Enzymatic dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 in organic solvents

Summary 4-Chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 showed dehalogenating activity in various organic solvents. In alcohols like methanol (150%) or ethanol (120%) higher activities than in water (100%) were obtained. In apolar solvents like petroleum ether (5%) and nhexane (5%) only trace activities were observed. The solvents did not increase the stability of the enzyme. 4-Chlorobenzoic acid methylester, a substance not soluble in water, was not dehalogenated in organic solvents.     

Myocardial S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia

The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinetic parameters of S-adenosylhomocysteine hydrolase. During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 microM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 microM) caused an increase of 0.37 and 4.17 nmol SAdoHcy/min per g wet weight during normoxia and ischemia, respectively. The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 11.5 nmol/g; P less than 0.05). Purine release was increased 4-fold during ischemia. The Km for hydrolysis of SAdoHcy was about 12 microM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 microM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. From the combined in vitro and perfusion studies, we conclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.     

Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.     

Exogenous glutathione induces sister chromatid exchanges,clastogenicity and endoreduplication in V79-E Chinese hamster cells

Glutathione (GSH) dissolved in Eagle's MEM and added to cultures o of V79-E cells in concentrations between 2.5 × 10^–4 and 10^–3 moles/l for 1 h induces a dose-dependent cell cycle delay, sister chromatid exchanges and clastogenic damage. 7–8% of the metaphases showed endoreduplication at a recovery phase of 25 and 30 h after treatment with 10^–3 molesll GSH. Higher concentrations were lethal. The highest tolerated dose corresponds to the intracellular GSH level in V79-E cells. In the same range of concentrations, glutathione disulfide was inactive. Endoreduplication induction by GSH is G2-phase specific and endoreduplication metaphases show a reduced occurrence of single SCEs when extrapolated to the diploid complement. The adverse effects of GSH are independent of the presence of serum in the culture fluid but completely abolished when the treatment is performed in Hank's solution instead of MEM. The mechanism of genotoxicity of exogenous GSH is discussed but, at present, no pertinent explanation can be given.Abbreviations BUdR 5-bromodeoxyuridine - GSH glutathione - GSSG glutathione disulfide - SCE sister chromatid exchange     

Myocardial S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia

(1) The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. (2) A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinteic parameters of S-adenosylhomocysteine hydrolase. (3) During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 μM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 μM) caused an increased of 0.37 and 4.17 nmol SAdony/soc per g wet weight during normaxia and ischemia, respectively. (4) The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 115 nmol/g; P < 0.05). Purine release was increased 4-fold during ischemia. (5) The K_m for hydrolysis of SAdoHcy was about 12 μM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 μM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. (6) From the combined in vitro and perfusion studies, we comclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.