Infrared spectroscopic studies on gramicidin ion-channels: relation to the mechanisms of anesthesia

Fourier transform infrared spectroscopic studies are reported on gramicidin ion-channels in phospholipid bilayers and the effects on the spectra of the anesthetics and related compounds (methoxyflurane, halothane, chloroform, carbon tetrachloride, n-pentane and n-decane) have been determined. The addition of anesthetics containing the 'acidic hydrogen' caused unique changes particularly on the amide I bands at 1639 cm-1 and 1670 cm-1. The 1639 cm-1 band became more intense while the intensity near 1670 cm-1 decreased dramatically. These effects were not observed with carbon tetrachloride, n-pentane and n-decane. The 1670 cm-1 band is interpreted as arising from the carbonyls involved in the head-to-head hydrogen-bonded dimerization where the relationship between chains is analogous to that of the antiparallel beta-pleated sheet structure and the anesthetics with 'acidic hydrogens' are considered to disrupt the hydrogen-bonded dimerization by competitive hydrogen bonding to the carbonyls at the head-to-head junction. As the dimer-monomer equilibrium is the 'on-off' mechanism for gramicidin ion-channel conductance, the results are considered in terms of the mechanism of action of anesthetics and are taken to suggest, for certain anesthetics, a hydrogen-bonding role to protein ion-channel components.     

Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.

In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.     

Subcellular origin of the oxalate- or inorganic phosphate-stimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin

Saponin, a cell-skinning reagent which perforates the cell membrane via its specific interaction with plasmalemmal cholesterol, was used to identify the subcellular origin of ATP-dependent Ca2+ accumulation in the presence and absence of inorganic phosphate and oxalate by microsomal fractions isolated from rat vas deferens and dog aorta. The purified plasma membranes from rat gastric fundus muscle, which elicit the stimulation of ATP-dependent Ca2+ accumulation by inorganic phosphate but not by oxalate, were used as a control reference. Saponin at concentrations effective for skinning smooth muscle fibres (10-50 micrograms/ml) inhibited Ca2+ binding in the absence of ATP to a similar extent in all fractions, but the inhibition of ATP-dependent Ca2+ accumulation was more pronounced in dog aorta microsomes and rat gastric fundus muscle plasma membranes than in rat vas deferens microsomes. The resistance of phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation to inhibition by saponin was much greater in rat vas deferens than in dog aorta microsomes. Our results suggest that phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation also occurs in plasma membrane vesicles isolated from smooth muscle and is by no means an unique property of endoplasmic reticulum.     

Acetate production from lactate and glucose fermentation by Gluconobacter oxydans

Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate.     

Seasonal distribution of enteroviruses and adenoviruses in domestic sewage

A seasonal distribution of enteroviruses and adenoviruses in raw sewage effluents of Athens, Greece, was observed over a 15-month surveillance period. All 36 samples tested were positive for both virus groups. Adenovirus concentration levels ranged from 70 to 3200 cytopathic units per litre of sample, whereas the corresponding values for enteroviruses were 90-900 cytopathic units per litre. Peak values for adenoviruses were recorded during the months of April and June 1983, whereas for enteroviruses the peak was recorded in September 1983. All three types of poliovirus were present. Coxsackievirus types B-1, B-2, B-4, and B-5 and echovirus type 7 were also isolated. Adenovirus types 1, 2, 3, 5, 7, and 15 were detected as well.     

Furosemide-sensitive Na+-K+ cotransport and cellular metabolism in human erythrocytes

Metabolic depletion of human red cells with 2-deoxy-D-glucose in the presence of EGTA decreased ATP to about 4% of the initial value and increased total ouabain- and furosemide-resistant Na+ and K+ effluxes by 20% and 100%, respectively, and furosemide-sensitive Na+ and K+ effluxes by 100% and 60%, respectively. When ATP was restored, all the components of Na+ and K+ fluxes measured returned to baseline levels suggesting a metabolic dependence.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed.     

Comparative in vitro and in vivo beta-oxidation of N-nitrosodiethanolamine in different animal species

The metabolism of N-nitrosodiethanolamine (NDELA) was studied to assess whether the formation of the beta-oxidated metabolites N-(2-hydroxyethyl)-N-(formylmethyl)nitrosamine (EFMN) and N-(2-hydroxyethyl)-N-(carboxymethyl)nitrosamine (ECMN) is involved in the mechanism of tumor induction in various animal species with different susceptibility to NDELA carcinogenicity. In vitro studies using liver S9 fractions from rats, hamster, B6C3F1 and CD-1 mice and rabbits showed that all the animal species metabolize NDELA through the beta-oxidation pathway, although to different extents. Urinary excretion of NDELA and its metabolite ECMN in rats, hamsters and mice after 5 mg X kg-1 NDELA i.p. confirmed these findings. The results suggest there is no correlation between carcinogenesis by NDELA and its beta-oxidation. The possibility that ECMN formation might represent a detoxifying metabolic pathway for NDELA is discussed.