Catalytic properties of NADPH-specific dihydropteridine reductase from bovine liver

The catalytic properties of a new type of dihydropteridine reductase, NADPH-specific dihydropteridine reductase [EC 1.6.99.10], from bovine liver, were studied and compared with those of the previously characterized enzyme, NADH-specific dihydropteridine reductase [EC 1.6.99.7]. With quinonoid-dihydro-6-methylpterin, approximate Km values of NADPH-specific dihydropteridine reductase for NADPH and NADH were estimated to be 1.4 micron and 2,900 microns, respectively. The Vmax values were 1.34 mumol/min/mg with NADPH and 1.02 mumol/min/mg with NADPH. With NADPH, the Km values of the enzyme for the quinonoid-dihydro forms of 6-methylpterin and biopterin were 1.4 micron and 6.8 microns, respectively. The enzyme was inhibited by its reaction product, NADP+, in a competitive manner, and the inhibition constant was determined to be 3.2 microns. The enzyme was severely inhibited by L-thyroxine and by 2,6-dichlorophenolindophenol.     

Random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsomes

The dilution of rabbit liver microsomes by soy-bean phospholipids was used as methodical approach to investigate the molecular organization of NADPH-dependent microsomal redox chain. The ultrastructural analysis of control and phospholipid diluted microsomes revealed that the incorporation of exogenous phospholipids into microsome membranes increased their surface area, as well as decreased the lateral density distribution and size of intramembrane particles. The dilution of microsome membranes by phospholipids slowed down the initial rate of cytochrome P-450 reduction by NADPH. The apparent second order rate constant of cytochrome P-450 reduction by NADPH: cytochrome P-450-reductase did not change in phospholipid-enriched microsomes. The results obtained provide strong evidence for the random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsome membranes.     

Identification of NADH-specific and NADPH-specific FMN reductases in Beneckea harveyi.

Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH-specific enzyme is more efficient than the NADPH-specific one with respect to the bioluminescent reaction.     

Purification and physicochemical properties of NADPH-specific dihydropteridine reductase from bovine and human livers

A new type of dihydropteridine reductase [EC 1.6.99.10], which is specific for NADPH as the substrate in the reduction of quinonoid-dihydropterin to tetrahydropterin, was purified to homogeneity from bovine liver and human liver. The molecular weight of the enzyme was determined to be 65,000-70,000. The enzyme was composed of two subunits with identical molecular weight of 35,000; the amino terminal residue was determined to be valine. The isoelectric point of the enzyme was 7.05. The physicochemical properties of this enzyme were quite different from those of bovine liver NADH-specific dihydropteridine reductase [EC 1.6.99.7]. NADPH-specific dihydropteridine reductase did not cross-react with an antiserum raised against the NADH-specific dihydropteridine reductase, nor did the latter enzyme react with an antiserum to the former enzyme, indicating that the two enzymes have no common antigenic determinants. NADPH-specific dihydropteridine reductase from human liver was shown to have properties similar to those of the bovine liver enzyme.     

Reconstitution of liver monooxygenase system in solution from cytochrome P-450 and NADPH-specific flavoprotein monomers

Microsomal monooxygenase system was reconstituted in the presence of non-ionic detergent Emulgen 913 from cytochrome P-450 and NADPH-specific flavoprotein isolated from phenobarbital-induced rabbit liver microsomes. At Emulgen 913 concentration of 0.05 g/l mixed complex between flavoprotein and cytochrome was formed with 5: 5 protein molar ratio and molecular weight of 700 kD. The 2-hour incubation of the enzymes with 0.25 g/l Emulgen 913 at 4 degrees C was accompanied by dissociation of protein oligomers to monomers. The reconstituted systems containing flavoprotein and cytochrome as mixed complexes or monomers were able to catalyze NADPH-dependent cytochrome P-450 reduction and benzphetamine N-demethylation. Taking into consideration the effective concentrations of the enzymes the apparent second order rate constants of these reactions with monomers were 100 times those with complexes.     

Microsomal hemoprotein reduction by superoxide radical formed on NADPH-specific flavoprotein]

The superoxide radicals formed on NADPH-specific flavoprotein of liver microsomes can reduce cytochromes c, b5, and P-450. This reaction is inhibited under aerobic conditions by a low molecular weight analog of superoxide dismutase, e.g. the copper-tyrosine complex. The inhibitory effect of the complex is not observed under anaerobic conditions. Based on the results obtained a scheme of the electron transfer between the flavoprotein and haemoproteins involving superoxide radicals is proposed.     

