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1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67–74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained α-D-N-acetylglucosamine and were fucosylated, sialy-lated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins. The two immunologically diverging mucins nucleated cholesterol from model bile 1–2 days earlier and also caused an almost threefold more rapid rupture of cholesterol/phospholipid vesicles than the other mucins. 3. We conclude that considerable differences exist between mucins derived from individual patients and that the heterogeneity in glycan composition might play a role in the pathogenesis of gallstone disease.

1. Human gallbladder mucin has been implicated to play a role in gallstone disease. In spite of this fact relatively little is known about the structure of human gallbladder mucin. In this study we have investigated the possible heterogeneity of mucin. For this purpose polyclonal and monoclonal antibodies against gallbladder mucin were raised. All antibodies reacted primarily with carbohydrate antigenic determinants. With these antibodies the immunoreactivity of gallbladder mucin from 60 patients with cholesterol gallstones and 20 subjects without stones was screened. In addition, reactivity with several lectins was studied. 2. Considerable heterogeneity was found with both antibody and lectin typing, but there was no significant difference in heterogeneity between mucin from patients with gallstones and control subjects. Immunoblotting revealed that there was similarity between the reaction of the polyclonal antibody and the Helix pomatia agglutinin. All mucin preparations reacting with the polyclonal antibody also bound to Helix pomatia agglutinin. Nineteen of the 21 reacting mucins (90%) were from patients with blood group A (18 patients) or AB (one patient) and expression of A antigen could be demonstrated on the mucin of these patients. The resulting two reacting mucins were from patients with type O. However, expression of the blood group antigen could not account for the lack of reactivity of the mucin of other patients. The Helix pomatia agglutinin partially blocked the reactivity of the polyclonal antibody, whereas anti-A antibody did not show inhibition, indicating that more then only blood group A epitopes were recognized by this antibody. 3. We conclude that considerable patient to patient heterogeneity of human gallbladder mucin exists. This may have functional consequences for the role of mucin in the pathogenesis of gallstone disease.

In order to locate sites of action of thyroid hormone on mitochondrial oxidative phosphorylation we have used an experimental application of control analysis as previously described [Groen, Wanders, Westerhoff, Van der Meer & Tager (1982) J. Biol. Chem. 257, 2754-2757]. Rat-liver mitochondria were isolated from hypothyroid rats or from hypothyroid rats 24 h after treatment with a single dose of 3,3′,5-triiodothyronine (T3). The amount of control exerted by four different steps on State-3 respiration with succinate as respiratory substrate was quantified by using specific inhibitors. The hormone treatment resulted in an increase in the flux control coefficient of the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase and a decrease in the flux control coefficient of the bc1-complex. The results of this analysis indicate that thyroid hormone treatment results in an activation of the bc1-complex and of at least one other enzyme, possibly succinate dehydrogenase. Measurement of the extramitochondrial ATP/ADP ratio at different rates of respiration (induced by addition of different amounts of hexokinase in the presence of glucose and ATP) showed that the adenine nucleotide translocator operates at a higher (ATP/ADP)out after T3 treatment, which supports previous reports on stimulation of this step by thyroid hormone.

Existing, qualitative notions with respect to the way in which enzyme properties control metabolism are discussed in the light of the control analysis developed by H. Kacser and J. A. Burns ((1973) in: Rate Control of Biological Processes, Davies DD, ed., Cambridge University Press, pp. 63–104) and R. Heinrich and T. A. Rapoport ((1974) Eur. 3. Biochem. 42, 89–95), and recent experimental data. Points at which the existing notions should be adjusted are: (i) Metabolic control is shared by enzymes rather than confined to one rate-limiting enzyme per pathway. (if) Whether an enzyme exercises strong control on a flux cannot be deduced solely from its own properties, nor is it directly related to its distance from equilibrium. With respect to metabolic control, enzymes should be classified into four groups, rather than two (reversible versus irreversible). (iii) The distribution of control among the enzymes depends on the metabolic conditions. (iv) Control structures of metabolic pathways probably differ with the function of that pathway.