30,OOOg for 30 min. Pellet fractions were suspended to their original volume and 50+1 aliquots of the pellet and supernatant fractions were assayed for enzyme activity in triplicate. Values expressed are counts per minute incorporated per milligram of protein during a 2-h incorporation. * Tergitol present in final concentration of 0.15% in reaction mixture.

pernatant fractions in the presence of calf fetuin (24,270 cpm) was greater than incorporation in both fractions of the control experiment in the presence of Tergitol (17,636 cpm). A possible explanation is that the time of exposure to Tergitol in the control was less than when the supernatant fraction was preincubated with Tergito1 for 15 min and subsequently fractionated. Results presented so far show that Tergitol affects the incorporation of sialic acid into endogenous acceptors and exogenous glycoproteins differently, endogenous activity being almost totally inhibited by Tergitol. For exogenous activity, the nonionic Tergitol is an obligatory requirement for incorporation. Sonication treatment as well as addition of 0.15% Triton X-100, showed similar effects on endogenous and activities exogenous sialyltransferase (data not presented). These results with fetal mouse liver in which the particulate preparation is active with exogenous glycoproteins in the presence of detergent are similar to those found with embryonic chick brain glucosyltransferase (16) and human serum N-acetylgalactosaminyltransferase (17). In Fig. 2, the enzymatic incorporation of sialic acid into endogenous acceptors, after heat inactivation, decreased rapidly with time of incubation and less than 20% of the initial activity

0"

0

2

4 TIME

IN

6 8 MINUTES

IO

FIG. 2. Effect of heat on enzymatic incorporation into endogenous and exogenous acceptor proteins as a function of time. A 10% homogenate of day-18.5 fetal liver was centrifuged at l2lg for 10 min. The supernatant solution was equally divided, and Tergitol (0.66%) was added to one part to yield a final concentration of 0.15%. Tergitol-treated and untreated enzyme preparations were then incubated for 15 min at room temperature and then centrifuged at 30,OOOg for 30 min. The supernatant solution from the Tergitol-treated preparation and the pellet fraction from the untreated sample were collected. The pellet fraction was then suspended in sucrose medium. Both preparations were incubated at 45”C, and 50-J aliquots were removed at various times. Thereafter endogenous enzyme activity (0) in the pellet preparation and exogenous enzyme activity (0) in the supernatant fraction (Tergitoltreated) were determined. Value for initial endogenous activity was 26,151 cpm per mg of protein per 2h incorporation while that of exogenous activity was 30,895.

SIALYTRANSFERASE

IN

TABLE OF PHOSPHOLIPASE

A, EDTA,

Additions

30,OOOg Endogenous ity

activ-

ZINC

IONS

7

TISSUES

The endogenous activity was less markedly inhibited by trypsin: 58% in the pellet and 28% in the supernatant fraction. It is difficult to explain the varying degrees of inhibition of endogenous and exogenous activities present in the pellet and supernatant fractions by trypsin. The greater inhibition of exogenous compared to endogenous activity might be explained by the partial protection of enzyme or acceptor by membranes in the endogenous system. In order to characterize further the nature of the endogenous acceptors, the 13Hlsialic acid-incorporated product was subjected to SDS -polyacrylamide-gel electrophoresis. Figure 3A shows that there are three main radioactivity peaks at gelslice numbers 8, 13 and 17. The peak at gel-slice number 13 has the same mobility as marker cY-fetoprotein (70,000 molecular weight). This radioactivity profile is fairly reproducible between experiments. However these proteins entering the gel represent only about 28% of the total radioactivity incorporated in endogenous acceptors (Table IV). It would seem likely that the majority of the sialylation occurs on membrane components which cannot enter the gel. In order to analyze the amount of labeled a-fetoprotein in the endogenous product more precisely, an immunoprecip-

was left after a lo-min incubation at 45°C. In contrast, the incorporation of radioactivity into the exogenous acceptor was not decreased during the same time interval. Table III shows the effects of various treatments on sialyltransferase activity. Phospholipase A inhibited endogenous activity by 43% and exogenous activity by 64%, suggesting that the enzyme is associated with membrane components under both conditions. Although there was a slight inhibitory effect of EDTA on both endogenous and exogenous activities (about 15%), it was not as marked as the effect of phospholipase A. A lack of effect of EDTA on sialyltransferase has been previously observed with enzyme bound to erythrocyte membranes (12) and that found in mammary glands (18). Zinc inhibited the exogenous sialyltransferase activity present in both the pellet and supernatant fractions by about 60%. Though there was a decrease in the endogenous activity of the pellet fraction (27%), the activity in the supernatant fraction increased by 55%. This increase was probably due to Zn2+ releasing endogenous activity from the pellet into the 30,OOOg supernatant fraction. Like Zn”+, trypsin markedly inhibited exogenous enzyme activity, though inhibition in the pellet fraction (89%) was greater than in the supernatant (69%).

