The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Anaerobic respiration in the Rhodospirillaceae: characterisation of pathways and evaluation of roles in redox balancing during photosynthesis

Abstract Recent discoveries relating to pathways of anaerobic electron transport in the Rhodospirillaceae are reviewed. The main emphasis is on the organism Rhodobacter capsulatus ** but comparisons are made with Rhodobacter sphaeroides ** f. sp. denitrificans and Rhodopseudomonas palustris . The known electron acceptors for anaerobic respiration in Rhodobacter capsulatus are trimethylamine- N -oxide (TMAO), dimethyl sulphoxide (DMSO), nitrate and nitrous oxide. In each case respiration generates a proton electrochemical gradient and in some cases can support growth on non-fermentable carbon sources. However, the principal objective of this review is to discuss the possibility that, apart from a role in energy conservation, anaerobic respiration in the photosynthetic bacteria may have a special function in maintaining redox balance during photosynthetic metabolism. Thus the electron acceptors mentioned above may serve as auxiliary oxidants: (a) to maintain an optimal redox poise of the photosynthetic electron transport chain; (b) to provide a sink for electrons during phototrophic growth on highly reduced carbon substrates.
Molecular properties of the nitrate reductase, nitrous oxide reductase and a single enzyme responsible for reduction of TMAO and DMSO are discussed. These enzymes are all located in the periplasm. Electrons destined for all three enzymes can originate from the rotenone-sensitive NADH dehydrogenase but do not proceed through the antimycin- and myxothiazol-sensitive cytochrome b/c₁ complex. It is likely, therefor, that the pathways of anaerobic respiration overlap with the cyclic photosynthetic electron transport chain only at the level of the ubiquinone pool. Redox components which might be involved in the terminal branches of anaerobic respiration are discussed.     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide     

Monoclonal antibodies to the two most basic papaya proteinases

The proteinases fromCarica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.     

Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells

BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.     

Change in cough reflex after treatment with enalapril and ramipril.

OBJECTIVE--To find out whether enalapril or ramipril causes the sensitivity of the cough reflex to change or symptomatic cough to develop in patients with hypertension. DESIGN--Prospective, placebo controlled, double blind, randomised crossover study. SETTING--Academic units of clinical pharmacology and medicine. PATIENTS--20 Patients (nine men and 11 women) who needed to take angiotensin converting enzyme inhibitors to control hypertension. INTERVENTIONS--All patients received enalapril 10 mg daily, ramipril 10 mg daily, or placebo daily for one week in random order, with a washout period of at least one week between treatments. For assessment of sensitivity of the cough reflex the patients inhaled various concentrations of capsaicin solution in random order. MAIN OUTCOME MEASURES--Measurement of the doses of capsaicin required to cause two or more and five or more coughs or the development of a symptomatic cough. RESULTS--Blood pressure, symptoms of cough, and the sensitivity of the cough reflex to inhaled capsaicin were recorded at the start of the study and before and at the end of each treatment period. Plasma urea and creatinine concentrations and angiotensin converting enzyme activity were measured at the start of the study and the end of each treatment period. Data were analysed by two way analysis of variance. Mean blood pressure was 159/97 mm Hg at the start of the study and 152/92, 143/88, and 147/86 mm Hg after treatment with placebo, enalapril, and ramipril respectively. Mean (SE) plasma angiotensin converting enzyme activity was 2.2 (0.2) mmol/l/h after treatment with placebo and fell significantly to 1.3 (0.1) mmol/l/h and to 0.4 (0.1) mmol/l/h after treatment with enalapril and ramipril respectively. No patient complained of cough while taking placebo but three women complained of cough when taking both enalapril and ramipril. The mean (95% confidence interval) lowest dose of capsaicin causing two or more coughs was 2.4 (1.5 to 4.0), 1.8 (1.12 to 2.82), and 2.2 (1.7 to 3.0) nmol after treatment with placebo, enalapril, and ramipril respectively; none of these changes were significant. The lowest dose of capsaicin causing five or more coughs was 18.9 (13.9 to 25.8), 14.4 (8.4 to 24.5), and 15.3 (10.8 to 21.2) nmol respectively; none of these changes were significant. The three patients who complained of cough had normal sensitivity to capsaicin after treatment with placebo but had a considerably increased sensitivity after treatment with enalapril and ramipril. CONCLUSIONS--Both enalapril and ramipril increase the sensitivity of the cough reflex appreciably in patients who complain of cough during treatment, but they do not change the se     

An estradiol-dependent protein from chicken liver binds single-stranded DNA and RNA

We have identified two estradiol-dependent single-stranded DNA binding proteins in the nucleus and cytoplasm of chicken hepatocytes that bind the sequence 5'TCACCTTCGCTATG3' in the first exon of the chicken vitellogenin gene. As judged by chromatography on heparin-Sepharose and by proteolytic clipping bandshift assay, the two proteins are different. Furthermore, they only bind to the oligonucleotide corresponding to the upper strand. Depurination and depyrimidination interference experiments with the cytoplasmic protein show that the bases CCTT-G are involved in the protein-DNA interaction. An RNA corresponding to the upper strand of the gene between nucleotide positions -73 and +53 competes for binding to the single-stranded DNA. UV cross-linking experiments performed with bromouridine-substituted single-stranded RNA reveal that an estradiol-dependent hepatocyte cytoplasmic protein with a Mr of 71,000 binds to the mRNA-like single-stranded RNA.     

L-Alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient

In human Caco-2 intestinal epithelial layers, xxxl-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na⁺-free media, is associated with a saturable net transepithelial absorption of xxxl-alanine. xxxl-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. xxxl-Alanine transport in Na⁺-free media is rheogenic, stimulating an inward short-circuit current in voltageclamped epithelial monolayers. By measurement of rapid xxxl-alanine influx across the apical membrane, xxxl-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate xxxl-alanine/proton stoichiometry to be 10.62 ±0.25 (xxxsd) (short-circuit current) or 10.73 ±0.19 (intracellular acidification). From competition studies, it is likely that xxxl-proline, -aminoisobutyric acid, and -alanine, but not xxxl-valine and xxxl-serine, are substrates for protonlinked, substrate transport in the brush border of Caco-2 cells.This study was supported by the Wellcome Trust (to D.T.T. and N.L.S.) and the LINK Programme in Selective Drug Delivery and Targeting (funded by the SERC/MRC/DTI and Industry). Charlotte Ward gave excellent technical assistance.