Arylsulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is ome evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Laminaria augmentation of intra-amniotic PGF2α for midtrimester pregnancy termination

In our hands, intra-amniotic PGF₂α 40 mg for midtrimester pregnancy termination had a mean infusion to abortion interval of 29.4 hr. However, pretreatment of 230 patients with laminaria tents inserted 14–18 hr before PGF₂α infusion resulted in a dramatically reduced time to abortion (14.3 hr mean) with a low incidence of gastrointestinal and other side effects. Laminaria tents inserted at the same time as PGF₂α infusion in 26 patients also resulted in reduced time to abortion (18.6 hr mean). In the laminaria pretreated group, the infusion to abortion interval was indirectly related to the number of laminaria inserted and not to the nulliparous or parous state. Although we have made significant strides in shortening the abortion interval in the hospital, retained placentae and blood loss persist as problems related to the use of prostaglandin for abortion.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Further characterization of the putative glycolipid receptor for mif: Role of fucose associated with an acidic glycolipid

Water soluble glycolipids were extracted from guinea pig macrophages. These glycolipids, when incubated with macrophages, augment the cells' response to migration inhibitory factor. The glycolipids were fractionated by diethylaminoethyl-Sephadex ion exchange chromatography into neutral and acidic fractions. Only the acidic glycolipid fraction was able to enhance the responsiveness of macrophages to migration inhibitory factor. Additional studies indicate that the enhancing activity of these glycolipid preparations can be abrogated by the removal of terminal fucose residues with α-L-fucosidase. The possibility that fucose functions as an essential component of a macrophage glycolipid receptor for migration inhibitory factor is discussed.     

Intestinal metallothionein and the mutual antagonism between copper and zinc in the rat.

A study has been made of the mechanism of the mutual antagonism between copper and zinc in rats. Dietary zinc concentrations of up to 450 mg/kg had no effect on intestinal 64Cu absorption but 900 mg/kg caused a 40% reduction. This was associated with an increase in the mucosal uptake of 64Cu in the small intestine. This occurred mainly in the form of metallothionein and it appeared that copper displaced zinc from the protein after its synthesis had been induced by zinc. Ths intestinal absorption of 65Zn was decreased by 20% when the dietary copper intake was increased from 3 to 24 mg/kg. Further increases in copper intake to 300 mg/kg did not cause any additional decrease in 65Zn absorption or any change in the association of intestinal 65Zn with metallothionein. Concentrations of this protein in the intestinal mucosa were not influenced by dietary copper intake.     

Effect of acute stress on the absorption and distribution of zinc and on Zn-metallothionein production in the liver of the chick.

A study has been made of the effects of chloroform inhalation, Escherichia coli endotoxin injection and hydrocortisone injection on the absorption of a single intragastric dose of 65Zn by the chick. Injection of hydrocortisone increased the absorption of the 65Zn by 30-55% in both Zn-deficient and Zn-supplemented chicks. The influence of chloroform and endotoxin was less consistent; the former treatment only increased 65Zn absorption and endotoxin was less consistent; the former treatment only increased 65Zn absorption in Zn-supplemented chicks fed ad libitum whereas endotoxin only increased that in Zn-supplemented chicks on a restricted food intake. Injection of endotoxin increased the hepatic uptake of the absorbed 65Zn in both Zn-deficient and Zn-supplemented chicks, whereas hydrocortisone had a similar effect in the Zn-supplemented birds only. Chloroform inhalation increased hepatic 65Zn uptake in Zn-deficient chicks only. The increase in hepatic Zn concentrations in the stressed chicks was mainly associated with a protein in the cytosol identified as metallothionein. Both endotoxin and hydrocortisone decreased total plasma Zn concentrations in Zn-supplemented and Zn-deficient chicks; chloroform decreased plasma 65Zn content only.     

Identification of beta-adrenergic receptors in pulmonary alveolar type II cells

Specific beta-adrenergic receptors have been identified in dissociated preparations of rabbit lung cells greatly enriched for alveolar type II cells and compared with receptors in preparations of mixed lung cells and erythrocytes. Freshly isolated type II cells as well as mixed dissociated lung cells and erythrocytes from fetal (28 days gestation) and adult rabbits contained high-affinity, low-capacity binding sites for [3H]dihydroalprenolol (DHA). Binding to all preparations was stereospecific and characteristic of the beta 1-subtype of beta-adrenergic receptors. The concentrations of the receptors were similar in mixed lung cells and alveolar type II cells, indicating that beta-adrenergic receptors are present not only in type II cells but also in other lung cell types. When the contribution of erythrocytes to receptor concentration observed in type II cells was determined, it was found to be insignificant. In mixed lung cells, both the affinity and concentration of the receptors were higher in adult than fetal preparations. The affinity of the receptors was also higher in adult than fetal type II cells, although we did not find a significant age-related difference in receptor concentrations in this cell type. These results suggest that stimulation of surfactant secretion observed after exposure of lung tissue to beta-adrenergic agonists is mediated by specific beta-adrenergic receptors on alveolar type II cells.     

