Isolation and Partial Characterization of the Membrane-Bound NADH Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

Abstract Chemotrophically grown cells of Rhodopseudomonas capsulata contain at least three different pyridine nucleotide dehydrogenases, i) a soluble, found in the supernatant (144000 × g) of cell free extracts, NADH-dependent, ii) a mem brane-bound, NADH-dependent, and iii) a soluble, found in the supernatant N AD PH dependent. The membrane-bound NADH dehydrogenase (E.C. 1.6.99.3) has been solubilized by sodium deoxycholate treatm ent of m em branes and purified 75 fold by column chrom atography on Sephadex G-150 and DEAE cellulose in the presence of sodium cholate. The native enzyme has an apparent molecular mass (Mr) of 97 000, containing polypeptides of Mr of about 15 000. The pH optim um was at 7.5. The enzyme was specific for NADH. The Michaelis constant for NADH and DCIP were 4.0 and 63 μm, respectively. The enzyme was inactivated by FMN, riboflavin and NADH. In contrast, the soluble NADH-dehydrogenase (i) was activated by FMN.

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Purification and Partial Characterization of the Soluble NADH Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

Zeitschrift für Naturforschung C ◽

10.1515/znc-1984-1-212 ◽

1984 ◽

Vol 39 (1-2) ◽

pp. 68-72 ◽

Cited By ~ 4

Author(s):

Toshihisa Ohshima ◽

Matsumi Ohshima ◽

Gerhart Drews

Keyword(s):

Cytochrome C ◽

Nadh Dehydrogenase ◽

Rhodopseudomonas Capsulata ◽

Subunit Structure ◽

Ph Optimum ◽

Single Polypeptide Chain ◽

Membrane Bound ◽

Single Polypeptide ◽

Sepharose 4B

Abstract Soluble NADH dehydrogenase was purified to homogeneity from chemotrophically grown cells of Rhodopseudomonas capsulata by ammonium sulfate fractionation, AH -Sepharose 4B chromatography and FMN-Sepharose 6B affinity chromatography. The enzyme contains a single polypeptide chain of an apparent M, of 37000, suggesting that the subunit structure is different from that of the membrane-bound enzyme. The purified soluble NADH dehydrogenase requires flavin compounds, e.g., FMN, FAD and riboflavin, for activity. Addition of FMN and FAD. but not riboflavin, to the enzyme solution stabilized the enzyme. The pH optimum for activity was at 7.5. The enzyme was specific for NADH as an electron donor while NADPH was inert. Menadione, ferricyanide, cytochrome c and DCIP served as an electron acceptor. The M ichaelis constants for NADH. DCIP, FM N. and cytochrome c were 45, 2.9. 7.9 and 15 μM, respectively. Many properties of soluble NADH dehydrogenase were substantially different from those of the membrane-bound enzyme, suggesting different functions.

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Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes

Biochemical Journal ◽

10.1042/bj1630211 ◽

1977 ◽

Vol 163 (2) ◽

pp. 211-217 ◽

Cited By ~ 26

Author(s):

I S Trowbridge ◽

M Nilsen-Hamilton ◽

R T Hamilton ◽

M J Bevan

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Membrane Vesicles ◽

Sodium Deoxycholate ◽

Pea Lectin ◽

Preliminary Characterization ◽

Low Concentrations ◽

Membrane Bound

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.

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Hydrolysis of L-Histidine Methyl Ester

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651211 ◽

1968 ◽

Vol 19 (03/04) ◽

pp. 321-333

Author(s):

E. R Cole

Keyword(s):

Methyl Ester ◽

Esterase Activity ◽

Sodium Citrate ◽

Deae Cellulose ◽

Sodium Cholate ◽

Michaelis Constant ◽

Bovine Thrombin ◽

Optimum Ph ◽

Autoprothrombin C ◽

Hydrolysis Of

SummaryThe hydrolysis of L-histidine methyl ester (HME) by bovine thrombin preparations has been investigated. Activation of purified bovine prothrombin in 25% sodium citrate solution resulted in the simultaneous development of fibrinogen clotting activity and of TAME and HME esterase activities. Most of the HME esterase activity was identified with fibrinogen clotting activity on sequential chromatography of activated prothrombin on DEAE-cellulose and Amberlite CG-50 resin columns, although some HME esterase activity could be demonstrated in concentrates of the autoprothrombin C fraction. The optimum pH for HME hydrolysis by thrombin was found at 7.6 in phosphate and Tris buffered reactions. Tris buffer and other amines depress HME esterase activity of thrombin, while sodium cholate accelerates the reaction. The Michaelis constant, Km, was estimated to be 0.134 M at pH 7.6 in phosphate buffer and at 37° C.

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