- Email: [email protected]
Characterization of sialyltransferases from serum of normal and hepatoma Mc-29 bearing chickens
hr. J. Biochm~. Vol. IX, No. 3. pp. 27 1-216, 19X6 Printed in Great Bntaln. All rights reserved
0020-711X/86
Copyrighl
CHARACTERIZATION FROM
SERUM
OF
OF NORMAL
BEARING H. Institute
CHELIBONOVA-LORER*,
of General and Comparative
$3.00+0.00
1986 Pergamon Press Ltd
SIALYLTRANSFERASES AND
HEPATOMA
MC-29
CHICKENS
S. IVANOV, E. GAVAZOVA
Pathology,
(Rewired
c
Bulgarian 17 April
Academy
and M.
ANTONOVA
of Sciences, Sofia
I II
3. Bulgaria
1985)
Abstract-l. Sialyltransferase was measured in serum of normal and hepatoma MC-29 bearing chickens. 2. By preparative isoelectric focusing the multiple forms of sialyltransferase from both kind of strums was studied as well. 3. By using influenza virus neuraminidase an attempt was made for partial structural characterization of the sialylation sites in asialofetuin applied as exogenous acceptor for sialytransferase determination. 4. It was established an elevated serum sialytransferase activity in tumor bearing chickens. In the pattern of the multiple enzyme forms separated from serum of chickens with tumor an enzyme form was detected with ~1-4.99 identical with an enzyme form described previously in solubilized plasma membrane preparations from hepatoma MC-29. 5. Monitoring of multiple forms of serum glycosyltransferases may be of value in answering the problem concerning the tissue origin of serum enzymes.
INTRODUCTION
(1975), Weiser (1973) who have presented data that glycosyltransferases are located on the cell plasma membrane, and could be released into the blood. In this report we have examined the activity of sialyltransferase in the serum of chickens bearing the virus MC-29 induced hepatoma. The multiple forms of serum sialyltransferase were determined as well in order to check the presence of forms which serve as an indicator of malignancy, taking into account that in a previous work (Ivanov et rrl., 1984) a tumor specific form of this enzyme was detected on the hepatoma MC-29 plasma membranes.
Soluble glycosyltransferases are detected in various animal fluids (Wagner and Cynkin, 1971; Kim et al., 1972; Nelson et al., 1973). The activity of some serum glycosyltransferases was found to be increased in tumor bearing animals and also in humans with neoplastic diseases (Podolsky and Weiser, 1975; Kesse1 and Allen, 1975; Bauer et al., 1977; Khilanani et al., 1978; Podolsky et al.. 1978; Chatterjee and Kim, 1978). As the glycosyltransferases are glycoproteins the abnormal levels of the serum activities were considered as a manifestation of some disorders in the glycoprotein metabolism in tumor cells and especially of plasma membrane glycoproteins. The problem concerning the mechanism by which these enzymes enter into blood circulation and which are the cells providing the serum enzyme activities in both normal and tumor bearing animals, and which is the role of these serum enzymes is still open to question. Yamada and Pouyssegur (1978) and Senger er al. (1979) have demonstrated an increased release of glycoproteins from the surface of the tumor cells. In contrast to Ip and Dao (1978), Kessel and Allen (I 975) and Evans et rd. (1980) who have revealed that the elevated serum activities were not connected with the presence of growing tumor, some other authors (LaMont et ul., 1977; Porter and Bernacki, 1975; Weiser. 1973) have shown that in metastasizing rat mammary tumor cells the cell surface shedding was much more higher than that from the surface of the benign tumor cells (Kim rt trl.. 1975). which was accompanied with an elevation of the sialyltransferase activity in the serum of the rats with the metastasizing tumor (Bernacki and Kim. 1977). These findings are consistent with the results published by LaMont rf c/l. (1977). Porter and Bernacki *Author
to whom
reprint
requests should
MATERIALS AND METHODS
Tumor Hepatoma MC-29 was obtained by injecting subcutaneously in 5.days old white Leghorn chickens a homogenous mince of hepatoma tumor suspended in saline. The hepatoma grow rapidly as a solid mass attaining a size from 3 4 g in about 10 days. Blood was collected without anticoagulant via heart puncture of 12 15 days old control and hepatoma bearing chickens and serum was obtained by centrifugation a1 3000~ for 15 min at 4 C.
[“‘CICMP-N-acetylneuraminic mmol) was purchased from Amersham, U.K.
acid (SA = 277 mCi. Radiochemical Center.
For rhe determination of the total serum activity of sialyltransfcrasc the method described by Ip and Dao (1978) was applied. Endogenous levels of the enzyme activity was established in the absence of added acceptor. When the exogenous activity was studied to the incubation media asialofctuin as acceptor a1 a saturation levels w’as added (Ip and Dao, 197X). The assay tubes w’ere incubated at 37 C for 60 min. Enzyme activity due to the transfer of the ,Vacetylneuraminic acid residue to exogenous acceptor was
be addressed. 271
calculated as the difference between the total activity with protein acceptor present minus the activity with only endogenous acceptors present in the serum. Protein concentrations were determined by the method of Lowry P/ trl. (1951) with bovine serum albumin as a standard.
Preparative isoelectric focusing was performed essentially as described previously (Ivtanov Ed (I/.. 1984) having in view the general considerati~)ns stated by Vesterberg (1981). The focusing was carried out with serum of normal and hepatoma bearing chickens (approx 20 mg protein) on I10 ml column (LKB 8101) using 20/b (v:v) ampholytes pH 3 -10 (LKB produkter. Stockholm. Sweden) and a gradient of O--SO% (w:v) sucrose. Electrofocusing was conducted for 12 hr at a starting voltage of 500 V which was increased to 600 V and the focusing was prolonged for another 28 hr while the column was kept at 4 C. Fractions (2 ml) were collected, their pH values were registered with Radiometer M83 digital pH-meter and aliquots (200 1’1) of each fraction were assayed for sialyltransferase activity in an incubation medium containing lXO/rl 0.5 mM Tris-I-ICI buffer (pH 6.6). 20~1 asialofetuin (lOmg~nl1) and IO//l [‘~CjCMP-iii-acetvlneuraminic acid (100.000 cpm approx). The samples ~ere.incnb~tcd for 60 min at 37 C, the reaction was stopped with 1% phosphotunstig acid (PTA) in 0.5 N HCI and the precipitate was subjected to the washing procedure and counting as described before (Ivanov L’I (il., 1984).
Morrin P$ al. (1983) have demonstrated that in the oligosaccharide chain of fetuin exist two r2 3 and one ~2-6 bonds between the terminal sialic acid and galactose and therefore asialofetuin could be SiaIyIated by the respective multiple forms orsiai~ltra~sr~rases at those three si~l~fl~tjon sites of the molecule. It was of interest to look which terminal sites of asialofetuin become sialylated by the rn~iti~le si~l~ltr~nferases by using in~uen~a virus n~ur~lrn~l~idase, which cleaves at a nearly 100% the r2 3 linkages. while the 32-6 and 12--g bonds between galactose and sialic acid are hydrolysed at a much lower degree (Schauer. 1983). Taking into consideration these properties of the influenza virus neuraminidase the following experimental scheme was adapted: aliquot samples (400 ~1) of the enzyme forms with approriate pl were Incubated for 60 min in the assay conditions for measurement of sialyltranferase activity (Ip and Dao. 1978). The reaction was stopped by addition of 0.4 ml 10% fw:v) aqueous suspension of Norit A. which preliminary experiments have shown, completely adsorbed the remained unreacted ~‘~C]CMP-~-~cetyineur~~minic acid. The tubes were shaken well for 20 min m an ice bath. after that were left to stay at 4 C in a refrigerator for IO min. The samples were centrifuged for 20 min at 5000 rpm. the supernatant containing the sialytated fetuin (sup,l) was collected. the sedimented charcoal was washed 2 times with vvatcr and the washings obtained after centrifupation were comhincd with tbc orginal supernatant-I.
