ABSTRACT
Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) and conjugation of glutathione to 1-chloro-2, 4-dinitrobenzene. We introduced site-directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH(2) in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased. Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione.
Subject(s)
Hematopoietic Stem Cells/enzymology , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Amino Acids/metabolism , Animals , Arginine/chemistry , Arginine/metabolism , Catalysis , Circular Dichroism , Crystallography, X-Ray , DNA Primers , Dinitrobenzenes/pharmacology , Escherichia coli/metabolism , Glutathione/metabolism , Glutathione Transferase/metabolism , Immunoblotting , Immunohistochemistry , Kinetics , Lipocalins , Models, Molecular , Mutagenesis, Site-Directed , Plasmids/metabolism , Polymerase Chain Reaction , Rats , Recombinant Proteins/metabolism , Time Factors , Tyrosine/chemistry , Tyrosine/metabolism , Ultraviolet RaysABSTRACT
To examine the function of prostaglandin (PG) D synthase (PGDS) gene, as well as endogenously produced PGD(2) in sleep regulation in vivo, we generated transgenic (TG) mice that overexpress human PGDS gene to study their sleep behavior. Although no difference was observed in the sleep/wake patterns between wild-type and TG mice, a striking time-dependent increase in non-rapid eye movement (NREM), but not in rapid eye movement (REM), sleep was observed in two independent lines of TG mice after stimulation by tail clipping. Concomitantly, the spontaneous locomotor activity of TG animals was drastically decreased in response to the tail clip. Induction of NREM sleep in TG mice was positively correlated with the PGD(2) production in the brain. Sleep, locomotion, and PGD(2) content were essentially unchanged in wild-type mice after tail clipping. The results with TG mice demonstrate the involvement of the PGDS gene in the regulation of NREM sleep.
Subject(s)
Intramolecular Oxidoreductases/genetics , Sleep Stages/genetics , Sleep, REM/genetics , Animals , Biological Clocks , Circadian Rhythm , Gene Expression Regulation, Enzymologic , Humans , Intramolecular Oxidoreductases/metabolism , Lipocalins , Male , Mice , Mice, Inbred Strains , Mice, Transgenic , Motor Activity , Sleep Stages/physiology , Sleep, REM/physiologyABSTRACT
Kinetic parameters of spontaneous lipid peroxidation (LP) in endoplasmic reticulum (ER) membranes isolated from the liver of healthy mice, tumour-host and ascitic Ehrlich tumour cells have been studied. The LP kinetics was characterized based on the primary LP products, dienic conjugates (DC), and the extent of lipid substrate unsaturation (double bond (DB) concentration per mg lipids). It was shown that the rate constants of DC formation and DB consumption were, respectively, (6.3 +/- 0.7).10(-2) and (4.5 +/- 0.3).10(-2) min-1 in the control; (2.5 +/- 0.3).10(-2) and (1.0 +/- 0.1).10(-2) min-1 in the ER from the tumour-host liver; (1.4 +/- 0.1).10(-2) and (0.38 +/- 0.01).10(-2) min-1 in the ER from the tumour cells. The initial DC and DB levels in membranes of the tumour cells were significantly (2 to 3 times) lower than in the ER isolated from the control liver. There was a correlation between the DB initial level and the constants of DB consumption during LP in vitro for all types of membranes studied.
Subject(s)
Carcinoma, Ehrlich Tumor/metabolism , Intracellular Membranes/metabolism , Lipid Peroxidation , Liver/metabolism , Animals , Endoplasmic Reticulum/ultrastructure , Kinetics , Mice , Microsomes, Liver/metabolismABSTRACT
We studied the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and protein kinase C (PKC) in a broad range of concentrations (10(-18)-10(-7) M) on lipid peroxidation (LP) in rat brain plasma membranes. TPA and PKC were shown to inhibit LP, the concentration curves had two maxima at 10(-15) M and 10(-12) M for TPA and at 10(-16) M and 10(-13) M for PKC. The combined action of TPA (10(-12) M) and PKC (10(-16) M) resulted in a synergic inhibition of LP. These data suggest PKC to have two (kinase and antioxidative) enzymatic activities. The properties of TPA as an LP inhibitor (via activation of PKC) and tumour promoter (inhibition of LP is a necessary step of tumour promotion) can be explained based on this suggestion.
