Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase.

An NAD(+)-dependent CDP-D-glucose oxidoreductase which catalyzes the first step of the biosynthesis of CDP-ascarylose (CDP-3,6-dideoxy-L-arabino-hexose), converting CDP-D-glucose to CDP-4-keto-6-deoxy-D-glucose, was isolated from Yersinia pseudotuberculosis. A protocol consisting of DEAE-cellulose, Matrex Blue-A, hydroxylapatite, DEAE-Sephadex, Sephadex G-100, and NAD(+)-agarose column chromatography was used to purify this enzyme 6000-fold to homogeneity. This enzyme consists of two identical subunits, each with a molecular weight of 42,500. Using CDP-D-glucose as the substrate, the Km and Vmax of this catalysis were determined to be 222 microM and 8.3 mumols mg-1 min-1, respectively. Unlike most other oxidoreductases of its class which have a tightly bound NAD+, this highly purified CDP-D-glucose oxidoreductase showed an absolute requirement of NAD+ for its activity. Using chemically synthesized (6S)- and (6R)-CDP-D-[4-2H,6-3H]glucose as substrates, a stereochemical analysis showed this enzymatic reaction involves an intramolecular hydrogen migration from C-4 to C-6, and the displacement of C-6 hydroxyl group by the C-4 hydrogen occurs with inversion. Thus, despite the low cofactor affinity, this enzyme undergoes a mechanism consistent with that followed by other members of its type. Such a mechanistic and stereochemical convergency found for all sugar oxidoreductases so far characterized suggests the presence of a common progenitor of this class of enzyme.     

The crystal structure of muscle phosphoglucomutase refined at 2.7-angstrom resolution.

A model of rabbit muscle phosphoglucomutase was refined at 2.7-A resolution by using two heavy atom derivatives for initial phasing and standard refinement procedures, including molecular replacement averaging about a 2-fold axis and dynamic simulation: final R-factor, 0.223 (no solvent modeling); RMS deviation from standard bond lengths and angles, 0.020 A and 3.6 degrees, respectively (all 8658 nonhydrogen atoms plus 36,953 reflections (F/sigma greater than or equal to 3) between 8- and 2.7-A resolutions); average of individually refined atomic B-factors, 40 A2 (all atoms) and 30 A2 (all atoms in domains I-III). An H-bonding scheme with 538 main chain H-bonds for the two monomers in the asymmetric unit and probable ligands for six uranyl ions in one heavy atom derivative is given. The monomer contains 42 strands/helices arranged into four alpha/beta-domains. Each of the first three domains contains an alpha 3 beta 4 alpha 1 motif, where the topology of beta 4 is 2,1,3,4:[arrows: see text] which is a topology not encountered in an extensive search among known protein structures. A spatial similarity is observed between corresponding residues in the three repetitions of this motif per monomer, but the minimal mutational distance between spatially corresponding residues is not statistically significant. The loop between the antiparallel strands in each of these domains is an important feature of the active site. In domain IV, beta-sheet topology is 2,1,3,4,5,6:[arrows:see text]. Noncovalent domain/domain interactions within the monomer are greatest between adjacent domains along the polypeptide chain, which are not substantially interdigitated and can be cleanly disengaged by altering the phi/psi torsional angles of three uniquely positioned residues in the model. The observed hierarchy of noncovalent interactions between structural units within the crystal, based on a semi-empirical paradigm, suggests that monomer-monomer contacts within the asymmetric unit are formed during growth of the lattice and provides a rationale for some of the diffraction characteristics of phosphoglucomutase crystals. An unusually deep crevice involving 58 residues is formed by the head-to-tail, twisted semicircular arrangement of the four domains of the monomer that places no atom more than 12 A from the water-accessible surface. The active site of the enzyme is extensively buried at the bottom of this crevice, at the approximate confluence of the four domains. Other features of the active site, including the surrounding helical dipoles, and the metal-ion binding pocket are described, together with structure/function comparisons with a number of other enzymes.     

Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.     

Possible involvement of protein kinase C and calcium in GSH efflux from Hep G2 cells.

We investigated the effects of protein kinase C modulations and calcium mobilization on GSH efflux in Hep G2 cells. GSH efflux from Hep G2 cells was increased by a phorbol ester. Staurosporine, an inhibitor of protein kinase C, diminished phorbol ester-stimulated GSH efflux from the cells. GSH efflux was negatively correlated with extracellular calcium concentrations. Verapamil enhanced GSH efflux, whereas ATP decreased GSH efflux. The latter effect was diminished in the absence of extracellular calcium. Protein kinase C and calcium mobilization may be crucial factors in GSH efflux from human hepatocytes.     

Inhibition of estradiol-induced early osteoarthritic changes by tamoxifen.

Previous studies on osteoarthritic changes induced by intraarticular injections of estradiol benzoate (EB) suggest estrogen as a risk factor in the development of knee osteoarthritis (OA). The present study observed the anti-arthritic effects of tamoxifen (TMX). Oophorectomized rabbits were subjected to intraarticular injections of vehicle control, EB, TMX, or EB/TMX for 3 weeks. The cartilage changes were assessed by light and scanning electron microscopic examination, enzyme histochemical analysis, and the amount of alcian blue stain binding to glycosaminoglycans. EB injections resulted in cell necrosis, chondrocyte clonings, and pittings, whereas the vehicle control, TMX, and EB/TMX-injected groups showed no histologic abnormalities. Histochemical analysis showed that the numbers of lactate dehydrogenase (LDH)-reactive chondrocytes in the EB-injected group were significantly reduced when compared to other groups (p less than 0.001). The injections of EB/TMX significantly reduced the chondrocyte numbers in the lateral superficial layer (p less than 0.05), compared with the vehicle injection. TMX-injected group revealed slight although insignificant decreases in chondrocyte numbers. The amount of alcian blue stains, representing the relative amount of proteoglycans, significantly decreased only in the superficial layer of the EB- and EB/TMX-injected groups (p less than 0.05). TMX, when concurrently injected with EB, antagonized the chondrodestructive effects of estradiol at the early stage of knee OA in rabbits. The results suggest the potential therapeutic use of TMX at the early stage of OA.     

Age-associated changes and sex differences in urinary 6-sulphatoxymelatonin circadian rhythm in the rat.

The circadian rhythm of 6-sulphatoxymelatonin (aMT6s) excretion has been determined in male and female rats at 3 weeks and at 2, 8, 14 and 20 months of age. All animals have a pronounced circadian pattern of aMT6s excretion under a 12 hour dark: 12 hour light cycle. A significant increase in aMT6s excretion is observed from 3 weeks to 14 months followed by a decrease at 20 months. There is a highly significant correlation between aMT6s excretion and body weight (r = 0.73 for female rats and r = 0.74 for male rats; p values are all less than 0.001). Thus, a decrease in aMT6s excretion associated with increasing age occurs when body weight is taken into consideration. aMT6s excretion is higher in males at 3 weeks and at 2 and 8 months of ages. Urinary testosterone in male rats and estradiol in female rats increase from 3 weeks to 8 months and decrease at older ages. These data suggest that increase of body weight from 3 weeks to 14 months is an important factor responsible for the age-related alteration. The sex differences in aMT6s excretion in younger rats may be associated with their sex hormonal milieu.