The experimental assay conditions described by Bendiak and Zalik (IY81) for optimal neuraminidasc action were applied To the supernatant containing the siaiylated [i~C~f~tuiil faClz and NaC’t solutions in a linal concentration corresponding to 0.1 and 0.9% respectively and enzyme concentration of 0.75 units (C~~lbi~~chcn~. LOT 003~4~. specilic activity 320 unitsml at pH S.6 and 37 0 were added. The incubation was carried out at 37 f in a buffered media with pH 5.6. The reaction was terminated by the addition of 0.1 ml 1% PTA in 0.5 N HCI tmd the tubes
were centrifuged at 3000 rpm for IO min. The supcrnatant (sup.11) was collected and the protein precipitate was washed twice with PT.4 0.5 N HCI and washings wcrc added to the first supernatant-Il. In an aliquot part of thi\ hydrolysdte the radioactivity due to the [“C’jsialic acrci splitted off by the Ileu~minidase was counted with II liqurtl scintilator (4.0 g PPQ and 0.15 g POPOP in 1 I ~oit~nl)IO which Triton X-100 was added (2: 1 v v). In order to avoid the error due tt> the pre\enec lit [‘Qialic acid liberated as a result of spontaneous hydraiysis of ~‘.~C]CMP-~-~cetyineur~minic acid during the mcubation of the samples for the si~ilyitransfer~~sc dctermination, a control was prepared taking an aiiyuot of the sample designed for neurammidase treatment, the protean was sedimented bv addine 0.1 ml PTA 0.5 N I-ICI. ccnir~fuged and the supernatant (sup.lll) was taken apart. 11le participate was washed two folds with PTA and the w:ishings were combined with the sup.111. The radioactivity \hli\ measured in a PPO:‘POPOP toluen based scintilator ct)irtaining Triton X-100 (2: I v L). The control values vterc subtracted from the sample values obtained as :t eonsequence of the neu~~n~inid~tsc treatment. The vvashctl protein precipitate obtained after the desialylati[)r~ ~)t‘fctuin hy the neuram~nidase was taken to dryness ov>ernight at 37 C’ and the dry residue was dissolved in I .Oml NC3 t~\av solubili~er (Amersh~~rn;Searle Corp.. Arlingt~~l~ Heights III L and placed in 5.0 ml scintill~iti~~n cocktail as dcccrihcd ahole without Triton X-100. The radioactivity of aI1 kind
Results in Table I concerning the sialyltranferase activity in serum of normal and hepdtoma Mc-29 bearing chickens are presented either as endogcnous activity expressing the transfer of N-acctytneurdminic acid residue to the glycoprotein acceptors existing in the serum, or as total activity est~~blished in the presence of the appropriate specific acceptor. The exogenous activity of sialylt~~nferasc is calculated h! the difference between the total and the cndngcnous activity. Our results disclose that in the serum of tht hepatoma bearing chickens the sialyltransferaso LICtivity is elevated compared to that found in the strum of control animals. Our results are in good agrecnient with data of some other authors who have reported elevation in serum activities of glycosyltransf~rasoc III both human (Baucr <‘I nl.. 1977; BhattacharyLt. (‘hatterjee and Barlow. 1976) and animal (Rernaoki and Kim, 1977: Bosmann and Hilf. 1974: Ip and Datr. IWZ) tumor bearing 197X: Podolsky and Wciscr. hosts. The causes ol‘ the incrcascd Icvcl:, o/‘ plvco\>itransferase activities in the strum of the anim;tl4 01 patients with tumors are not W/I tmcicr~tood. The question which is discussed in the Iitcr;tturc concorns the sour-cc of the serum activities. Arc the cn/ymc< relcascd or “shed” from the growing tumor mass. :I\ several authors have suggested (Podolsky 1’1(I/.. 19?7: Bhattacharya, Chattcrjec and Barlou, 1976: Hosmann and Hiif. 1974). or the cnzymc actrvitic\ derived directly from the iivcr or other organ4 :t\ some other investigators have postulated (Hosm~mn clt II!., 1975: Ip and &to. tY77). Bernacki rind Kim (1977) have tbund that in thu serum of fats bearing rn~t~~st~isi~jn~ rn~irnln~lry tumars the sii~lyltransfer~~se activity was ~~~nsid~r~lbiv increased and they suggested that the highor lcvcls d this cnqme activity wcrc probahi\: due to :~n in-
fable
1
Slalyllransferase aclivity in the serum of normal and hepatoma MC-29 bearmg chickens Enzyme act&y
Normal chickens Tumor bearing chickens
(cpm’mp pr;hr)
ElldOgellOUl activity
With asialofetuin
Exogenous activily
x2x * 100 (4) 1417+ 13717Y
2095 2 196 (4) 5844 i 420 171**
1267i 14X(4) 4427 + 278 171**
V&es are the mean t SE of the number assays presented in parentheses. The exogenous enzyme wct~wy is calculated as a difference between the achvity measured in the presence of asialofetun and the endogenou~ acti\lty. 'P < 0.05:**p < 0.01.
creased ceil surface shedding of ectosialyltransferase into the blood stream. One of the most convincing arguments in favour of the statement that the serum glycosyltransferases are derived from tumor cells comes from the studies of Podolsky and Weiser (1975) and Podolsky t’t ul. (1977). They have revealed in the serum of cancer patients a specific isoenzyme of galactosyltransferase (GT II) that was absent in the healthy donors. The cancer associated isoenzyme (Podolsky et al., 1977) has been detected also in the serum of hamsters with tumor provoked after inoculation of BHK cells, transformed with the polyoma virus (BHK-PY). The same isoenzyme form was demonstrated in the cutture media of BHK-PY cells, but not in the media from the nontransformed BHK cells. Taking into consideration these findings we attempt to answer the question about the tissue origin of the serum sialyltransferase activity by detailed study on the multiple forms of the serum enzyme transferase by preparative isoelectrofocusing because in a previous work (Ivanov et al., 1984) it was shown that on the hepdtoma plasma membranes is located a specific form of sialyltransferase with ~1-5.00, which was not detected in the profile of the solubilized plasma membrane preparations from chicken liver. The results obtained in this study arc shown on Figs I and 2 and Table 2. Each point represents the relative enzyme activity expressed as a percentage of the total counts per min in all fractions taken as 100.
