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Florisil (activated magnesium silicate) (Saito & Hakomori, 1971) or silicic acid (Vance & Sweeley, 1967). The glycolipids recovered from the membranes of unfractionated erythrocytes accounted for 33 %of the galactosamine, 6 % of the glucosamine, but less than 2 of the sialic acid of erythrocyte membranes. When erythrocyte populations from single individuals were fractionated on the basis of density differences it was observed that there was marked heterogeneity in membrane carbohydrate content. The oldest (most dense) cell fractions contained substantially smaller amounts of sialic acid, galactosamine, glucosamine, galactose and glucose when compared with younger cells (Table 1). The decrease in sialic acid content on cell aging must be mainly attributable to changes in cell-surface glycoprotein, because there is insufficient sialic acid in the glycolipid to account for a change of this magnitude. However, the suggestion that there may also be loss of glycolipid carbohydrate is supported by the observation that less hexosamine was obtained in the glycolipid fractions from the membranes of aged cells than from young cells. The population of circulating erythrocytes appears to be heterogeneous in both glycoprotein and glycolipid components, loss of carbohydrates contained in both types of molecule being associated with aging in uiuo. This project was supported by a grant from the Medical Research Council. Aminoff, D. (1961) Biochem. J. 81,384-392 Balduini, C., Balduini, C. L. & Ascari, E. (1974) Biochem. J. 140,557-560 Baxter, A. & Beeley, J. G. (1975) Biochem. SOC.Trans. 3, 134-1 36 Beeley, J. G. (1976) Biochem. J . 159,335-345 Chambers, R. E. &Clamp, J R. (1971) Biochem. J . 125,1009-1018 Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10,260&2617 Ghamberg, C. G. & Hakomori, S. (1973)J. Biol. Chem. 248,43114317 Greenwalt, T. J. & Steane, E. A. (1973) Br. 3. Haematol. 25,207-215 Saito, T. & Hakomori, S. (1971) J. Lipid Res. 12,257-259 Vance, D. & Sweeley, C. C. (1967) J. Lipid Res. 8,621-630

The Uptake of [l-3H]Glucosamine and Sodium I3’S]Sulphate into Glycoproteins Secreted by Mucus Glands of the Common Sea Mussel Mytilus edulis MICHAEL COOK,* SUSAN CROSBY,* M. SHEILA JEFFERY* and PAUL W. KENT? Markham Laboratories, Dental School, University of Newcastle upon Tyne, Newcastle upon Tyne NEl 7RU, U.K. and Glycoprotein Research Unit, University of Durham, Durham DH1 3LH, U.K.

The mussels were anaesthetized with 10 % (v/v) Nembutal solution, producing some relaxation of their adductor muscle, so allowing access for injection of the radioactive isotope into the soft tissues of the base of the ‘foot’. Each mussel, injected with Na235S04(0.I ml, 0.15 mCi), was kept in a tank containing artificial sea water at 15°C.Plates of poly(tetrafluoroethy1ene) were placed on the floor of the tank to collect trails deposited by the ‘foot’ of the animal (Cook, 1970; Lane & Nott, 1975). Mucus was collected from excised ‘feet’ by washing with EDTA (tetrasodium salt; 1 %, w/v, at pH 7) for intervals of time between 60min and 5 days (at 8°C). Mussels were again anaesthetized and injected with 0.1 ml of a solution of D - [ ~ - ~ H ] glucosamine hydrochloride (0.05 mCi) in sterile saline. They were again allowed to recover for various times before the ‘foot’ was removed to collect the mucus. Collected solutions of mucus were separately dialysed for 16 h at 2°C against excess distilled water, centrifuged to remove small amounts of suspended matter and then evaporated to 2 ml volume. 1977

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Fraction no Fig. 1 . Fractionation of glycoproteins of Mytilus ediilis on Sepharose 4 B The column dimensions were lOOcmx 1 cm and the eluent was 50mhl-sodium phosphate buffer, pH 7. Fractions (volume 5ml) were collected. Vi, included volume; V , ,excluded volume.