EFFECT

FETAL

III AND

TRYPSIN

ON SIALYLTRANSFERASE

Pellet Exogenous ity

ACTIVITY”

30,OOOg Supernatant activ-

Endogenous ity

activ-

Exogenous

activity

None 2,523 (lOOjb 9,449 (100) 841 (100) 1,457 (100) Phospholipase A 1,441 (57) 3,424 (36) EDTA 2,180 (86) 8,361 (89) Zinc ions 1,850 (73) 3,450 (37) 1,302 (155) 584 (40) Trypsin 1,069 (42) 607 (72) 999 (11) 445 (31) ’ The 10% homogenate of day-17.5 fetal liver (frozen for 2 weeks) was successively centrifuged at SOOg for 10 min and at 30,OOOg for 30 min. The pellet fraction from the second centrifugation was suspended in sucrose medium and was used in this study. The effects of 1 mM zinc acetate and 0.25% trypsin on enzyme activity were studied by preincubating the enzyme preparations for 1 h at room temperature and 15 min at 37”C, respectively. After preincubation the preparation were again centrifuged at 30,OOOg. Endogenous and exogenous activities in the appropriate pellet and supernatant fractions were determined and are expressed as in Table II. * The percentages of initial enzyme activity left after various additions are given in parentheses.

MADAPPALLY,

o-

1

IO

26

I

I

GEL

SLICE

30

I6

20

WILSON

30

I

NUMBER

FIG, 3. Sodium dodecyl sulfate (SDSkpolyacrylamide-gel electrophoresis of L3Hlsialic acid incorporated into endogenous acceptors. Replicate samples were pooled and solubilized as described in Materials and Methods. Aliquots (100 ~1) of a control sample (A) or that immunoprecipitated with 150 ~1 of anti-a-fetoprotein (B) were subjected to SDSpolyacrylamide-gel electrophoresis (19). Samples were first solubilized with 8 M urea and 1% SDS. The arrows indicate the mobility of a-fetoprotein as determined by parallel electrophoresis of purified Fp5 and its subsequent staining with Coomassie blue. Radioactivity (0) in each appropriate gel slice was determined.

itation of this preparation with mouse anti-cu-fetoprotein was carried out. When the immunoprecipitate was also subjected to SDS-polyacrylamide-gel electrophoresis, a predominant labeled peak is observed that has the same mobility as marker a-fetoprotein. There is some background radioactivity, and there is a small labeled peak that slightly contaminates the a-fetoprotein immunoprecipitate. After integration of labeled cw-fetoprotein in the gel, it was determined that 7% of the total endogenous radioactivity was in CXfetoprotein (Table IV). Exogenous and Endogenous Activity Sialyltransferase with Development

of

Since it has been previously shown that the mouse cY-fetoproteins are progressively sialylated with time of development, we have studied the developmental changes in both exogenous and endogenous sialyltransferase activity. Figure 4 shows the differences in developmental patterns of

AND

ZIMMERMAN

exogenous and endogenous sialyltransferase activities in fetal liver, yolk sac, placenta and embryo (minus layer). Figure 4A shows that the exogenous sialyltransferase activity, assayed with desialylated calf fetuin, progressively increased both in fetal liver (1Bfold) and yolk sac (Cfold) from day 13.5 to 18.5 of gestation. However, the increase in liver was more marked, increasing rapidly from day 13.5, while yolk sac lagged behind liver and started to increase significantly from day 14.5. Furthermore the enzyme level in fetal liver at day 13.5 was twice that of the yolk sac at this time. The enzyme activity did not markedly change in embryo (minus liver). Although the enzyme activity increased 2.9-fold from day 13.5 to 17.5 in placenta, it started decreasing thereafter. The endogenous activity with time of development (Fig. 4B) did not differ significantly in yolk sac and embryo from day 12.5 to 18.5. On the other hand, fetal liver and placenta showed an initial increase in endogenous activity starting at day 13.5 but the activity decreased at later times of TABLE INCORPORATION FETOPROTEIN Fraction Total homogenate 105,OOOg supernatant Fp immunoprecipitate* Soluble proteins SDS gel’ Fp immunoprecipitate in SDS geld

IV

OF [%]SIALIC OF ENDOGENOUS

in

ACID INTO aACCEPTORS

Incorporationn (dpm)

Percentage of tutal

203,745 174,545 21,951 56,618

100 86 11 28

14,339

7

a Values are disintegrations per minute incorporated in 5-ml total volume of the pooled and detergent-solubilized reaction mixture. Disintegrations are presented to compensate for different counting efficiencies of various systems. b Immunoprecipitation of a-fetoprotein is described in Materials and Methods. Equivalence reached at 200 ~1 of antibody preparation. c Profile of sodium dodecyl sulfate (SDSkolubilized proteins entering the gel is indicated in Fig. 3A. d Profile of cr-fetoprotein immunoprecipitate subjected to SDS-polyacrylamide gel electrophoresis is indicated in Fig. 3B.