It is evident that the profile of the multiple forms of sialyltransferase from the serum of healthy chickens (Fig. I) and that of chickens with hepatoma (Fig. 2) is similar-more active forms in the alkaline region of the gradient especially for the serum of the hepatoma bearing chickens and presence of nearly the same forms in the acid region in both serums under investigation. There are also some other well pronounced differences-the appearance of a fraction with ~14.99 and disappearance of the fraction with ~19.49 from the serum of the animals with tumor. One comparison between the distribution of the liver and hepatoma MC-29 plasma membrane associated multiple forms of sialyltransferasc is presented on Table 2. It is evident that among the pl values of the membrane and serum multiple forms of the enzyme some differences could be delineated which are cxpressed at a different degree. For the better evaluation of these differences we defined three comparative criteria: (a) as identical with the plasma
lZr
11 -
IO -
9-
87-
2 -
6-
x t .> ':
7-
?_ E6 al ;
5
z G I?? 4-
3-
2-
1-
I
,
/
!
I
I
1
I
3
4
5
6
7
8
3
IO
I 3
I. Sialyltransfcrasc multiple pattern normal chickens.
/ 5
!
6
I
/
/
7
8
9
I 10
PH
PH Fig.
, 4
of
swum
from
Fig.
2. Sialyltransferase multiple pattern of serum hepatoma MC-29 bearing chickens.
from
H.
~‘H~~I.I~oN(~~A-Lo~~:K PI d.
membrane were accepted those serum forms whose pl were either equal with them or the differences in the pI values are not higher than 0.05; (h) analogous with the plasma membrane are serum forms with a difference in the p1 values which reach not more than 0.20; (c) as a typical serum forms were considered those of them whose pf values markedly deviated when compared to the plasma membrane forms and the differences are in the range between 0.40 and 0.60. On the basis of these criteria of similarity one can assume from the data of Table 2 that between the liver plasma membrane enzyme forms and the forms established in the normal serum there are 3 identical forms (Nos 5, 7 and S), 3 analogous (Nos 3.4 and 6) and 2 typical serum forms with pI 4.05 and 5.40 (Nos 1 and 2). The analysis of the multiple forms from hepatoma plasma membranes and the serum of the hepatoma bearing chickens demonstrates that forms Nos 2 and 5, and also this with pl 9.80 are identical, while forms with pI 4.15 and 4.03 (No. I) are analogous. A considerable differences were detected in the pf values of the forms No. 3 and No. 6 of the serum from the chickens with tumor and hepatoma plasma membranes. As a typical might be considered for the both kind of serums forms with ~14.05 and 4.03: 5.40 and 5.38; 6.61 and 6.60 and they outlined the similarity between the Fattern of the multiple enzyme forms in the two kind of serums. The differences
appearing in the acid region of the cnzymc protile arc due to the presence of the form with pl 4.W and in the alkaline zone to the disappearance of the form with pl 9.49. Present in the normal serum the identification of a form with ~14.99 exclusively in the serum of chickens with hepatoma allow to assume that this form might be released without any modification from the tumor cell surface, whet-c as it has been already described a form with p1 5.00 was detected. This selec!ive release might suggest that this enzyme form is more loosely bound to cell mcmbrane, so that it can be more easily disassociated at the plasma membrane. If it is true the data imply that at feast some of the strum glycosyltransferases in tumor bearing animals and patients is derived by the cell plasma membranes and the increased levels of these enzymes in the serum support this assumption. The mechanism of the enzytne release and the nature of the releasing process is far to be understood. The results listed in Table 3 show that the influenza virus neliranlinidase hydrolyses almost completeI> the fetuin sialylatud by the forms Nos I. 7. 4. 5. 7 and 8 separated by isoelectrofocusing of normal serum and desialylates at a negligible rate the product obtained after the incubation for sialylation with forms Nos 3 and 6 with pl 6.61 and 8.63. The sialylated fctuin obtained after incubation with forms Nos I. 3. 5. 6 and 8 from serum of tumor hearing
Sialyltran:ifefases chickens are predominantly cleaved by the neuraminidase, while the ability of this enzyme to desialylate the fetuin molecules obtained after incubation with sialyltransferase forms with pI 4.99 (No. 2). 6.60 (NO. 4) and 8.70 (No. 7) is considerably lower. The fact that the fetuin sialylated by the analogous forms detected in both kind of serums is cleaved at nearly the same rate by neuraminidase could be interpreted in a favour of the assumption that for the forms Nos 1.2.4, 5,7 and 8 from normal chicken serum and for forms Nos 1, 3, 5, 6 and 8 for the serum from the animals with tumor whose pl values are very close to each other the sites in sialofetuin, which are predominantly sialylated formed with the penultimate sugar residue 12-3 linkages. At the same time the forms 3 and 4 for the normal serum and 4 and 7 for the serum of the tumor animals sialylate probably only few sites for binding with x2-3 bonds which is well evident as by the low values of the radioactive sialic acid released after neuraminidase treatment, as well as by the relatively high level of the residual radioactivity, One can postulate that the form No 2 with pf 4.99 established in the serum of the tumor bearing animals catalyzes predominantly the sialylation of the oligosaccharide chains by forming x2-6 linkages with the penultimate galactose residues. Tbis assumption is in accordance with the data presented by Schauer (I 983) that influenza virus neuraminidase does not cleave at a considerable rate the sialic acid residues linked to galactose with ~2-6 bonds and it is also confirmed by the low ratio between the radioactivity of the sialic acid released to the residual radioactivity. This study shows that the activity of sialyltransferase is elevated in the serum of chickens with MC-29 viral induced transplantable hepatoma. More detailed studies were conducted in order to disclose the cause of this elevation and some experiments were undertaken in an effort to elucidate if this abnormal level of the enzyme in the circulation is by any manner due to “shedding” of plasma membrane matoriai from neoplastic cells (Bhattacharya. Chatterjee and Barlow, 1976; Henderson and Kessel, 1977; Weiscr, Podolsky and Jsselbacher, 1976). It appears likely from our results on the pattern of the multiple forms of sialyltransferase in the serum from tumor bearing animals and hcpatoma plasma membranes that the identical with the membrane forms and especially that with pJ 4.99 in the serum of tumor animals could be released from the tumor cell surface without any modification in the pf value. The appearance of the serum forms with analogous pJ values with those found in the plasma membranes or the presence only of specific serum forms could be a consequence of a postsynthetic modification of the enzyme molecules passing from insoluble to soluble form as it has been proposed by Fraser and Mookerejea (1976) for the serum galactosyltransferrase. On the basis of the fact described in this study. that the individual forms of serum sialyltranferasc show specificity in relation to the sialytation sites of the acceptor molecule, a conclusion could be drawn that some of the multipic forms arc isoenzyme and rcprcsents specific expressed gene products and are not enzyme molecules modified only in the oligosaccharidc side chain. Besides, the appearance in the
275
serum of the tumor bearing animals of an enzyme form identical by p1 with that in the hepatoma plasma membranes suggests that at least a part of serum multiple forms is of tumor origin and this form shows a capacity to sialylate galactose residues predominantly by x2-6 linkages in the asialofetuin.
