Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.5.3 (complex I)
8,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.
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PMID:The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme. 0 8

The hemocytes of the hard clam M. mercenaria were of three types: an agranulocyte, a small, and a large granulocyte. The agranulocyte, with only a thin periphery of cytoplasm surrounding the nucleus, had no visible cytoplasmic granules in living preparations but did exhibit a few centers of nonspecific esterase activity. This cell type represented 2% of the hemocyte population. The small granulocyte possessed four distinct granule types and comprised 61% of the total cell population. Large granulocytes accounted fro 37% of all hemocytes. While they contained the same four granule types identified in the small granulocyte, only one-third the total number were present. The nucleus of all three hemocyte types appeared morphologically similar. The four types of granules observed were a blunt, dot-like, a refractile and a filamentous granule. Blunt granules were identified as mitochondria, based on their ability to reduce Janus Green B to diethyl safranin, the presence of NADH dehydrogenase activity and boundary staining with Sudan black B. Dot-like granules were identified as lysosomes on the basis of neutral red staining, localization of acid phosphatase and nonspecific esterase activity and staining with Sudan black B. Refractile granules were demonstrated to be membrane-bound, lipid-filled structures that reacted positively with Sudan black B and Oil red O, respectively; these granules act as lipid storage centers. Nuclear similarity of the three cell types suggest that these cells might represent different stages of maturity, rather than three distinct cell lines. This was also indicated by the similar yet graded cytochemical reactions and the varying degree of motility and phagocytic activity demonstrated by hemocyte types.
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PMID:Cytochemical aspects of Mercenaria mercenaria hemocytes. 6 87

Measurement of certain membrane-bound enzymic activities was used to study the orientation of the outer membrane of the double-membraned forespore of Bacillus megaterium KM. 2. Adenosine triphosphatase, NADH dehydrogenase and L-malate intact protoplasts, but were readily detected in intact stage II or IV forespores, consistent with reversed polarity of the outer forespore membrane relative to the mother-cell plasma membrane. 3. Measurement of NADH oxidase activity revealed that intact stage III forespores had the same high affinity for NADH as protoplast membrane preparations and protoplast lystates, consistent with ready access of NADH to oxidation sites on the outer forespores membrane. 4. Forespores and protoplasts showed osmometric behaviour in solutions of non-permanent solutes consistent with the presence of an intact permeability barrier in these structures.
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PMID:Biochemical evidence for the reversed polarity of the outer membrane of the bacterial forespore. 13 69

The phospholipid requirement of membrane-bound enzymes may depend on several reasons. In our laboratory we have investigated lipids (1) as a bidimensional medium required for the movement of Coenzyme Q, a lipid-soluble cofactor of the mitochondrial respiratory chain, and (2) as a hydrophobic environment necessary to impose the proper conformation to membrane-bound enzymic proteins. We have found that Coenzyme Q, once reduced by NADH dehydrogenase, must cross the inner mitochondrial membrane; only quinones having long isoprenoid side chains can easily cross phospholipid bilayers, and this is the reason why a short chain quinone such as CoQ-3 inhibits NADH oxidation. The incapability of short quinones to cross lipid bilayers is due to their disposition in the lipid bilayer, stacked within the phospholipids. The conformational role of lipids has been investigated indirectly observing the kinetics of membrane-bound enzymes, e.g. the mitochondrial ATPase, and directly by circular dichroism. Lipid removal or lipid perturbation with organic solvents induce a decrease of alpha-helical content in mitochondrial proteins, and give rise to a series of kinetic changes in ATPase, including uncompetitive inhibition, increased activation energy, and loss of cooperativity in oligomycin inhibition. The recognition of a conformational role of lipids has allowed us to postulate a working hypothesis for the mechanism of action of general anesthetics. Such drugs have been found by us, by means of spin labels and fluorescent probes, to disrupt lipid protein interactions in several membranes, including synaptic membranes. The loosening of such interactions is believed to induce conformational changes, which will alter ion transport systems necessary to the propagation of neural impulses. Conformational changes induced by anesthetics have been found by us both directly by circular dichroism and indirectly by enzyme kinetics. The conformational effect of anesthetics is not directly exerted on the proteins but is mediated through the lipids. In agreement with this hypothesis we have found that membrane-bound acetylcholinesterase is inhibited by anesthetics, whereas the solubilized enzyme is not inhibited. However, binding of the solubilized enzyme to phospholipids restores anesthetic inhibition.
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PMID:Biophysical studies on agents affecting the state of membrane lipids: biochemical and pharmacological implications. 15 58

An NADH dehydrogenase possessing a specific activity 3-5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0% ethanol at 43 degrees C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1:2 FAD to FMN and 30-40% lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O2, ubiquinone Q10 or cytochrome c. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0% ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar Km's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller V's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg2+, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O2 uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.
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PMID:The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes. 17 76

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.
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PMID:Functional mosaicism of membrane proteins in vesicles of Escherichia coli. 19 Feb 12