_These were fractionated on a column (85cm x 1.4cm) of Sephadex G-50 and eluted with 50rnhl-sodium phosphate buffer, pH7, containing NaN, (0.02%). The elution profile of non-radioactive samples was followed by measuring the protein content as described by Lowry et al. (1951), and the radioactive materials were detected by liquidscintillation counting. Two principal fractions were obtained, one just before the void volume (peak A) and the other at the included volume (peak B). In 35S-labellingexperiments when mucus was collected 1 day after injection, peak B alone was radioactive; when collected 5 days after injection, the label in peak B was small, the radioactivity being predominantly located in peak A. Electrophoresis on cellulose acetate (pH 8.4) of peak-A material gave a broad Nigrosine-positive band in which the radioactivity was coincident. However, in 3H-labelling experiments only peak A was radioactive (8300000d.p.m./mg of protein) up to 5 days after injection, peak B being unlabelled throughout. Peak-A fractions were pooled, dialysed for 16h against distilled water, evaporated to 2ml and fractionated on a Sepharose 4B column by elution with 50m~-sodiumphosphate buffer, pH7. A typical profile of the non-radioactive material eluted from Sepharose 4B is shown in Fig. 1. However, when the 35S-labelledmaterial in peak A was fractionated thus, a reproducible profile was obtained, but all the 35Swas concentrated in the first component, although the third peak also contained ester sulphate (unlabelled). Analysis showed all these components to be glycoproteins in which about SO% peptide was present. The monosaccharide constituents included N-acetylglucosamine, N-acetylgalactosamine and neutral sugar. Ester sulphate was present in peaks 1 and 111. The original secreted mucus left in the collecting vessel was dissolved in I M-HCI(it did not dissolve in 0. I M-HCI) and immediately neutralized with NaHCO,. Comparison with material from peaks I and I1 by cellulose acetate (low-voltage) electrophoresis showed very similar patterns. Both sets stained with Nigrosine and periodate/Schiff reagent, showing protein and carbohydrate to be present in all components. The mussels have also been allowed to attach to Teflon plates placed in their tanks for several days. The Teflon was then removed and dried. The attachment discs were carefully removed and the material adhering to the surface of this Teflon was scraped off, VOl. 5

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dissolved in 1M-HCI and then neutralized with NaHC03. After dialysis the material was concentrated and then examined electrophoretically. A range of components similar to those eluted earlier from Sepharose column was observed. A preliminary study has been made of the action of proteinases on these glycoproteins. Cook, M. (1970) in Adhesion in BiologicalSystems (Manley, R. S.,ed.), pp. 139-150, Academic Press, New York and London Lane, D. J. W. & Nott, J. A. (1975) J. Mar. Biol. Assoc. U.K. 55,477495 Lowry, 0. H., Rosebrough,N.J., Farr, A. L. &Randall, R. J. (1951)J.Biol.Chem.193,265-275

The Effect of Autologous Serum on Phytohaemagglutinin-Induced Lymphocyte Transformation ALAN H. JOHNSON and PATRICK B. COLLINS Division of Biochemistry, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2, Ireland T lymphocytes have an essential role in cell-mediated immunity, and their transformation by phytohaemagglutinin is accepted as a means of measuring, at least in part, their effectiveness to participate in cell-mediated immune responses. Normal human serum contains immunosuppressive factors, one of which is a peptide that is associated with the a-globulin fraction, namely the immunoregulatory a-globulin (Occhino et al., 1973). This peptide, which inhibits phytohaemagglutinin-induced lymphocyte transformation, is found in increased amounts in tumour-bearing patients (Nimberg et al., 1975). The blastogenic response of lymphocytes to phytohaemagglutinin involves culturing the cells in a balanced nutritional medium and serum. The function of the serum in the culture medium is not well understood although it does contain nutritional factors (Burger, 1977). In the present study we have examined further the effects of serum on mitogen-induced lymphocyte blastogenesis. Peripheral venous blood (10ml) was collected in heparinized tubes from normal healthy donors (20-25 years). The blood was left to settle for 1 h a t 37°C and lymphocytes from the upper layer were purified on a Ficoll/Hypaque gradient (specific gravity 1.007) by isopycnic centrifugation. The cells were washed twice with Hanks balanced-salt solution and adjusted to a concentration of 5 x 105/ml in RPMI 1640 culture medium (Gibco Bio-Cult, Glasgow, Scotland, U.K.) containing 25 mM-Hepes [4-(2-hydroxyethyl)- 1-piperazine-ethanesulphonicacid] buffer, pH7.6, 4m~-~-glutamine, penicillin/ streptomycin (100units/ml) and 10% (v/v) serum. The lymphocytes were cultured in 200pl samples (lo5 cells) in microtitre plates with 0-500ng of purified phytohaemagglutinin (Wellcome Laboratories, Beckenham, Kent, U.K.) at 37°C for 48 h. [3H]Thymidine (0.5pCi; 1 Ci/2mmol) was added to each well 4 h before harvesting. The cells were harvested on glass-fibre filters (Whatman GF/C) and were washed twice with icecold trichloroacetic acid ( 5 %, w/v) and twice with methanol. The filters were dried and their radioactivity was counted in scintillant [0.5 % (w/v) 2,5-diphenyloxazole in toluene] with a Packard Tri-Carb 3380 liquid-scintillation spectrometer. The effects of preincubation of lymphocytes and phytohaemagglutinin, lymphocytes and serum, and phytohaemagglutinin and serum at 37°C for 30min on lymphocyte transformation are shown in Table 1. When serum and phytohaemagglutinin were preincubated, [3H]thymidine incorporation into lymphocytes from our donors was quite varied. Because of the many factors that influence lymphocyte activity such variation is not surprising. [3H]Thymidine incorporation was appreciably higher with autologous serum than with foetal calf serum, although with the foetal calf serum a more uniform mitogenic response was obtained. Incubating the lymphocytes with phytohaemagglutinin before the addition of autologous serum resulted in increased mitogenic stimulation with five of the lymphocyte samples. In contrast, the incubation of lymphocytes and autologous serum before the addition of mitogen dramatically inhibited the 1977