NADPH-specific and NADH-specific xylose reduction is catalyzed by two separate enzymes in Pachysolen tannophilus

Summary Pachysolen tannophilus contains — in addition to an NADPH-linked xylose reductase — a separate NADH-linked one, in this respect differing from the yeast Pichia stipitis. Both enzyme proteins can conveniently be separated from each other by either ion exchange chromatography or chromatofocusing.     

Interactions of some acceptors with superoxide anion radicals formed by the NADPH-specific flavoprotein in rat liver microsomal fractions.

In rat liver microsomal fractions oxidation of adrenaline was effected by superoxide anion radicals (O2-), whereas cytochrome c, 2,6-dichlorophenol-indophenol and ferricyanide accepted electrons from NADPH-specific flavoprotein only directly. Nitro Blue Tetrazolium was reduced both by O2- and by the direct acceptance of electrons. Elevation of pH and addition of menadione shift the Nitro Blue Tetrazolium reduction towards the O2--dependent pathway. From the values of the kinetic constants for interaction of adrenaline and Nitro Blue Tetrazolium with NADPH-specific flavoprotein, the rates of generation of O2- in rat liver microsomal fraction were determined.     

Purification of NADPH-specific indole-3-acetaldehyde reductase from Cucumis sativus by two-dimensional native polyacrylamide gel electrophoresis

NADPH-specific indole-3-acetaldehyde (IAAId) reductase from cucumber ( Cucumis sativus L. 相似文献   

Evidence for a second microsomal trans-2-enoyl coenzyme A reductase in rat liver. NADPH-specific short chain reductase

Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase has a specific requirement for NADPH, is unable to utilize NADH, and catalyzes the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic acid at a rate of 5 and 65 nmol per min per mg of microsomal protein, respectively. Highly purified NADPH cytochrome P-450 reductase incorporated into liposomes prepared from dilauroyl phosphatidylcholine in the presence or absence of cytochrome P-450 possesses no SC reductase activity. These liposomal preparations did, however, catalyze mixed function oxidations of benzphetamine and testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450 reductase had little to no effect on the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts reducing equivalents directly from NADPH. When acetoacetyl-CoA was incubated with hepatic microsomes and either NADH or NADPH, no formation of butyrate was detected; however, when both cofactors were present, a rate of formation of 3 nmol of butyrate was determined per min per mg of microsomal protein. These results suggest the presence of a previously unknown short chain beta-ketoreductase which catalyzes the reduction of short chain beta-keto acids, only in the presence of NADH. Our results also indicate that the electrons from NADH to the beta-ketoreductase bypass cytochrome b5. The physiological significance is discussed in terms of lipogenesis and ketone body utilization by the liver.     

Determination of NADPH-specific dihydropteridine reductase in extract from human, monkey, and bovine livers by single radial immunodiffusion: selective assay differentiating NADPH- and NADH-specific enzymes

It has been difficult to determine exactly NADPH-specific dihydropteridine reductase [EC 1.6.99.10] in samples which also contain NADH-specific dihydropteridine reductase [EC 1.6.99.7], because the latter enzyme interferes with the activity measurement of the former. We have devised a method to measure selectively the NADPH-specific reductase in crude extracts of bovine, human and monkey livers by the single radial immunodiffusion method using specific antiserum against the enzyme. This method makes it possible to determine the enzyme amount in 5 microliters of the 3-volume extracts of the livers. The amounts of NADPH-specific dihydropteridine reductase were calculated to be 0.252, 0.296, and 0.583 munits/5 microliter of the extracts of bovine, human, and monkey livers, respectively.     

Improving isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with over-expressing NADPH-specific glucose-6-phosphate dehydrogenase (Zwf1)

Isobutanol is a more promising biofuel than ethanol due to its higher energy density and lower hygroscopicity. Saccharomyces cerevisiae, as a model eukaryote, has the potential advantage to produce isobutanol because of its greater tolerance to higher alcohols. NADPH is a key cofactor for isobutanol synthesis, and glucose-6-phosphate dehydrogenase (Zwf1) is one of the main NADPH-supplying sources in S. cerevisiae. In this study, we investigated the effects of over-expressing ZWF1 on isobutanol titers. Our results showed that engineered strain HZAL-7023 produced 6.22 mg isobutanol per g glucose, which increased by 6.64-fold compared with the parent strain, while engineered strain HZAL-7023 22-ZWF1 produced 11.46 mg isobutanol per g glucose, which increased by 1.82-fold compared with engineered strain HZAL-7023. These results suggested that improvement of NADPH supply through over-expressing ZWF1 contributed to isobutanol biosynthesis in S. cerevisiae. These results also verified the proposed concept of increasing isobutanol titers in S. cerevisiae by resolving cofactor imbalance. Finally, this study provides a new strategy for enhancing isobutanol biosynthesis.     

