Enzymatic transformation of PGH2 to PGF2 alpha catalyzed by glutathione S-transferases

Glutathione S-transferases (GSTs) purified from both rat liver cytosol and microsomes catalyzed the direct reduction of PGH2 to PGF2 alpha. As much as 40% of the substrate was transformed into a prostanoid whose Rf value corresponded to that of PGF2 alpha. The identification of the reaction product as PGF2 alpha was confirmed by TLC and reverse-phase HPLC as well as by mass spectral analysis. In the absence of GSTs, PGH2 was found to be primarily converted to PGE2 and PGD2. Also, PGF2 alpha formation was completely abolished by decylglutathione, a potent inhibitor of both peroxidase and transferase activity associated with GSTs. These results indicate that the direct reduction of endoperoxide moiety of PGH2 to form PGF2 alpha is an enzymatic process. Interestingly, selenium-dependent glutathione peroxidase (Se-GSH-Px) showed very little PGF2 alpha formation from PGH2. However, this enzyme was very active in the reduction of PGG2 to PGH2. In contrast, GSTs were very poor in the conversion of PGG2 to PGH2. Therefore, it is possible that the relative tissue distribution of Se-GSH-Px and GSTs might play an important role in the tissue specific synthesis of PGF2 alpha.     

Increased efflux rather than oxidation is the mechanism of glutathione depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP+-induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechanism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a time-dependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.     

Involvement of erythrocyte skeletal proteins in the modulation of membrane fluidity by phenothiazines

The effects of phenothiazines (chlorpromazine, chlorpromazine sulfoxide, and trifluoperazine) and antimitotic drugs (colchicine and vinblastine) on the erythrocyte membrane have been investigated. Chlorpromazine and trifluoperazine induced a dose-dependent increase in the freedom of motion of stearic acid spin-labels bound to both intact erythrocytes and ghosts, but did not affect the freedom of motion of stearic acids bound to vesicles depleted of spectrin and actin or of ghosts resealed with anti-spectrin antibodies. Further, chlorpromazine and trifluoperazine were able to eliminate a protein 4.1 dependent membrane thermal transition detected by stearic acid spin-labels at 8.5 +/- 1.5 degrees C. Antimitotic drugs and chlorpromazine sulfoxide did not change either the freedom of motion of stearic acid spin-labels or the 8.5 degrees C membrane thermal transition. Results indicate the involvement of skeletal proteins as possible membrane target sites of biologically active phenothiazines and suggest that the control of stearic acid spin-label freedom of motion is mediated by the spectrin-actin network and the proteins that link the skeletal network to the membrane.     

Properties of carboxymethylated cross-linked hemoglobin A

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: differential effects of monensin

Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37 degrees C. Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-[3H]Phe for up to 30 min at 37 degrees C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 microM. 50% inhibition was observed at 1-10 microM monensin with no significant change in fMet-Leu-Phe dose dependency. Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent. The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected. Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology. Incubation of cell suspensions with up to 30 microM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation. Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations. These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production. They also are consistent with a role for receptor modulation and processing in the chemotactic response.     

The nature of phonons and solitary waves in alpha-helical proteins.

A parametric study of the Davydov model of energy transduction in alpha-helical proteins is described. Previous investigations have shown that the Davydov model predicts that nonlinear interactions between phonons and amide-I excitations can stabilize the latter and produce a long-lived combined excitation (the so-called Davydov soliton), which propagates along the helix. The dynamics of this solitary wave are approximately those of solitons described using the nonlinear Schr?dinger equation. The present study extends these previous investigations by analyzing the effect of helix length and nonlinear coupling efficiency on the phonon spectrum in short and medium length alpha-helical segments. The phonon energy accompanying amide-I excitation shows periodic variation in time with fluctuations that follow three different time scales. The phonon spectrum is highly dependent upon chain length but a majority of the energy remains localized in normal mode vibrations even in the long chain alpha-helices. Variation of the phonon-exciton coupling coefficient changes the amplitudes but not the frequencies of the phonon spectrum. The computed spectra contain frequencies ranging from 200 GHz to 6 THz, and as the chain length is increased, the long period oscillations increase in amplitude. The most important prediction of this study, however, is that the dynamics predicted by the numerical calculations have more in common with dynamics described by using the Frohlich polaron model than by using the Davydov soliton. Accordingly, the relevance of the Davydov soliton model was applied to energy transduction in alpha-helical proteins is questionable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose oxidation in a dual hollow fiber bioreactor with a silicone tube oxygenator

A dual hollow fiber bioreactor, consisting of an outer silicone membrane for oxygen supply and an inner polyamide membrane for substrate permeation, was used as an immobilized enzyme reactor to carry out enzymatic glucose oxidation. Attaching a silicone tube oxygenator to provide an additional oxygen supply improved the conversion in glucose oxidation when the oxygen supply was rate-limiting. The reactor was operated in both diffusion and ultrafiltration modes. In the latter case, the conversion was much higher, but the stability of the immobilized enzyme was better maintained in the diffusion mode. As the inlet glucose concentration increased from 10mM to 500mM, the conversion decreased from 70 to 20%.     

Successful pregnancy in a woman with congenital factor XIII deficiency treated with substitutive therapy. Report of a second case

A syndrome of marked fetal wastage is associated with congenital factor XIII deficiency in adult women. A previously unreported case of a woman with factor XIII deficiency is described, in which substitutive treatment with normal plasma or placental factor XIII concentrate permitted two normal pregnancies. Factor XIII activity was maintained above 1-2% with intermittent infusion of 300 ml to 450 ml of plasma every 14 days or of 500 units of concentrate every 21 days. This case confirms the only other case so far reported in which factor XIII substitutive therapy was able to permit a normal pregnancy in a woman with factor XIII deficiency and seems to suggest factor XIII to be involved in the process of annidation.     

Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties.

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.