The number of cell surface and total asialoglycoprotein receptors was investigated in normal and diabetic rat hepatocytes using 2 methods: ligand and polyclonal antibody binding. An identical number of immunoreactive receptors was found in both types of cells, while the ligand binding activity of cell surface receptors was reduced by 58% in diabetic rats compared with normal ones, or by 33% for total cell receptors.

Rats with Jejunoileal bypass were used to study the biological activity of the hepatic binding protein. Hepatocytes were prepared 11 weeks after surgical procedure, and presence of asialoo-rosomucoid in serum has been determined. Compared to control rat hepatocytes, endocytosis of [3H]asialoorosomucoid by bypassed rat hepatocytes was decreased by about 60%. This was due to a decreased number of total and surface receptors. In most sera, an accumulation of asialoorosomucoid was found. A reduction of protein synthesis or turnover could be considered.

The kinetic constants of internalization of asialoorosomucoid were determined for normal and jejuno-ileal by-passed rat hepatocytes. In by-passed rats the maximum velocity of asialoorosomucoid internalization is decreased 3-fold, without any modification of apparant constant of internalization. Moreover, the rate constant of internalization was the same in the two groups of rats. These data suggest that the process of asialoorosomucoid internalization is not altered in by-passed hepatocytes and that the decrease of maximal velocity is only due to a decrease of total uptake receptor number.

Using high concentrations of extracellular [3H]asialoorosomucoid we obtained the steady-state level of [3H]asialoorosomucoid endocytosis by isolated hepatocytes from normal and streptozotocin diabetic rats. At the steady-state of the overall reaction, the intracellular amount of [3H]asialoorosomucoid did not change with time, the apparent overall rate of [3H]asialoorosomucoid degradation was close to that of [3H]asialoorosomucoid internalization, in both normal and diabetic rat hepatocytes. Although in diabetic cells the intracellular amount of [3H]asialoorosomucoid was about three-times lower than in normal cells, the same fraction of intracellular asialoorosomucoid was degraded per time interval by both normal and diabetic cells. The apparent first-order rate constant of steady-state degradation was about 0.011 min-1 for both normal and diabetic cells. In diabetic rat hepatocytes, the decrease of the clearance of serum asialoglycoproteins was directly correlated to the variation of cell surface receptor number, without any modification of internalization and degradation steps.

The membrane lipid composition of isolated hepatocytes, Kupffer cells and endothelial cells was determined. The hepatocytes are characterized by a lower quantity of gangliosides, cholesterol, sphingomyelin and a reduced cholesterol/phospholipid molar ratio when compared to the two other liver cell types. The main gangliosides of Kupffer and endothelial cells are the GM3 species, and those of hepatocytes are of the polysialogangliosides species.

The total capacity of hepatocytes to bind asialoorosomucoid was measured on normal and streptozotocin diabetic rats. 4 days after the streptozotocin injection, a slight decrease of total receptor concentration was observed while a more marked reduction of cell surface receptor occurred. In animals sacrificed 11 days after the streptozotocin injection, the total capacity of hepatocytes to bind asialoorosomucoid was about 70% of the normal level.

The initial velocity of asialoorosomucoid internalization is determined for normal and diabetic rat hepatocytes. The analysis of results according to Woolf-Hofstee's method, showed no modification of the endocytosis constant. In contrast, the maximum velocity of asialoorosomucoid internalization is decreased by threefold in diabetic rat hepatocytes as compared to normal rat hepatocytes. No modification of internalization constant is observed between the two groups of rats. This suggests that the decrease of asialoorosomucoid total uptake, previously reported for diabetic rat hepatocytes is directly related to a decrease of total surface receptor number.

The binding and the total uptake of [3H]asialoorosomucoíd was studied in normal rats, streptozotocïn‐diabetic rats and insulin‐treated diabetic rats. The binding of [3H]asialoorosomucoi'd to normal rat hepatocytes gave a curvilinear plot when analysed by the Scatchard method, suggesting either two classes of independent receptor sites with different apparent association constants or a negative cooperativity in binding. The data, plotted according to the method of De Meyts and Roth showed a decrease of the affinity constant with respect to site occupancy. The Scatchard plot obtained for the [3H]asialoorosomucoid binding to streptozotocin‐diabetic rat hepatocytes was curvilinear. According to the independent site model, the receptor number of each class was decreased, without any change of the apparent association constants. According to the model of De Meyts and Roth, no modification of the interaction factor and apparent affinity constants was observed, whilst the total number of receptor sites was decreased by half in streptozotocin‐diabetic rats as compared with normal rats. Study of the total uptake of [3H]asialoorosomucoId by hepatocytes from each group of rats showed a decrease by half of the endocytosis in streptozotocin‐diabetic rats as compared with normal rats. Insulin treatment of the streptozotocin‐diabetic rats restored both parameters studied to normal level, whilst the withdrawal of insulin treatment decreased their level to that of streptozotocin‐diabetic rats.These results are related to possible modifications of the hepatic binding protein, receptor for the plasma asialoglycoproteins, in the diabetic state.