KEFERENCES
Athar S., Hwang E. and Reaven P. (1983) Effect of antimicrotubule agents on terminal gfycosyltransferascs and other enzymes associated with rat liver subcellular fractions. Biochznt. J. 212, 721 731. Bauer C.. KKttgen E. and Reutter W. (1977) Elevated activities of r-2 and x-3 fucosyltransfcrases in human serum as a new indicator of malignancy. Bioc~hmr. hio~/!I’.F. Rrs. C’orwnur~. 76, 488-494. Bendiak B. and Zalik S. E. (1981)The specificity of sialyltransferase activity in smooth memdrane fractions -of embrvonic chicken liver. Cirn. J. &o&m. 59, I7 I t80. Bernacki R. J. and Kim U. (1977) Concomitent elevation in serum sialyltransferase activity and sialic acid content in rats with metastasizing mammary tumors. Scic>lxxJ 195, 571.-580. Bhattacharya M., Chatterjee S. K. and Barlow J. J. ( f 976) Uridine 5’-diphosphate-galactose-glycoprotein galactosyltransferase activity in the ovarian cancer patients. Cantor Rw. 36, 2096-2103. Bosmann H. B. and Hilf R. (1974) Elevation of serum glycoprotein-N-acetylneuraminic acid transferase in rats bearing mammary tumors. FE&S Le/f. 44, 313 316. Bosmann H. B.. Spataro A. C., Myers M. W.. Bernacki R., Hillman M. J. and Caputi S. (1975) Serum and host liver activities of glycosidases and sialyltransferases in animal bearing transplantable tumors. Ru. Comnnrrr. Cilc~n~. Putftnf. Pharnracol. i 2, 499 5 1I. Chatterjee S. K. and Kim G. (197X) Fucosyltransfcrase activity in metastasizing and nonmetastasizing mammary carcinoma. J. N~rn. Currc~r I~st. 61, 151 i 57. Evans I. M.. Hilf R., Murphy M. and Bosmann H. 8. (1980) Correlation of serum, tumor and litier serum glycoprotein: it’-acctylneuraminic acid transferax activity with growth of the R 323OAC mammary tumor in rats and relationship of the serum activity to tumor burden. Ci~nc,rr Re.c. 40, 3 IO3 1 I I I Fraser I. H. and Mookerejea S. (1976) Studies on the purification and properties of Il[)P-galactose-trlycoprotein galactosyltransferasc from rat liver and serum. Bio&ern. J. 156, 347 -355. Gavazoia E.. lvanov S. and Chclibonovri-Lorcr H (19X0) Isolation and partial characterization of plasma memhranes from chicken liver and from Mc-29 GINS induced transplantable hepatoma. ~~~,~~/~/~~.s~~~~/ 27, 399 408. Henderson M. and Kessel D. (1977) Alterations in plasma sialyllransfcrase levels in palxnts with neoplastic diseases. CaY?cc,r 39, I 1’9 I i34. ip C. and Dao T. L. 119771 lncrcase in serum and tissue glycosyltransferases and glycosidascs in tumor hearing rats. Cu8?cer &F. 37, 3442 3447. Ip C. and Dao T. L. (197X) Alterations in serum glycosyltransferascs and 5’-nucleotidasc in breask cancer patlcnts. Ctr~c,c~ Rcs. 38, 723 73X.
Ivanov S.. Antonova M.. Gavazova E. and ChelibonovaLorer H. (1984) Multiple lixms of chicken hver and hepatoma MC-29 micrhsomal and plasma membrane siaiyl and fucosyttransferaser. INI. J. Ric~&,rn. IZ, 1307 1311. Judah J. D., Gamble M. and Stcadman J. H. (19731 Biosynthesis of serum albumm in rat liter. Evidence for the existancc of “Pro;tlbunrin”. BincG,,r. J. 134, 10x3 1091.
Kessel D. and Allen J. (1975) Elevated plasma sialyltransferase in cancer patients. Canc,cv Rc.Y. 35, 670 672. Khhilanani P.. Chou T. H., Lomen P. L. and Kessel D. (1977) Variation of levels of plasma guanosinc diphosphate-L-fucose-/~-galactosyl-r-2-L-fucosyltran~ferase in acute adult leukemia. Cut~cv,- Rev. 37,?557 2559. Khllanani P.. Chou T. H. and Kessel D. (1978) Guanosine N-acetylglucoramine fudiphosphate-L-fucose plasma cosyltransferase as an index of bone marrow hqpcrplasia after chemotherapy. C‘crnc,cr Re.v. 38, 181 1x4. Kim Y. S.. Perdomo J.. Whitehead J. S. and Curtls K. J. (1972) Glycosyltransferases in human blood. II. Study of serum galactosyltransfcrase and N-acetylgalactosaminyltransferase in patients with liver diseases. .I (,/it! Irtre.v/. 51, 2033 2039. Kim U.. Baumler A., Carruthers C. and Bielat K. (1975) Immunological escape mechanism in spontaneously metastasizing mammary tumors. Proc,. nu/n .4cud. .%i. C1.S.A. 72, 1012 1016. LaMont J. T.. Gammon M. T. and lsselbacher K. J. (lY77) Cell surface glycosyltransferases in cultured tibroblasts: increase activity and release during serum stimulation of growth. Pro<,. nurn. Acud. Sci. C.;.S..4. 74, 1086 1090. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. hio/. C11cn1. 193, 265 275. Mookerejea S.. Michaels M. A., Hudgin R. L.. Moscarello A. A., Chow A. and Schachter H. (1972) The levels of nucleotide-sugar:glycoprotein sialyl and N-acetylglucosaminyltransferases in normal and pathological human sera Ccm. J. Riochm. 50, 738 740. Mookerejea S. and Young W. M. (1974) A study on the effect of lysolecitin and phospholipase A on membranebound galactosyltransferase. Can. J. Biochem. 52, 1053 1066. Morrin M. J.. Porter C. W.. Petrie C. P., Korytnyck W. and Bernacki R. J. (1983) Effect of a membrane sugar analogs h-deoxy-6-fluoro-[I-galactose on the L-1210 cell cctosialyltransferase system. Biochcn7. Phclrn~rrc. 32, 553 563. Nelason J. D.. Jato-Rodriguez J. T. and MookereJea S. ( 1973) Occurence of soluble glyco~yltransferasea in human amniotic fluid. Bio&r)r. hiop/ll,.\. Rc.v. ~‘orw~w~. 55, 530 537.
Okamoto Y.. Ito E. and Akamatsu N. (197X) UDP-.%acetylglucosamine-glycoprote~n-~V-acetylglucosam~~~yltransferase in regenerating rat hver. Bio&rn~. hro/r/f\,\ .4<,/0 542, 2 I 27. Podolsky D. K. and Welser M. M. (lY75) GaIacto\>l~ transfcrasc activities In human sera: detection of a cancc~ associated isocnlymc Hioc~hcw~. hoph~ s Rc\ ~‘o~w~~~H 65, 545 55 I. Podolskq D. K.. Welacr M. M., West~~ood .I (..\ irl /hc L.irw (Edited by Popper 1I _). pp. Xi 97 MPT Press Limited. Senger D. R., Writh I). I’. and Hyncs K. 0. (1970~ Transformed mammalian cells hecrcte specific protcIn\ and phosphoprotelns. Cc,// 16, X85 X93. Vcsterherg 0. (19X1 ) Isoclectrlc focusing of protcln\ III .\~~~/hot/.\ r~/ 01:~~~vr1/og1~(Edited hq Jacohq W B.) 22, 389 412. Wagner R. R. and C‘ynkin M. A (lY71) Glycoprorcln metabolism: .A UDP-galactose:gIycoprotc~~~ pal,lctosyltransferase of rat scrunl. Nioc /fc,nl. h,r~/~lri~\ KC,! C‘on1tmr,1. 45, 57 62 Wciser M. M. (1973) Intestinal epithehal cell surf‘tcc m~‘n,brane glycoprotein synthesis. II. Glycosyltransfcr~l~c\ and endogenous acceptor5 of the u~~differcntinted cell \urfaci, membrane. J. h/d. C‘lwnr. 248, 2542 254X. Weiscr M M.. Podolskq D. K. and Isselhacher K J. (1’)71>1 Cancel- associated isoenrymc r>f \crum gnl;~c~ tosvltransferase Pr0,. tzrrlfr .lctrr/ .Sri C 5 I 73. 131.9 Ii’? Yamada K. M and Pouysaegur J (197X) (‘cl1 \urfa~c glycoproteins and malignant transli)rmation. N~o&v,r~~~ 60, 1211 1.233.