The site of Na+-dependent activation in the respiratory chain of the marine bacterium, Vibrio alginolyticus, was investigated. The respiratory chain system contained ubiquinones (Q), menaquinones (MK), cytochromes b(560), c(553), d(630), and o(560). The membrane-bound and partially purified NADH dehydrogenase was stimulated 2- to 3-fold by the addition of 0.2 M Na+ or K+ and no specific requirement for Na+ was observed in this reaction step. The cytochrome oxidase showed no requirement for monovalent cations. The respiratory activity (NADH oxidase) of the membrane was lost on removal of the quinones, and the reincorporation of authentic Q-10 or MK-4 restored the activity. The rate of MK-4 reduction by NADH (menaquinone reductase) as measured using MK-4 incorporated membrane was activated by Na+, but only slightly by K+. The apparent Ka for Na+ was 78 mM for both menaguinone reductase and NADH oxidase. The requirement for Na+ of menaquinone reductase was greatly reduced in the presence of 0.2 M K+. Ubiquinone reductase as measured by using Q-10 incorporated membrane was also activated more effectively by Na+ than by K+. These results strongly suggested that the site of Na+-dependent activation in the respiratory chain of marine V. alginolyticus was at the step of NADH; quinone oxidoreductase.
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PMID:NADH: quinone oxidoreductase as a site of Na+-dependent activation in the respiratory chain of marine Vibrio alginolyticus. 45 42

A membrane-bound NADH dehydrogenase, solubilized and partially purified from a marine bacterium Photobacterium phosphoreum, contains FAD as the prosthetic group, and is specific for NADH. Ferricyanide, various other redox dyes and cytochrome c can act as electron acceptors. The enzymatic activity when assayed with electron acceptors other than cytochrome c, is activated by monovalent cations (Na+ and K+) and deactivated by high concentrations of monovalent anions (SCN-, NO3-, and Cl-) but not by phosphate ions. The enzymatic reaction follows a ping-pong mechanism and kinetic analysis of the enzyme showed that the activation by monovalent cations is due to increase of affinity of the enzyme for substrates; Vm was not affected. The increase of affinity was 62- and 46-fold for NADH and 57- and 31-fold for 2,6-dichlorophenol indophenol in the presence of Na+ and K+, respectively. On the other hand, NADH-cytochrome c reductase activity of the enzyme was strongly inhibited by these cations.
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PMID:Properties and kinetics of salt activation of a membrane-bound NADH dehydrogenase from a marine bacterium Photobacterium phosphoreum. 72 93

The NADH dehydrogenase of the Escherichia coli respiratory chain has been identified by the following properties: (a) its location in membrane vesicles; (b) its inhibition by AMP in a fashion similar to that of the NADH oxidase; (c) its specificity for NADH, but not NADPH, with the same Km for NADH as that of the NADH oxidase; (d) its sensitivity when membrane-bound to inhibition by dicoumarol, rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide, which are also inhibitors for the NADH oxidase. The NADH-dehydrogenase of the cytosol fraction (assayed as NADH-dichlorphenolindophenol reductase activity) differs substantially from the membrane-bound activity both in substrate specificity and in the inhibitors of the reaction. The respiratory chain NADH dehydrogenase was extracted from isolated membrane vesicle preparations by solubilization in Triton X-100, and was purified in buffers containing that detergent. The purification employed chromatography on DEAE-cellulose, precipitation by 30% ethanol, and chromatography on hydroxyalapatite and DEAE-agarose. The most highly purified preparations of the enzyme were homogeneous in migration on polyacrylamide gels containing Triton X-100, at pH 9.5, where one band accounted for all of the protein and activity. Electrophoresis on polyacrylamide gels containing sodium dodecul sulfate showed 1 band of molecular weight 38,000, which accounted for over 75% of the protein on the gel. Because of requirements for either Triton X-100 or phospholipid for activity of the purified enzyme, it is difficult to estimate the level of purification achieved over isolated membrane vesicles. However, we estimate that the enzyme was purified some 30-fold over membrane vesicles, or some 300-fold over whole cells.
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PMID:The NADH dehydrogenase of the respiratory chain of Escherichia coli. I. Properties of the membrane-bound enzyme, its solubilization, and purification to near homogeneity. 78 86

The ethanol-extracted respiratory chain-linked NADH dehydrogenase of Acholeplasma laidlawii has been purified 25-35-fold. This purification involved delipidation of the ethanol-extracted minute non-sedimentable membrane fragments by detergent treatment and gel filtration on Bio-Gel P-200. Sodium deoxycholate-sucrose density gradient centrifugation was followed by dialysis of the active NADH dehydrogenase fractions which caused flocculation of 60% of the membrane proteins while the NADH dehydrogenase remained suspended. Poylacrylamide gel electrophoresis of the purified NADH dehydrogenase gave one major and two minor bands after staining with Coomassie Blue. The purified enzyme gave straight line kinetics in Lineweaver-Burk plots and a Km = 0.510 mM and V = 0.236 mumol/min. Fatty acid supplementation of A. laidlawii membranes had negligible effect on the membrane-bound or ethanol-extracted dehydrogenase, but substantiated the values of the Km and V. Purification, however, altered the constants by 2-4-fold, suggesting that alteration of the microenvironment or fragmentation of the dehydrogenase was significant. The purified dehydrogenase was very susceptible to a rapid inhibition was much slower (90 min) and less complete. Consideration of published purification procedures of NADH dehydrogenase strongly suggested that the purified A. laidlawii respiratory chian-linked NADH dehydrogenase was over 90% pure and certainly one of the most purified respiratory chain-linked bacterial NADH dehydrogenases.
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PMID:Purification of the reduced nicotinamide adenine dinucleotide dehydrogenase from membranes of Acholeplasma laidlawii. 99 Mar 15


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