Boyle, F

Duane, P

Peters, T

Snape, S

Kent Academic Repository

Kent Academic RepositoryFull text document (pdf)Copyright & reuseContent in the Kent Academic Repository is made available for research purposes. Unless otherwise stated allcontent is protected by copyright and in the absence of an open licence (eg Creative Commons), permissions for further reuse of content should be sought from the publisher, author or other copyright holder. Versions of researchThe version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record.EnquiriesFor any further enquiries regarding the licence status of this document, please contact: researchsupport@kent.ac.ukIf you believe this document infringes copyright then please contact the KAR admin team with the take-down information provided at http://kar.kent.ac.uk/contact.htmlCitation for published versionBoyle, F and Snape, S and Duane, P and Peters, T  (1987) Further evidence that galactosyltransferaseis not located to the brush border membrane of human duodenum.    In: Biochemica Society Transactions,1987, Canterbury, Kent.DOILink to record in KARhttp://kar.kent.ac.uk/49260/Document VersionPublisher pdf1082 BIOCHEMICAL SOCIETY TRANSACTIONS cell-retained fraction includes an unusually small heparan sulphate proteoglycan, less than M ,  30000. This is less than Levitt, D. & Ho, P. L. (1983) J .  Cell BioL 97, 351-358 Hart, G .  w. (1982) 24, 6088-6096 half the size of the cell-surface heparan sulphate proteogly- *Idberg, Kjellen, L. & Hook, M. (1979) J .  Chem. 254, 8505-8670 can from liver (Oldberg et 1979), previously the Rider, C,  C, & Hart, G ,  W, (1987) Mo/,  Immune[. in the press known molecule of this type. Wasteson, A. (1971) J .  Chromaiogr. 59, 87-97 Received 5 June 1987 I gratefully acknowledge the financial support of the Central Research Fund of London University and the Smith Kline Foundation. Further evidence that galactosyltransferase activity is not localized to the brush border membrane of human duodenum FRANCES BOYLE, SUSAN SNAPE, PAUL DUANE and TIMOTHY PETERS Division of Clinical Cell Biology, M.R.C. Clinical Research Centre, Harrow, H A 1  3UJ, U . K .  Galactosyltransferase (EC. 2.4.1.22) catalyses the transfer of galactose from UDP-galactose to glycoproteins with a terminal N-acetylglucosamine residue. It is considered as a suitable marker enzyme for Golgi [l]. It has been claimed, using immunocytochemical techniques with an antibody raised against galactosyltransferase partially purified from milk, that galactosyltransferase is located at  the liminal surface of absorptive enterocytes in human duodenal biopsies [2 ] .  Using analytical subcellular fractionation of human jejunum biopsy specimens by a single-step sucrose density gradient centrifugation, we demonstrated that galactosyl- transferase activity Is located in the Golgi fraction and not in the brush border membrane fraction [3]. In this paper we present the results of three separate fractionation experi- ments, together with fraction mixing and time-course experiments further substantiating our previous report. Human jejunal biopsies were homogenized and frac- tionated in a single-step sucrose density gradient centri- Density (g . cm > )  fl 1 2 3 4 5 Assay time ( h )  Fig. 1. Fractionation of human jejunum biopsy (a) and time courses of galactosyltransferasr activity (b) (a)  Relative concentration - density distributions [7] of galactosyltransferase (0 )  and a-glucosidase (0) .  Averaged data of duplicate assays for three experiments. Mean recovered enzyme activities are 77 and 71 % for galactosyltransferase and a-glucosidase, respectively. (b) Galactosyltransferase activity assayed in the peak fraction (see a) den- sities l .14g .cm-3(0)and  1.23g.~rn-~(.). 1987 623rd MEETING, CANTERBURY I083 fugation procedure as before [3]. Galactosyltransferase activity, using ovalbumin as an acceptor in a 4 h assay [4], and brush border a-glucosidase activity [5] were assayed in each gradient fraction. Fig. I(a) shows the averaged distribution of galactosyl- transferase and a-glucosidase activities from three separate experiments. Galactosyltransferase shows a peak relative concentration at a density of 1.14g - cm-’, typical of Golgi distribution [6]. a-Glucosidase activity was well separ- ated from galactosyltransferase, with a peak activity at 1.22 g cm373 typical of brush border membrane [5]. Fig. I(b) shows a time course of galactosyltransferase activities in each of these two peak fractions, which were linear over 5 h. Galactosyltransferase activity in the peak a-glucosidase