0020-711X/86
Copyrighl
CHARACTERIZATION FROM
SERUM
OF
OF NORMAL
BEARING H. Institute
CHELIBONOVA-LORER*,
of General and Comparative
$3.00+0.00
1986 Pergamon Press Ltd
SIALYLTRANSFERASES AND
HEPATOMA
MC-29
CHICKENS
S. IVANOV, E. GAVAZOVA
Pathology,
(Rewired
c
Bulgarian 17 April
Academy
and M.
ANTONOVA
of Sciences, Sofia
I II
3. Bulgaria
1985)
Abstract-l. Sialyltransferase was measured in serum of normal and hepatoma MC-29 bearing chickens. 2. By preparative isoelectric focusing the multiple forms of sialyltransferase from both kind of strums was studied as well. 3. By using influenza virus neuraminidase an attempt was made for partial structural characterization of the sialylation sites in asialofetuin applied as exogenous acceptor for sialytransferase determination. 4. It was established an elevated serum sialytransferase activity in tumor bearing chickens. In the pattern of the multiple enzyme forms separated from serum of chickens with tumor an enzyme form was detected with ~1-4.99 identical with an enzyme form described previously in solubilized plasma membrane preparations from hepatoma MC-29. 5. Monitoring of multiple forms of serum glycosyltransferases may be of value in answering the problem concerning the tissue origin of serum enzymes.
INTRODUCTION
(1975), Weiser (1973) who have presented data that glycosyltransferases are located on the cell plasma membrane, and could be released into the blood. In this report we have examined the activity of sialyltransferase in the serum of chickens bearing the virus MC-29 induced hepatoma. The multiple forms of serum sialyltransferase were determined as well in order to check the presence of forms which serve as an indicator of malignancy, taking into account that in a previous work (Ivanov et rrl., 1984) a tumor specific form of this enzyme was detected on the hepatoma MC-29 plasma membranes.
Soluble glycosyltransferases are detected in various animal fluids (Wagner and Cynkin, 1971; Kim et al., 1972; Nelson et al., 1973). The activity of some serum glycosyltransferases was found to be increased in tumor bearing animals and also in humans with neoplastic diseases (Podolsky and Weiser, 1975; Kesse1 and Allen, 1975; Bauer et al., 1977; Khilanani et al., 1978; Podolsky et al.. 1978; Chatterjee and Kim, 1978). As the glycosyltransferases are glycoproteins the abnormal levels of the serum activities were considered as a manifestation of some disorders in the glycoprotein metabolism in tumor cells and especially of plasma membrane glycoproteins. The problem concerning the mechanism by which these enzymes enter into blood circulation and which are the cells providing the serum enzyme activities in both normal and tumor bearing animals, and which is the role of these serum enzymes is still open to question. Yamada and Pouyssegur (1978) and Senger er al. (1979) have demonstrated an increased release of glycoproteins from the surface of the tumor cells. In contrast to Ip and Dao (1978), Kessel and Allen (I 975) and Evans et rd. (1980) who have revealed that the elevated serum activities were not connected with the presence of growing tumor, some other authors (LaMont et ul., 1977; Porter and Bernacki, 1975; Weiser. 1973) have shown that in metastasizing rat mammary tumor cells the cell surface shedding was much more higher than that from the surface of the benign tumor cells (Kim rt trl.. 1975). which was accompanied with an elevation of the sialyltransferase activity in the serum of the rats with the metastasizing tumor (Bernacki and Kim. 1977). These findings are consistent with the results published by LaMont rf c/l. (1977). Porter and Bernacki *Author
to whom
reprint
requests should
MATERIALS AND METHODS
Tumor Hepatoma MC-29 was obtained by injecting subcutaneously in 5.days old white Leghorn chickens a homogenous mince of hepatoma tumor suspended in saline. The hepatoma grow rapidly as a solid mass attaining a size from 3 4 g in about 10 days. Blood was collected without anticoagulant via heart puncture of 12 15 days old control and hepatoma bearing chickens and serum was obtained by centrifugation a1 3000~ for 15 min at 4 C.
[“‘CICMP-N-acetylneuraminic mmol) was purchased from Amersham, U.K.
acid (SA = 277 mCi. Radiochemical Center.
For rhe determination of the total serum activity of sialyltransfcrasc the method described by Ip and Dao (1978) was applied. Endogenous levels of the enzyme activity was established in the absence of added acceptor. When the exogenous activity was studied to the incubation media asialofctuin as acceptor a1 a saturation levels w’as added (Ip and Dao, 197X). The assay tubes w’ere incubated at 37 C for 60 min. Enzyme activity due to the transfer of the ,Vacetylneuraminic acid residue to exogenous acceptor was
be addressed. 271
calculated as the difference between the total activity with protein acceptor present minus the activity with only endogenous acceptors present in the serum. Protein concentrations were determined by the method of Lowry P/ trl. (1951) with bovine serum albumin as a standard.
Preparative isoelectric focusing was performed essentially as described previously (Ivtanov Ed (I/.. 1984) having in view the general considerati~)ns stated by Vesterberg (1981). The focusing was carried out with serum of normal and hepatoma bearing chickens (approx 20 mg protein) on I10 ml column (LKB 8101) using 20/b (v:v) ampholytes pH 3 -10 (LKB produkter. Stockholm. Sweden) and a gradient of O--SO% (w:v) sucrose. Electrofocusing was conducted for 12 hr at a starting voltage of 500 V which was increased to 600 V and the focusing was prolonged for another 28 hr while the column was kept at 4 C. Fractions (2 ml) were collected, their pH values were registered with Radiometer M83 digital pH-meter and aliquots (200 1’1) of each fraction were assayed for sialyltransferase activity in an incubation medium containing lXO/rl 0.5 mM Tris-I-ICI buffer (pH 6.6). 20~1 asialofetuin (lOmg~nl1) and IO//l [‘~CjCMP-iii-acetvlneuraminic acid (100.000 cpm approx). The samples ~ere.incnb~tcd for 60 min at 37 C, the reaction was stopped with 1% phosphotunstig acid (PTA) in 0.5 N HCI and the precipitate was subjected to the washing procedure and counting as described before (Ivanov L’I (il., 1984).