SIALYTRANSFERASE

IN 1

FIG. 4. Sialyltransferase activity in various tissues with time of development were measured. Pregnant mice from day 12.5 to 18.5 of gestation were sacrificed in the morning between 10 and 11 AM on the day of the experiment. The uterus was removed and placenta, yolk sac, liver and embryo (minus liver) were separated. Tissues from several embryos were pooled to obtain adequate amounts of tissue to make a 10% homogenate. The sialyltransferase activity in 600g pellet, 30,OOOg pellet and supernatant fraction was determined separately in duplicate. Since the distribution of the enzyme activity in each fraction varied slightly with time of development, the activities in all the fractions were combined and the results are expressed as counts per minute incorporated per gram of tissue during a 2-h incubation. Six to eight experiments were performed to obtain the average & SE values for sialyltransferase activity on any day of development. The procedures for the preparation of tissue homogenates, subcellular fractionation and enzyme assay are described in Materials and Methods. (A), Exogenous enzyme activity; (B), endogenous enzyme activity. Values for exogenous sialyltransferase activity of fetal liver and yolk sac previously presented (20) are reproduced here for comparison.

development. Endogenous and exogenous activities did not differ significantly in placenta. In fetal liver the endogenous activity was significantly lower on day 18.5 compared to exogenous activity. Yolk sac and embryo (minus liver) demonstrated high endogenous activity compared to exogenous activity. DISCUSSION

Developmental changes in the activities of glycosyltransferases in chicken brain (21, 22) and rat pancreas (23) have been shown to be high during early embryonic stages and decreased by birth or shortly thereafter. Since mouse a-fetoprotein

FETAL

TISSUES

9

shows a developmental microheterogeneity due to differences in sialic acid content, we have examined sialyltransferase activity in different embryonic tissues including fetal liver and yolk sac, the tissues where the synthesis of a-fetoprotein occurs (1, 24). As found in chicken brain and rat pancreas, there was a very high level of endogenous activity in all the fetal mouse tissues tested. We have examined this activity as well as exogenous enzyme activity since endogenous activity would partially reflect the endogenous acceptor levels during development and morphogenesis of mouse fetal tissues. When the developmental changes in sialyltransferase activity were measured by using desialylated calf fetuin as the sialic acid acceptor, fetal liver and yolk sac showed a many-fold increase in enzyme activity (20). Enzyme activity in placenta also increased during development, but activity in embryo (minus liver) remained essentially constant (Fig. 4A). Thus the progressive sialylation of a-fetoprotein found in fetal plasma and amniotic fluid (2) could derive from the temporal increase in sialyltransferase activity in fetal liver and yolk sac. Alternatively, the twofold greater activity in fetal liver than in yolk sac at day 13.5 of gestation could indicate that the enzyme in fetal liver is not limiting, while the yolk-sac enzyme is limiting. As a result, fetal liver would not show the developmental changes in sialylation of (Yfetoprotein, while the yolk sac could contribute to the production of incompletely sialylated a-fetoproteins. Although endogenous enzyme activity increased with time in fetal liver, yolk sac, placenta and embryo (minus liver), the magnitude was much less than that observed by the assay of exogenous activity. Furthermore, a maximum level was observed by day 15.5 to 17.5 and then decreased towards the late fetal period. The temporal differences between the exogenous and endogenous activity levels may reflect changes in the endogenous acceptor levels. Such biochemical changes could play an important role in tissue development and morphogenesis. It is known that, during the process of differentiation of intestinal epithelial cells, the incorpora-

10

MADAPPALLY,

WILSON

tion of sialic acid into endogenous acceptor increases and reaches a maximum just prior to full differentiation; thereupon activity started to decrease (25). We have compared the properties of exogenous sialyltransferase activity with the endogenous activity. Tergitol was an obligatory requirement in the incubation mixture for incorporation of sialic acid into exogenous acceptor (Table I, Fig. 1). Little radioactivity was incorporated in the absence of Tergitol. The enzyme is bound to the membrane as are most other glycosyltransferases, and in order for the enzyme to catalyze the sialic acid incorporation into exogenous acceptor it has to be made accessible by methods such as detergent treatment (Table II). Endogenous enzyme activity was highly sensitive to inhibition by Tergitol (Table I> and heat (Fig. 21, possibly inactivating the endogenous acceptor protein or the putative dolichollipid complex (26-28) or upsetting their close spatial relationship to the membrane-bound enzyme. Another possibility, although less likely, is that the endogenous and exogenous sialyltransferase activities represent two enzyme systems, one being labile and the other being stable towards treatments with heat, detergents, etc. However, further evidence that the endogenous activity is associated with membranes is shown in Table III. Zinc ions transferred the endogenous enzyme activity from the particulate fraction to ‘the supernatant fraction. Similarly, phospholipase inhibited the endogenous enzyme system. A limitation to measuring endogenous enzyme activity during development is that cw-fetoprotein would be only one of many glycoproteins sialylated. However, since a-fetoprotein is a major secreted protein in liver and yolk sac (1, 23), it would be expected to constitute a significant portion of the endogenous glycoproteins sialylated. That such an expectation was fulfilled comes from the observation that 7% of total sialic acid incorporation was into endogenous a-fetoprotein. I. (1971)

Advan.

5. 6. 7. 8. 9.

10. 11. 12.

13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26.

27. 28.

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