fraction was < 10% of that in the peak galactosyltrans- ferase fraction. Mixing these two peak fractions together caused no inhibition of galactosyltransferase activity com- pared to an equivalent dilution of the peak galactosyl- transferase fraction with buffer alone. Thus, the absence of galactosyltransferase activity in the brush border membrane fraction cannot be attributed to the presence of an inhibitor, or inactivation of the enzyme substrates. These studies provide further evidence confirming that enzymically active galactosyltransferase is localized to the Golgi and is not found significantly in the brush border membrane. We are grateful to Amersham International for financial support (F.B.). 1. Ram, B. P. & Munjal, D. D. (1985) CRC Crii. Rev. Biochem. 17, 2. Roth, J. ,  Lentze, M. J .  & Berger, E. G. (1985) J .  Cell. B i d .  100, 3. Boyle, F., Snape, S., Duane, P., Cook, N. & Peters, T. (1986) Bio- 4. Leake, D., Lieberman, G. E. & T. J. (1983) Biochim. Biophys. Acia 5. Peters, T. J. (1976) Clin. Sci. Mol. Med. 51, 557-574 6 .  Smith, G. & Peters, T. J .  (1980). Eur. J .  Biochem. 104, 305-31 1 7. Smith, G .  D., Osterloh, K. R. S. & Peters, T. J. (1987) A n d .  Bio- 257-3 1 I 118-125 .rci. Rep. 6, 171-175 162, 52-57 chem. 160, 17-23 Received 18 June 1987 Binding and endocytosis of exogenous glycosaminoglycans in cultures of rat rhabdomyosarcoma cells FRANCOISE REDINI, MARIE-FRANCE POUPON* and ELEMER MOCZAR Laboratoire de Biochimie du Tissu Conjonctif, U A  C N R S  No. 1174, Facultk de Mkdecine, Universitt! Paris XII ,  F-94010 CrPteiI Cedex, and *IRCS du CNRS, BP no. 8, VilIejuif Cedex, France Recent studies suggest that interactions of proteoglycans or glycosaminoglycans secreted by malignant cells or by the adjacent host cells may be involved in the control of tumour growth and metastasis formation (Iozzo, 1985; Redini et al., 1986). Exogenous heparin, and to a lesser extent heparan sulphate, inhibits specifically the growth of strongly metastatic rat rhabdomyosarcoma cells (F. Redini, E. Moczar & M.-F. Poupon, unpublished results). In this paper we examine the binding and mode of internalization of glycosaminoglycans by strongly (RMSO) and weakly (RMS8) metastatic rat rhabdomyosarcoma cells in culture. The cell lines were obtained by cloning from a nickel- induced rat rhabdomyosarcoma (Sweeney el a/., 1982). Heparin (Choay, Paris); heparan sulphate (from ovine pla- centa, Bioetica, Lyon and from bovine kidney, Miles); dermatan sulphate (pig skin, Sigma); chondroitin 4- and 6-sulphates (Sigma) were N-acetylated with [’ Hlacetic anhydride to yield [’ H]glycosaminoglycans (Hook et al., 1982). The time course of the binding and internalization of the glycosaminoglycans were studied in confluent cultures of the rhabdomyosarcoma cells (3 x 10‘ cells/dish of 6cm diameter). The cells were incubated for 1, 2, 6 and 24 h with the labelled glycosaminoglycans (4 pg/ml) in Dulbecco’s minimum essential medium with 7.5% (v/v) fetal calf serum at 37°C. The radioactivities were determined in the extra- cellular (culture medium), pericellular (trypsinate) and intracellular (cell residue) compartments. Heparin, heparan sulphate and dermatan sulphate were taken up and internalized in a time-dependent way by both cell types. The internalization of the glycosaminoglycans reached an equilibrium after 6 h for the RMSO and after 24 h for the RMS8 cells. At 24 h, the amount of the cell- associated heparin, heparan and dermatan sulphates was respectively: 2-, 1.6- and 2,6-fold higher in the pericellular and 1.5-, 1.6- and 5-fold higher in the intracellular compart- Vol. 15 .......... -** ..... ............. \\ , 24 (h) 6 1 2  (6) Incubation time ...... ... ....... ...... 0’ 1 I I 1 3 6  10 15 Exogenous glycosaminoglycan concn. (pgiml) Fig. 1. Internalization of exogenous [’H Jglycosaminoglycans by rat rhabdomyosarcoma cells in culture (a) Time course of  the internalization (4pg/ml) and (b)  dose dependency o f  the internalization of the glycosaminoglycans in 6 h: V-V, heparin; U, heparan sulphate (ovine placenta); A+, dermatan sulphate; M, chondroitin 4- and 6-sulphates; for RMSO cells (open symbols) and RMS8 cells (closed symbols). Results are means & S.D. > 15% (n = 3). 

Further evidence that galactosyltransferase is not located to the brush border membrane of human duodenum

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Further evidence that galactosyltransferase is not located to the brush border membrane of human duodenum

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