Anthropology and genetics of the caucasus peoples and the problem of the origin of the caucasoids

The electrophoretical polymorphisms of some blood proteins were studied in the Talysh population of Pirasora situated in South-East Azerbaidjan. We calculated the gene frequencies of these polymorphisms and determined the genetic distances between the Talyshes and some Iranian populations of North, Central and South Iran, Afghans, and three populations of Azerbaijan. The Talyshes are very close to Iranians of Shiraz, whereas they are distant from the Azerbaijanians. Anthropological investigations showed that the Caucasoids and Mongoloids lived in the Aragvi Basin since the Eneolithic period. This was stated by Alexeev (1974), who emphasized the mixture of the Caucasus populations from ancient times on. We calculated the genetic distances between the Caucasus populations and numerous populations of other geographic regions, considering 28 alleles of 12 loci of blood group, serum protein and red cell enzyme polymorphisms and constructed the dendrogram of these populations. The position of the Caucasus populations in the dendrogram corresponds on principle to the earlier anthropological observations. The clustering of the Caucasoid populations corresponds completely with anthropological and historical data, and supports our earlier hypothesis (Nazarova 1999) concerning the differentiation of Caucasoids, Northern Mongoloids and Amerinds from the populations, which inhabitated Asia in palaeolithic times.     

On the structure and dynamics of the complex of the nucleosome and the linker histone

Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.     

Kinematics and dynamics of the temporomandibular joint and of the movements of the mandible

In order to analyze the complicated movements of the mandible as the open-closing movement and the protrusio are, it is useful to evaluate the basic kinematic principles and reduce them to simple technical constructions. Both the open-closing movement and the protrusio could be reduced to 4-bar links, which were used to simulate the movements with help of a computer. Besides, the polodes and the curves of points in the muscular attachments could be constructed. The 2 entirely different 4-bar links have 3 things in common: The resting system - cranium, the moving system - mandibula, and 1 of the 2 arms connecting these 2 systems - the ligamentum laterale. As this ligament is taut during movements it can be considered a "guiding ligament" representing 1 of the 3 determining components of the mandibular movements. The other of the 2 arms has no anatomical equivalent; this arm, however, is "replaced" by the 2 other determining components of the mandibular movements: the joint and the muscles. The curves, which the Caput mandibulae describes, are practically identical for the open-closing movement and the protrusio despite of the different 4-bar links and these curves exactly correspond to the Discus articularis, taut by the upper part of the M. pterygoideus lateralis. The muscles do not only just move the mandibula, but they are also the component, which can choose between the different mandibular movements. By means of the curves, which points in the muscular attachments describe, the function of the masticatory muscles could be analyzed exactly.     

Biophysics and Structure of the Patch and the Gigaseal

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 μm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E_a), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer (∼10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E_a seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E_a and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E_a and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of ∼7 gigohm/μm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E_a and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.     

Serology and systematics of the Ebenales and the Theales

The systematic position ofthe Ebenaceae, Sapotaceae, Styracaceae, Ochnaceae, Stachyuraceae, Dipterocarpaceae, Clusiaceae and Hypericaceae has been investigated using serological comparisons of sets of antigenic determinants. The results show that the Sytracaceae and Sapotaceae are undoubtedly more closely associated with the Actinidiaeceae and Theaceae, respectively, than with each other. We found no corresponding determinants betnween antigen systems from the Ebenaceae and systems from any other family whose relations to this family have been proposed. As discovered previously, investigations of antigen systems from the Ochnaceae, Dipterocarpaceae, Stachyuraceae, Clusiaceae and Hypericaceae are against the idea of a natural order “Theales” in which these families, or at least some of them, are combined with the Theaceae and Actinidiaceae. This paper completes our previous investigations which largely support a superorder Ericanae sensu Ehrendorfer and Takhtajan. We propose to include the Actinidiaceae and Theaceae in this superorder, assigning them a central position laong with the Sapotaceae and Sytracaeae on one side and the Primulales and Ericales on the other. Another most interesting finding is that there are corresponding determinants between antigen systems from the members of the Ericanae and representatives of the Polemoniaceae and Loasaceae.