ABSTRACT
The parameters affecting the crystal quality of complexes between p21(H-ras) and caged GTP have been investigated. The use of pure diastereomers of caged GTP complexed to the more stable p21(G12P)' mutant of p21 and the addition of n-octyl-beta-D-glucopyranoside improved the reproducibility and decreased the mosaicity of the crystals significantly. Furthermore, the crystallization technique was changed from the batch method to the sitting-drop technique. With the availability of a larger yield of well ordered crystals, it was possible to extend the time-resolved crystallographic investigations on p21(H-ras). A structure of p21(G12P)':GTP could be obtained 2 min after photolytic removal of the cage group and led to the identification of a previously unidentified conformation for the so-called catalytically active loop L4. The refinement of five data sets collected within 2 min at different times (2-4, 11-13, 20-22, 30-32 and 90-92 min) after the initiation of the intrinsic GTPase reaction of the protein indicates that the synchrotron Laue method can be used to detect small structural changes and alternative conformations, but is presently limited in the analysis of larger rearrangements since these produce diffuse and broken electron density.
ABSTRACT
beta-N-Acetylglucosaminidase and beta-galactosidase activities were determined in serum and liver from guinea pigs fed "toxic oil" (related to cases of TOS) under different experimental conditions. The results obtained were compared with those of guinea pigs fed non "toxic oil" (case-unrelated oil; controls 1) and animals fed no oil (controls 2). In serum, both activities were significantly increased after all treatments with case-related oil as compared with controls 1 and 2. In the liver, beta-galactosidase activity did not show significant differences in any of the treatments when compared with controls 2. However, NAG activity decreased significantly after 7 days of treatment with non-heated oil--either case-related or not--when compared with controls 2; it also decreased significantly after 28 days of treatment with heated case-unrelated oil, both with respect to controls 2 and the animals fed case-related oil. Liver weights tended to increase in the animals fed oil--toxic or not--with respect to those of the livers from untreated animals. Morphologically, a slight vacuolization of the hepatocytes was observed in some of the samples from the treated animals.
Subject(s)
Acetylglucosaminidase/metabolism , Brassica , Liver/drug effects , Liver/enzymology , Lysosomes/drug effects , Lysosomes/enzymology , Plant Oils/poisoning , beta-Galactosidase/metabolism , Acetylglucosaminidase/blood , Animals , Behavior, Animal/drug effects , Body Weight/drug effects , Fatty Acids, Monounsaturated , Foodborne Diseases/etiology , Guinea Pigs , Liver/anatomy & histology , Male , Organ Size/drug effects , Proteins/metabolism , Rapeseed Oil , Syndrome , beta-Galactosidase/bloodABSTRACT
1. Comparative studies of beta-N-acetylglucosaminidase (NAG, EC 3.2.1. 52) and beta-galactosidase (EC 3.2.1. 23) activities, protein concentration and creatinine clearance, were carried out in urine and kidney from guinea pigs treated with "toxic oil", orally administered under different conditions, related to controls. 2. Enzyme activities did not vary significantly in urine with any of the studied conditions of oil administration. By contrast, in kidney, beta-D-galactosidase disclosed a significant increase in all the treatments studied when compared to controls without treatment. 3. Urinary protein excretion, creatinine concentration and creatinine clearance were significantly greater in treated animals than in controls after 4 weeks of treatment. 4. Relative kidney weight (g/100 g body wt), was significantly lower in animals treated for 28 days with previously heated oil.