Morrin P$ al. (1983) have demonstrated that in the oligosaccharide chain of fetuin exist two r2 3 and one ~2-6 bonds between the terminal sialic acid and galactose and therefore asialofetuin could be SiaIyIated by the respective multiple forms orsiai~ltra~sr~rases at those three si~l~fl~tjon sites of the molecule. It was of interest to look which terminal sites of asialofetuin become sialylated by the rn~iti~le si~l~ltr~nferases by using in~uen~a virus n~ur~lrn~l~idase, which cleaves at a nearly 100% the r2 3 linkages. while the 32-6 and 12--g bonds between galactose and sialic acid are hydrolysed at a much lower degree (Schauer. 1983). Taking into consideration these properties of the influenza virus neuraminidase the following experimental scheme was adapted: aliquot samples (400 ~1) of the enzyme forms with approriate pl were Incubated for 60 min in the assay conditions for measurement of sialyltranferase activity (Ip and Dao. 1978). The reaction was stopped by addition of 0.4 ml 10% fw:v) aqueous suspension of Norit A. which preliminary experiments have shown, completely adsorbed the remained unreacted ~‘~C]CMP-~-~cetyineur~~minic acid. The tubes were shaken well for 20 min m an ice bath. after that were left to stay at 4 C in a refrigerator for IO min. The samples were centrifuged for 20 min at 5000 rpm. the supernatant containing the sialytated fetuin (sup,l) was collected. the sedimented charcoal was washed 2 times with vvatcr and the washings obtained after centrifupation were comhincd with tbc orginal supernatant-I.
The experimental assay conditions described by Bendiak and Zalik (IY81) for optimal neuraminidasc action were applied To the supernatant containing the siaiylated [i~C~f~tuiil faClz and NaC’t solutions in a linal concentration corresponding to 0.1 and 0.9% respectively and enzyme concentration of 0.75 units (C~~lbi~~chcn~. LOT 003~4~. specilic activity 320 unitsml at pH S.6 and 37 0 were added. The incubation was carried out at 37 f in a buffered media with pH 5.6. The reaction was terminated by the addition of 0.1 ml 1% PTA in 0.5 N HCI tmd the tubes
were centrifuged at 3000 rpm for IO min. The supcrnatant (sup.11) was collected and the protein precipitate was washed twice with PT.4 0.5 N HCI and washings wcrc added to the first supernatant-Il. In an aliquot part of thi\ hydrolysdte the radioactivity due to the [“C’jsialic acrci splitted off by the Ileu~minidase was counted with II liqurtl scintilator (4.0 g PPQ and 0.15 g POPOP in 1 I ~oit~nl)IO which Triton X-100 was added (2: 1 v v). In order to avoid the error due tt> the pre\enec lit [‘Qialic acid liberated as a result of spontaneous hydraiysis of ~‘.~C]CMP-~-~cetyineur~minic acid during the mcubation of the samples for the si~ilyitransfer~~sc dctermination, a control was prepared taking an aiiyuot of the sample designed for neurammidase treatment, the protean was sedimented bv addine 0.1 ml PTA 0.5 N I-ICI. ccnir~fuged and the supernatant (sup.lll) was taken apart. 11le participate was washed two folds with PTA and the w:ishings were combined with the sup.111. The radioactivity \hli\ measured in a PPO:‘POPOP toluen based scintilator ct)irtaining Triton X-100 (2: I v L). The control values vterc subtracted from the sample values obtained as :t eonsequence of the neu~~n~inid~tsc treatment. The vvashctl protein precipitate obtained after the desialylati[)r~ ~)t‘fctuin hy the neuram~nidase was taken to dryness ov>ernight at 37 C’ and the dry residue was dissolved in I .Oml NC3 t~\av solubili~er (Amersh~~rn;Searle Corp.. Arlingt~~l~ Heights III L and placed in 5.0 ml scintill~iti~~n cocktail as dcccrihcd ahole without Triton X-100. The radioactivity of aI1 kind
Results in Table I concerning the sialyltranferase activity in serum of normal and hepdtoma Mc-29 bearing chickens are presented either as endogcnous activity expressing the transfer of N-acctytneurdminic acid residue to the glycoprotein acceptors existing in the serum, or as total activity est~~blished in the presence of the appropriate specific acceptor. The exogenous activity of sialylt~~nferasc is calculated h! the difference between the total and the cndngcnous activity. Our results disclose that in the serum of tht hepatoma bearing chickens the sialyltransferaso LICtivity is elevated compared to that found in the strum of control animals. Our results are in good agrecnient with data of some other authors who have reported elevation in serum activities of glycosyltransf~rasoc III both human (Baucr <‘I nl.. 1977; BhattacharyLt. (‘hatterjee and Barlow. 1976) and animal (Rernaoki and Kim, 1977: Bosmann and Hilf. 1974: Ip and Datr. IWZ) tumor bearing 197X: Podolsky and Wciscr. hosts. The causes ol‘ the incrcascd Icvcl:, o/‘ plvco\>itransferase activities in the strum of the anim;tl4 01 patients with tumors are not W/I tmcicr~tood. The question which is discussed in the Iitcr;tturc concorns the sour-cc of the serum activities. Arc the cn/ymc< relcascd or “shed” from the growing tumor mass. :I\ several authors have suggested (Podolsky 1’1(I/.. 19?7: Bhattacharya, Chattcrjec and Barlou, 1976: Hosmann and Hiif. 1974). or the cnzymc actrvitic\ derived directly from the iivcr or other organ4 :t\ some other investigators have postulated (Hosm~mn clt II!., 1975: Ip and &to. tY77). Bernacki rind Kim (1977) have tbund that in thu serum of fats bearing rn~t~~st~isi~jn~ rn~irnln~lry tumars the sii~lyltransfer~~se activity was ~~~nsid~r~lbiv increased and they suggested that the highor lcvcls d this cnqme activity wcrc probahi\: due to :~n in-
fable
1
Slalyllransferase aclivity in the serum of normal and hepatoma MC-29 bearmg chickens Enzyme act&y
Normal chickens Tumor bearing chickens
(cpm’mp pr;hr)
ElldOgellOUl activity
With asialofetuin
Exogenous activily
x2x * 100 (4) 1417+ 13717Y
2095 2 196 (4) 5844 i 420 171**
1267i 14X(4) 4427 + 278 171**
V&es are the mean t SE of the number assays presented in parentheses. The exogenous enzyme wct~wy is calculated as a difference between the achvity measured in the presence of asialofetun and the endogenou~ acti\lty. 'P < 0.05:**p < 0.01.
creased ceil surface shedding of ectosialyltransferase into the blood stream. One of the most convincing arguments in favour of the statement that the serum glycosyltransferases are derived from tumor cells comes from the studies of Podolsky and Weiser (1975) and Podolsky t’t ul. (1977). They have revealed in the serum of cancer patients a specific isoenzyme of galactosyltransferase (GT II) that was absent in the healthy donors. The cancer associated isoenzyme (Podolsky et al., 1977) has been detected also in the serum of hamsters with tumor provoked after inoculation of BHK cells, transformed with the polyoma virus (BHK-PY). The same isoenzyme form was demonstrated in the cutture media of BHK-PY cells, but not in the media from the nontransformed BHK cells. Taking into consideration these findings we attempt to answer the question about the tissue origin of the serum sialyltransferase activity by detailed study on the multiple forms of the serum enzyme transferase by preparative isoelectrofocusing because in a previous work (Ivanov et al., 1984) it was shown that on the hepdtoma plasma membranes is located a specific form of sialyltransferase with ~1-5.00, which was not detected in the profile of the solubilized plasma membrane preparations from chicken liver. The results obtained in this study arc shown on Figs I and 2 and Table 2. Each point represents the relative enzyme activity expressed as a percentage of the total counts per min in all fractions taken as 100.