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.     

The copper-enzyme dopamine beta-monooxygenase: studies on the ability of several metals to inhibit the enzyme activity and to replace the copper

The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme.     

The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by ascorbic acid

Purified commercial hyaluronic acid contains significant amounts of iron. Addition of Fe2+ to solutions of it causes depolymerization, which is inhibited by catalase and scavengers of the hydroxyl radical (. OH) but not by superoxide dismutase. Fe3+ is ineffective. Ascorbic acid also depolymerizes hyaluronic acid, apparently because it can reduce Fe3+ in the reaction mixtures to Fe2+. Ascorbate-induced depolymerization is inhibited by the specific iron chelator desferrioxamine, by catalase, and by scavengers of the hydroxyl radical. The relevance of these observations to rheumatoid arthritis and inflammatory joint diseases is discussed.     

Kinetic studies on the diaqua form of cis-platin and various nucleobases

Kinetic studies on cis-[Pt(NH3)2(OH2)2]2+ and various nucleobases show that this ion reacts more quickly with guanosine than with adenosine, cytidine, and thymidine, and that a monophosphoric acid unit considerably enhances the rate of reaction of guanosine; the kinetic preference of 5'-GMP over 5'-AMP may point to a greater thermodynamic selectivity.     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with Mercury(II) and Methylmercury(II)

Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH₃Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by ¹H and ¹³C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH₃Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH₃Hg(II) bind to N₃ of thymidine in preference to N₁ of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.     

Hyperfine coupling of 17O to Mo(V) in the rapid epr signal of xanthine oxidase: Evidence for an oxygen ligand of the metal

The spectrum of the Rapid Mo(V) electron paramagnetic resonance signal from xanthine oxidase dissolved in ¹⁷O-enriched water is presented. Difference technqiues have been used to eliminate the ¹⁶O contribution. Clearly observed structure in the spectrum is attributed to moderately strong hyperfine coupling of one oxygen atom to molybdenum. Though complete interpretation of the spectrum has not been attempted, one component of A(¹⁷O) is about 1.6 mT. The possibility that the oxygen is present in a MoOH group, whose proton is the strongly-coupled proton of the Rapid signal, is discussed.     

Evidence for a shift from IgD to IgA synthesis in the maturation of human B lymphocytes.

A case of chronic lymphatic leukemia (CLL) is described in which IgD and IgA are the copredominant membrane immunoglobulins. Since CLL represents malignant proliferations of B lymphocytes arrested at discrete points during maturation, the findings in this case suggest that at least some of the developing cells destined to synthesize IgA for secretion pass through a stage in which immunoglobulins D and A are present together on the cell membrane.     

The effects of age,sex, and zinc status on the accumulation of (copper,zinc)-metallothionein in rat kidneys

A study has been made of the distribution of copper in the kidneys of growing rats. Renal copper concentrations increased steadily with age and were greater in female than in male animals. Most of the copper was present as (copper, zinc)-metallothionein and two forms of this protein were isolated and characterized from the kidneys of mature female rats. That copper metabolism in kidneys is subject to hormonal influence was indicated by a reduction in the concentrations of copper and (copper, zinc)-metallothionein in ovariectomized rats and by an increase in their concentrations after the administration of progesterone. Concentrations of renal (copper, zinc)-metallothionein were less in zinc-deficient than in zinc-adequate rats during pregnancy and after progesterone administration.     

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.     

Raman and infrared spectroscopy of the oxo-bridged iron(III) complex, [Cl3Fe-O-FeCl3]-2 as a spectroscopic model for the oxo bridge in hemerythrin and ribonucleotide reductase

Vibrational spectroscopic data were collected on the salt [C5H6N]2[Cl3FeOFeCl3] . C5H5N, which has previously been structurally characterized by X-ray crystallography. The modes associated with the oxo bridge were identified by experiments on the 18O-containing species. Spectra for the mu-16O complex contain Raman bands at 870, 458, and 203 cm-1 that shift to 826, 440, and 198 cm-1 in the mu-18O complex. These are respectively assigned to the asymmetric, symmetric, and angle deformations of the bent Fe-O-Fe moiety. A normal mode vibration analysis based on a simple valence force field for the Fe-O-Fe portion of the molecule provides surprisingly good agreement with these experimental frequencies and their assignments. The vibrational data for this simple inorganic complex confirm the assignment of a resonance Raman band around 500 cm-1 in the oxygen-carrying protein hemerythrin and enzyme ribonucleotide reductase as the symmetric stretch of an oxo bridge between two iron(III) centers.