With regard to the toxic effects of Ricinus lectin, neuraminidase-treated hepatoma cells have been found to be the most sensitive, and untreated hepatoma cells the least. Cells treated with neuraminidase and galactose oxidase exhibited an intermediate sensitivity. At 37 degrees C, the number of Ricinus lectin molecules bound to untreated, neuraminidase-treated and neuraminidase and galactose oxidase-treated cells required to being about 30% toxicity within 2 h was 15 . 10(5), 7.5 . 10(5) and 11.5 . 10(5) molecules/cell, respectively. This difference was rather small and suggests that the additional binding sites exposed following enzyme treatment were as efficient in mediating lectin toxicity as those present before enzyme treatment. Positive cooperativity was observed during Ricinus lectin binding to enzyme-treated cells at 37 degrees C and the apparent association constant increased with the increase of binding site occupancy. The binding sites on enzyme-treated cells appeared to be homogeneous since under different physical conditions (4 degrees C) the shape of the Scatchard plot could be altered in such a way as to produce a single line of slope. In contrast to enzyme-treated cells, untreated cells did not exhibit a positive cooperative process either at 37 degrees C or at 4 degrees C. We found that the toxicity of Ricinus lectin paralleled the irreversible specific binding of lectin, suggesting that only this was able to mediate the toxic effect. Our results are discussed in terms of the possible entry into the cells of Ricinus lectin and this occurs more rapidly in enzyme-treated than in untreated cells. This difference agrees with the sequence of events proposed: (i) Binding of Ricinus lectin; (ii) Clustering of lectin binding sites; and (iii) Endocytosis.

The effects of several detergents commonly used to solubilize membrane glycoproteins have been investigated on the binding of hepatoma cell surface [H]-galactoglycoproteins to, and their elution from, concanavalin A or Ricinus communis lectins conjugated to Sepharose 4B. The optimum conditions (pH, ionic strength) in the presence of ionic [sodium deoxycholate (DOC) and sodium dodecyl sulphate (SDS)] and non-ionic detergents (Triton X-100) at a constant concentration were determined in order to ascertain which would yield the better efficiency. The effects of different detergent concentrations on binding and elution were then studied. The range of concentrations for each detergent to be used without modifying efficiency was determined. Triton X-100 and DOC (0.1–1%) did not change the efficiency on Ricinus lectin-Sepharose, whereas SDS, at a concentration greater than 0.05%, caused a dramatic decrease in efficiency. On concanavalin A-Sepharose, by contrast, the non-ionic detergent had no effect on the efficiency at all the concentrations tested (0.1–1%), while concentrations of more than 0.5% DOC and 0.1% SDS significantly decreased both binding and elution.

The glycoconjugates on Toxoplasma gondii membrane have been investigated in several ways, usually used for the study of cell surfaces: binding of |14C| lectins, Concanavalin A (Con A) and Wheat germ agglutinin (WGA) to parasites; parasites membrane labelling, using galactose oxidase/tritiated sodium borohydride (NaB|3H4|); determination of sialic acid amount, sensitive to the neuraminidase. Toxoplasma gondii binds Con A but did not bind significantly WGA. The affinity constant for Con A binding was on the same order of magnitude as for other cell systems, but the number of sites per parasite was low (3×104 sites per parasite). In contrast, with other cell systems, no surface incorporation of tritium was obtained using galactose oxidase/NaB|3H4|. Moreover the determination of sialic acid, sensitive to the neuraminidase gave a negative result. These results shown that Toxoplasma gondii surface do not contain all sugars, usually found in oligosaccharidic chains of membrane glycoconjugates and suggest the presence of low amount of surface glycoconjugates.

The cell-surface glycoproteins have been involved in growth control, and it is assumed that alterations of these substances may represent key steps in cell transformation. Mitogenic lectins, which bind to glycoprotein receptors, bring about a stimulation of the growth of normal cells and a reduction of that of transformed cells, suggesting a difference in the cell-surface glycoproteins. Thus, lectins can be used as powerful tools for cell-membrane glycoprotein studies. This chapter discusses two methods of isolation and characterization of lectin-binding glycopeptides and glycoproteins. In the experiments discussed in the chapter, the released material consisted of cell-surface glycoproteins, and very less radioactive labeling was released by the two methods employed. The results indicate that the glycoprotein and glycopeptides material removed from the cell surface possesses receptor activities for several lectins (Con A, WGA, RSA, Robinia lectin). The unexpected presence of bound glucose was observed and affinity-chromatography purification of the glycoprotein fraction was also performed.

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.