It is evident that the profile of the multiple forms of sialyltransferase from the serum of healthy chickens (Fig. I) and that of chickens with hepatoma (Fig. 2) is similar-more active forms in the alkaline region of the gradient especially for the serum of the hepatoma bearing chickens and presence of nearly the same forms in the acid region in both serums under investigation. There are also some other well pronounced differences-the appearance of a fraction with ~14.99 and disappearance of the fraction with ~19.49 from the serum of the animals with tumor. One comparison between the distribution of the liver and hepatoma MC-29 plasma membrane associated multiple forms of sialyltransferasc is presented on Table 2. It is evident that among the pl values of the membrane and serum multiple forms of the enzyme some differences could be delineated which are cxpressed at a different degree. For the better evaluation of these differences we defined three comparative criteria: (a) as identical with the plasma
lZr
11 -
IO -
9-
87-
2 -
6-
x t .> ':
7-
?_ E6 al ;
5
z G I?? 4-
3-
2-
1-
I
,
/
!
I
I
1
I
3
4
5
6
7
8
3
IO
I 3
I. Sialyltransfcrasc multiple pattern normal chickens.
/ 5
!
6
I
/
/
7
8
9
I 10
PH
PH Fig.
, 4
of
swum
from
Fig.
2. Sialyltransferase multiple pattern of serum hepatoma MC-29 bearing chickens.
from
H.
~‘H~~I.I~oN(~~A-Lo~~:K PI d.
membrane were accepted those serum forms whose pl were either equal with them or the differences in the pI values are not higher than 0.05; (h) analogous with the plasma membrane are serum forms with a difference in the p1 values which reach not more than 0.20; (c) as a typical serum forms were considered those of them whose pf values markedly deviated when compared to the plasma membrane forms and the differences are in the range between 0.40 and 0.60. On the basis of these criteria of similarity one can assume from the data of Table 2 that between the liver plasma membrane enzyme forms and the forms established in the normal serum there are 3 identical forms (Nos 5, 7 and S), 3 analogous (Nos 3.4 and 6) and 2 typical serum forms with pI 4.05 and 5.40 (Nos 1 and 2). The analysis of the multiple forms from hepatoma plasma membranes and the serum of the hepatoma bearing chickens demonstrates that forms Nos 2 and 5, and also this with pl 9.80 are identical, while forms with pI 4.15 and 4.03 (No. I) are analogous. A considerable differences were detected in the pf values of the forms No. 3 and No. 6 of the serum from the chickens with tumor and hepatoma plasma membranes. As a typical might be considered for the both kind of serums forms with ~14.05 and 4.03: 5.40 and 5.38; 6.61 and 6.60 and they outlined the similarity between the Fattern of the multiple enzyme forms in the two kind of serums. The differences
appearing in the acid region of the cnzymc protile arc due to the presence of the form with pl 4.W and in the alkaline zone to the disappearance of the form with pl 9.49. Present in the normal serum the identification of a form with ~14.99 exclusively in the serum of chickens with hepatoma allow to assume that this form might be released without any modification from the tumor cell surface, whet-c as it has been already described a form with p1 5.00 was detected. This selec!ive release might suggest that this enzyme form is more loosely bound to cell mcmbrane, so that it can be more easily disassociated at the plasma membrane. If it is true the data imply that at feast some of the strum glycosyltransferases in tumor bearing animals and patients is derived by the cell plasma membranes and the increased levels of these enzymes in the serum support this assumption. The mechanism of the enzytne release and the nature of the releasing process is far to be understood. The results listed in Table 3 show that the influenza virus neliranlinidase hydrolyses almost completeI> the fetuin sialylatud by the forms Nos I. 7. 4. 5. 7 and 8 separated by isoelectrofocusing of normal serum and desialylates at a negligible rate the product obtained after the incubation for sialylation with forms Nos 3 and 6 with pl 6.61 and 8.63. The sialylated fctuin obtained after incubation with forms Nos I. 3. 5. 6 and 8 from serum of tumor hearing
Sialyltran:ifefases chickens are predominantly cleaved by the neuraminidase, while the ability of this enzyme to desialylate the fetuin molecules obtained after incubation with sialyltransferase forms with pI 4.99 (No. 2). 6.60 (NO. 4) and 8.70 (No. 7) is considerably lower. The fact that the fetuin sialylated by the analogous forms detected in both kind of serums is cleaved at nearly the same rate by neuraminidase could be interpreted in a favour of the assumption that for the forms Nos 1.2.4, 5,7 and 8 from normal chicken serum and for forms Nos 1, 3, 5, 6 and 8 for the serum from the animals with tumor whose pl values are very close to each other the sites in sialofetuin, which are predominantly sialylated formed with the penultimate sugar residue 12-3 linkages. At the same time the forms 3 and 4 for the normal serum and 4 and 7 for the serum of the tumor animals sialylate probably only few sites for binding with x2-3 bonds which is well evident as by the low values of the radioactive sialic acid released after neuraminidase treatment, as well as by the relatively high level of the residual radioactivity, One can postulate that the form No 2 with pf 4.99 established in the serum of the tumor bearing animals catalyzes predominantly the sialylation of the oligosaccharide chains by forming x2-6 linkages with the penultimate galactose residues. Tbis assumption is in accordance with the data presented by Schauer (I 983) that influenza virus neuraminidase does not cleave at a considerable rate the sialic acid residues linked to galactose with ~2-6 bonds and it is also confirmed by the low ratio between the radioactivity of the sialic acid released to the residual radioactivity. This study shows that the activity of sialyltransferase is elevated in the serum of chickens with MC-29 viral induced transplantable hepatoma. More detailed studies were conducted in order to disclose the cause of this elevation and some experiments were undertaken in an effort to elucidate if this abnormal level of the enzyme in the circulation is by any manner due to “shedding” of plasma membrane matoriai from neoplastic cells (Bhattacharya. Chatterjee and Barlow, 1976; Henderson and Kessel, 1977; Weiscr, Podolsky and Jsselbacher, 1976). It appears likely from our results on the pattern of the multiple forms of sialyltransferase in the serum from tumor bearing animals and hcpatoma plasma membranes that the identical with the membrane forms and especially that with pJ 4.99 in the serum of tumor animals could be released from the tumor cell surface without any modification in the pf value. The appearance of the serum forms with analogous pJ values with those found in the plasma membranes or the presence only of specific serum forms could be a consequence of a postsynthetic modification of the enzyme molecules passing from insoluble to soluble form as it has been proposed by Fraser and Mookerejea (1976) for the serum galactosyltransferrase. On the basis of the fact described in this study. that the individual forms of serum sialyltranferasc show specificity in relation to the sialytation sites of the acceptor molecule, a conclusion could be drawn that some of the multipic forms arc isoenzyme and rcprcsents specific expressed gene products and are not enzyme molecules modified only in the oligosaccharidc side chain. Besides, the appearance in the
275
serum of the tumor bearing animals of an enzyme form identical by p1 with that in the hepatoma plasma membranes suggests that at least a part of serum multiple forms is of tumor origin and this form shows a capacity to sialylate galactose residues predominantly by x2-6 linkages in the asialofetuin.
