A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (Rfme 0.248) and 0.1 82 (Rfree 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ( ( B ) room temperature GPb = 34.9 A2; ( B ) 1 0 0 K GPb = 23.4 A*). The glucopyranose spirohydantoin inhibitor (K, = 3.0 p M ) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 28 1-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of IO3, makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.Keywords: anti-hyperglycaemic agent; glucopyranose spirohydantoin; glycogen phosphorylase; hydantocidin; inhibitor binding; water structure Glycogen phosphorylase (GP) catalyzes the first step in the phosphorolysis of glycogen to yield glucose-1-phosphate which, in muscle, is utilized via glycolysis to provide energy for muscle contraction and, in the liver, is converted to glucose to supply
Rhodamine‐labelled epidermal growth factor (Rh‐EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(‐10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(‐9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh‐EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(‐10) M Rh‐EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees‐37 degrees C. The low affinity receptors (labelled with 2.4 X 10(‐7) M Rh‐EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(‐10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.
The order of addition of substrates and release of products in the reaction catalyzed by rat-liver glucokinase has been studied by measurements of isotope exchange. Experiments at chemical equilibrium showed some degree of randomness, but steady-state experiments showed a predominantly ordered process with glucose binding first and glucose 6-phosphate released last. Experiments to trap binary complexes in the steady state demonstrated the existence of complexes of the enzyme with glucose and with glucose 6-phosphate but gave no evidence for the occurrence of corresponding complexes with ATP or ADP. Flux ratios measured in both the forward and reverse reactions provided a more rigorous and quantitative confirmation of these characteristics of the reaction. These observations support the interpretation of glucokinase cooperativity in terms of a "mnemonical" mechanism and conflict with an alternative interpretation in terms of a random addition of substrates.
The expression of epidermal growth factor receptor (EGF-R) was studied immunohistochemically in 72 meningiomas using two monoclonal antibodies with specificities to protein and carbohydrate components, respectively, of the external domain of the EGF-R. One third of the tumors had cytoplasmic and membrane positivity with the protein-specific antibody but in none were there positive tumor cells with the carbohydrate-specific antibody which recognizes the blood group A antigen. There was no difference in EGF-R expression between typical and aggressive meningiomas. No evidence was found to support previous reports of specific EGF-R immunoreactivity in the vascular endothelial cells of meningiomas. The authors believe this discrepancy to be due to detection of normal blood group A antigen attached to endothelial cells in patients of blood group A or AB. This occurs because many monoclonal anti-EGF-R antibodies are specific for A antigen which is found on the EGF-R of A431 cells but has not been reported on EGF-R elsewhere.
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