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.     

Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues

Ovomucoids were isolated from egg whites of 100 avian species and subjected to limited proteolysis. From each an intact, connecting peptide extended third domain was isolated and purified. These were entirely sequenced by single, continuous runs in a sequencer. Of the 106 sequences we report (five polymorphisms and chicken from the preceding paper [Kato, I., Schrode, J., Kohr, W. J., & Laskowski, M., Jr. (1986) Biochemistry (preceding paper in this issue)]), 65 are unique. In all cases except ostrich (which has Ser45), the third domains are either partially or fully glycosylated at Asn45. The majority of the third domain preparations we isolated are carbohydrate-free. Alignment of the sequences shows that their structurally important residues are strongly conserved. On the other hand, those residues that are in contact with the enzyme in turkey ovomucoid third domain complex with Streptomyces griseus proteinase B [Read, R., Fujinaga, M., Sielecki, A. R., & James, M. N. G. (1983) Biochemistry 22, 4420-4433] are not conserved but instead are by far the most variable residues in the molecule. These findings suggest that ovomucoid third domains may be an exception to the widely accepted generalization that in protein evolution the functionally important residues are strongly conserved. Complete proof will require better understanding of the physiological function of ovomucoid third domains. This large set of variants differing from each other in the enzyme-inhibitor contact area and augmented by several high-resolution structure determinations is useful for the study of our sequence to reactivity (inhibitory activity) algorithm. It is also useful for the study of several other protein properties. In the connecting peptide fragment most phasianoid birds have the dipeptide Val4-Ser5, which is absent in most other orders. This dipeptide is often present in only 70-95% of the molecules and appears to arise from ambiguous excision at the 5' end of the F intron of ovomucoid. Connecting peptides from the ovomucoids of cracid birds contain the analogous Val4-Asn5 peptide. In laughing kookaburra ovomucoid third domain we found (in 91% of the molecules) Gln5A, which we interpret as arising from ambiguous intron excision at the 3' end of the F intron.     

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%.     

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.     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

Natural killer (NK) cell immunodeficiency in patients with chronic myelogenous leukemia

Summary Defective natural killer (NK) cell populations from patients with chronic myelogenous leukemia (CML), that reacted with both HNK-1⁺ and B73.1⁺ antibodies, were obtained by a flluorescence-activated cell sorter (FACS). These fractions, along with NK fractions from normal donors which reacted with both antibodies, were expanded as bulk cultures or clones by limiting dilution, for 4 weeks in the presence of 10% interleukin 2 (IL 2), human type AB plasma, and irradiated human allogeneic mononuclear cells. Successfully established clones from patients with CML, with lytic activity against autologous and more differentiated neoplastic granulocytes, were generated more efficiently from B73.1⁺ than from HNK-1⁺ subsets. However, there were no significant differences among the generations of B73.1⁺ and HNK-1⁺ clones for both patients and normal donors with lytic activity against NK susceptible K-562 targets. Fresh myeloblast preparations from a blast crisis were found to be more susceptible to lysis by IL 2-proliferative B73.1⁺ and HNK-1⁺ clones than were fresh myelocyte preparations from a chronic phase CML patient, which were lytically susceptible to only B73.1⁺ clones. B73.1⁺ and HNK-1⁺ subsets from CML patients demonstrated major histocompatibility complex nonrestricted killing, and showed the following predominant phenotypes: B73.1⁺T3⁺T8⁺ or B73.1⁺T3⁺T8^– from B73.1⁺ subsets; and HNK-1^–T3⁺T8⁺ (initially HNK-1⁺) from HNK-1⁺ subsets. In contrast, B73.1⁺ and HNK-1⁺ clones from normal donors showed the following predominant phenotypes: B73.1⁺T3^–T8^–; and HNK-1^–T3^–T8^– or HNK-1^–T3^–T8⁺ (initially all HNK-1⁺). Short-term in vitro IL 2 or interferon treatment of fresh NK defective subsets from CML patients resulted in minimal cytotoxic augmentation. In contrast, defective NK cells from CML patients, whether HNK-1⁺ or B73.1⁺ subsets, proliferated with complete regeneration of cytolytic activity after a 3–4 week exposure to IL 2, but differed in phenotypic profiles as compared to those of normal donors. These observations imply that not only fresh defective NK cells but also the cytotoxically restored clones from CML patients are derived from different NK subsets and may represent undifferentiated forms of NK cells that may be arrested at an early stage of development by yet unknown mechanism(s). In vitro substantiation of autologous leukemia cell killing by IL 2-proliferative NK cell clones is encouraging and may allow for new in vivo immunotherapeutic modalities in CML patients.     

Effect of biological toxins on gap-junctionai intercellular communication in Chinese hamster V79 cells

Since chemical modulation of gap junctional intercellular communication has been implicated in several toxicological endpoints, a study to examine the ability of several biological toxins to inhibit this process was undertaken. Eight biological toxins were tested for their ability to inhibit metabolic cooperation, a measure of gap-junctional intercellular communication, in the Chinese V79 cell system. Aplysiatoxin, anhydrodebromoaplysiatoxin and debromoaplysiatoxin showed the strongest ability to inhibit metabolic cooperation while T2-toxin and vomitoxin inhibited metabolic cooperation to a lesser degree. Afatoxin B₁, afatoxin B₂ and palytoxin were inactive in the Chinese V79 system. Palytoxin, which was extremely cytotoxic, might act as a tumor promoter if it induces compensatory hyperplasia in vivo.Abbreviations 6-TG 6-thioguanine - TPA 12-0-tetradecanoylphorbol-13-acetate