KEFERENCES
Athar S., Hwang E. and Reaven P. (1983) Effect of antimicrotubule agents on terminal gfycosyltransferascs and other enzymes associated with rat liver subcellular fractions. Biochznt. J. 212, 721 731. Bauer C.. KKttgen E. and Reutter W. (1977) Elevated activities of r-2 and x-3 fucosyltransfcrases in human serum as a new indicator of malignancy. Bioc~hmr. hio~/!I’.F. Rrs. C’orwnur~. 76, 488-494. Bendiak B. and Zalik S. E. (1981)The specificity of sialyltransferase activity in smooth memdrane fractions -of embrvonic chicken liver. Cirn. J. &o&m. 59, I7 I t80. Bernacki R. J. and Kim U. (1977) Concomitent elevation in serum sialyltransferase activity and sialic acid content in rats with metastasizing mammary tumors. Scic>lxxJ 195, 571.-580. Bhattacharya M., Chatterjee S. K. and Barlow J. J. ( f 976) Uridine 5’-diphosphate-galactose-glycoprotein galactosyltransferase activity in the ovarian cancer patients. Cantor Rw. 36, 2096-2103. Bosmann H. B. and Hilf R. (1974) Elevation of serum glycoprotein-N-acetylneuraminic acid transferase in rats bearing mammary tumors. FE&S Le/f. 44, 313 316. Bosmann H. B.. Spataro A. C., Myers M. W.. Bernacki R., Hillman M. J. and Caputi S. (1975) Serum and host liver activities of glycosidases and sialyltransferases in animal bearing transplantable tumors. Ru. Comnnrrr. Cilc~n~. Putftnf. Pharnracol. i 2, 499 5 1I. Chatterjee S. K. and Kim G. (197X) Fucosyltransfcrase activity in metastasizing and nonmetastasizing mammary carcinoma. J. N~rn. Currc~r I~st. 61, 151 i 57. Evans I. M.. Hilf R., Murphy M. and Bosmann H. 8. (1980) Correlation of serum, tumor and litier serum glycoprotein: it’-acctylneuraminic acid transferax activity with growth of the R 323OAC mammary tumor in rats and relationship of the serum activity to tumor burden. Ci~nc,rr Re.c. 40, 3 IO3 1 I I I Fraser I. H. and Mookerejea S. (1976) Studies on the purification and properties of Il[)P-galactose-trlycoprotein galactosyltransferasc from rat liver and serum. Bio&ern. J. 156, 347 -355. Gavazoia E.. lvanov S. and Chclibonovri-Lorcr H (19X0) Isolation and partial characterization of plasma memhranes from chicken liver and from Mc-29 GINS induced transplantable hepatoma. ~~~,~~/~/~~.s~~~~/ 27, 399 408. Henderson M. and Kessel D. (1977) Alterations in plasma sialyllransfcrase levels in palxnts with neoplastic diseases. CaY?cc,r 39, I 1’9 I i34. ip C. and Dao T. L. 119771 lncrcase in serum and tissue glycosyltransferases and glycosidascs in tumor hearing rats. Cu8?cer &F. 37, 3442 3447. Ip C. and Dao T. L. (197X) Alterations in serum glycosyltransferascs and 5’-nucleotidasc in breask cancer patlcnts. Ctr~c,c~ Rcs. 38, 723 73X.
Ivanov S.. Antonova M.. Gavazova E. and ChelibonovaLorer H. (1984) Multiple lixms of chicken hver and hepatoma MC-29 micrhsomal and plasma membrane siaiyl and fucosyttransferaser. INI. J. Ric~&,rn. IZ, 1307 1311. Judah J. D., Gamble M. and Stcadman J. H. (19731 Biosynthesis of serum albumm in rat liter. Evidence for the existancc of “Pro;tlbunrin”. BincG,,r. J. 134, 10x3 1091.
Kessel D. and Allen J. (1975) Elevated plasma sialyltransferase in cancer patients. Canc,cv Rc.Y. 35, 670 672. Khhilanani P.. Chou T. H., Lomen P. L. and Kessel D. (1977) Variation of levels of plasma guanosinc diphosphate-L-fucose-/~-galactosyl-r-2-L-fucosyltran~ferase in acute adult leukemia. Cut~cv,- Rev. 37,?557 2559. Khllanani P.. Chou T. H. and Kessel D. (1978) Guanosine N-acetylglucoramine fudiphosphate-L-fucose plasma cosyltransferase as an index of bone marrow hqpcrplasia after chemotherapy. C‘crnc,cr Re.v. 38, 181 1x4. Kim Y. S.. Perdomo J.. Whitehead J. S. and Curtls K. J. (1972) Glycosyltransferases in human blood. II. Study of serum galactosyltransfcrase and N-acetylgalactosaminyltransferase in patients with liver diseases. .I (,/it! Irtre.v/. 51, 2033 2039. Kim U.. Baumler A., Carruthers C. and Bielat K. (1975) Immunological escape mechanism in spontaneously metastasizing mammary tumors. Proc,. nu/n .4cud. .%i. C1.S.A. 72, 1012 1016. LaMont J. T.. Gammon M. T. and lsselbacher K. J. (lY77) Cell surface glycosyltransferases in cultured tibroblasts: increase activity and release during serum stimulation of growth. Pro<,. nurn. Acud. Sci. C.;.S..4. 74, 1086 1090. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. hio/. C11cn1. 193, 265 275. Mookerejea S.. Michaels M. A., Hudgin R. L.. Moscarello A. A., Chow A. and Schachter H. (1972) The levels of nucleotide-sugar:glycoprotein sialyl and N-acetylglucosaminyltransferases in normal and pathological human sera Ccm. J. Riochm. 50, 738 740. Mookerejea S. and Young W. M. (1974) A study on the effect of lysolecitin and phospholipase A on membranebound galactosyltransferase. Can. J. Biochem. 52, 1053 1066. Morrin M. J.. Porter C. W.. Petrie C. P., Korytnyck W. and Bernacki R. J. (1983) Effect of a membrane sugar analogs h-deoxy-6-fluoro-[I-galactose on the L-1210 cell cctosialyltransferase system. Biochcn7. Phclrn~rrc. 32, 553 563. Nelason J. D.. Jato-Rodriguez J. T. and MookereJea S. ( 1973) Occurence of soluble glyco~yltransferasea in human amniotic fluid. Bio&r)r. hiop/ll,.\. Rc.v. ~‘orw~w~. 55, 530 537.
Okamoto Y.. Ito E. and Akamatsu N. (197X) UDP-.%acetylglucosamine-glycoprote~n-~V-acetylglucosam~~~yltransferase in regenerating rat hver. Bio&rn~. hro/r/f\,\ .4<,/0 542, 2 I 27. Podolsky D. K. and Welser M. M. (lY75) GaIacto\>l~ transfcrasc activities In human sera: detection of a cancc~ associated isocnlymc Hioc~hcw~. hoph~ s Rc\ ~‘o~w~~~H 65, 545 55 I. Podolskq D. K.. Welacr M. M., West~~ood .I (.