NADH- and NADPH-linked aquacobalamin reductases occur in both mitochondrial and microsomal membranes of rat liver

To elucidate the mechanism for the synthesis of the coenzyme forms of cobalamin in mammals, rat liver aquacobalamin reductase was partially characterized. Rat liver contained both NADH- and NADPH-linked aquacobalamin reductases. The NADH-linked enzyme was distributed in the mitochondria (approx. 40%) and microsomes (60%), identical to the distribution of the NADPH-linked enzyme. The two mitochondrial NADH- and NADPH-linked enzymes were located inside of the outer membrane.     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Evidence for NADH- and NADPH-linked glutamate dehydrogenases in Trypanosoma cruzi epimastigotes.

Two glutamate dehydrogenases, NADH-linked (EC 1.2.1.2) and NADPH-linked (EC 1.2.1.4) were isolated from the epimastigote forms of Trypanosoma cruzi and purified. Both enzymes exist as hexamers. The molecular weights of the native NADH-and NADPH-linked glutamate dehydrogenases were estimated to be 360,000 and 265,000, respectively, and those of the subunits to be 58,000 and 43,000, respectively. The isoelectric point of the NADH-linked dehydrogenase is at pH 5.25 and that of the NADPH-linked enzyme at pH 5.1. The activities of both enzymes are regulated by product inhibition. In addition, purine nucleotides were shown to be potent inhibitors of the NADH-linked glutamate dehydrogenase.     

Evidence for NADH- and NADPH-linked Glutamate Dehydrogenases in Trypanosoma cruzi Epimastigotes*

SYNOPSIS Two glutamate dehydrogenases, NADH-linked (EC 1.2.1.2) and NADPH-linked (EC 1.2.1.4.) were isolated from the epimastigote forms of Trypanosoma cruzi and purified. Both enzymes exist as hexamers. The molecular weights of the native NADH-and NADPH-linked glutamate dehydrogenases were estimated to be 360,000 and 265,000, respectively, and those of the subunits to be 58,000 and 43,000, respectively. The isoelectric point of the NADH-linked dehydrogenase is at pH 5.25 and that of the NADPH-linked enzyme at pH 5.1. The activities of both enzymes are regulated by product inhibition. In addition, purine nucleotides were shown to be potent inhibitors of the NADH-linked glutamate dehydrogenase.     

NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes.

To elucidate the mammalian system for synthesis of cobalamin coenzymes, microsomal NADPH-linked aquacobalamin reductase was purified and characterized. The enzyme was purified about 534-fold over rat liver microsomal fraction in a yield of about 32%. The purified enzyme was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a monomeric molecular weight of 79,000. The purified aquacobalamin reductase showed a high specific activity (about 55 mumol/min per mg protein) of NADPH-cytochrome c (P-450) reductase. About 33% of the NADPH-cytochrome c reductase activity found in the microsomal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked aquacobalamin reductase was about 5.0 through the purification steps, indicating that the rat liver microsomal NADPH-linked aquacobalamin reductase is the NADPH-cytochrome c reductase.     

Distribution and properties of NADPH-linked aldehyde reductases from rat brain synaptosomes

Studies on the subcellular distribution of NADPH-linked aldehyde reductase from rat brain showed that 10% of the total reductase activity is located in the mitochondrial-synaptosomal fraction. There are differences in the percentages of reductase activity found in the synaptosomes compared to cytosol in various regions of the brain. The NADPH-linked aldehyde reductase from the synaptosomal fraction exhibited a nonlinear Lineweaver-Burk plot. This nonlinearity is due to the presence of two distinct aldehyde reductases, which can be distinguished by Michealis constants forp-nitrobenzaldehyde of 4.1×10^–5 M and 2.6×10^–6 M. The two NADPH-linked aldehyde reductases isolated from synaptosomes were further characterized according to pH optima, andK _i values for inhibition by barbiturates. In addition regional distributions for the two enzymes were determined. TheK _i values for pentobarbital for the highK _m enzyme and the lowK _m enzyme were estimated to be 2×10^–5 M and 6×10^–5 M, respectively. It was concluded from the above studies that the lowK _m reductase is probably responsible for 3,4-dihydroxyphenylglycoaldehyde (derived from norepinephrine) reduction in brain and a role of the highK _m enzyme for protection of neurons from high concentrations of chemically reactive aldehydes was proposed.This work was supported in part by Grants from the National Institute of Mental Health, MH 18948 from the University of Colorado Council on Research and Creative Work and by an MBS Program Grant #081-39.This work was performed in partial fulfillment of the requirements for the Ph. D. thesis.     

NADH- and NADPH-dependent formation of superoxide radicals in liver nuclei

A method for the quantitation of the superoxide radical generation rate (V) in murine liver nuclei by the oxidation of 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine O2-. radicals with the formation of a stable nitroxyl radical recorded by the EPR method, has been developed. It was shown that NADP- and NADPH-dependent superoxide radical generation is suppressed by superoxide dismutase (approximately by 90%). The Km values for NADH and NADPH are 1.5 x 10(-6) and 4.4 x 10(-7) M, respectively; the maximal rate (0.2 nmol.min-1.mg protein-1) is equal for both substrates. Cyanide (greater than 2 mM) causes a practically complete inhibition of the O2-. generation by both substrates. It is suggested that there exists a single readily autooxidized site of O2-. generation by both substrates for NADH- and NADPH-dependent site of the electron transport chain in nuclear membranes.     

Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils.

The subcellular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotaurine and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50:1. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiring cells by exposure to opsonized zymosan. These results prompted in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypotaurine competes with 5,5'-dimethyl-1-pyrroline N-oxide) (DMPO) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.     

Tonoplast subcellular localization of maize cytochrome b5 reductases

Plant cytochrome b5 reductases (b5R) are assumed to be part of an ER-associated redox chain that oxidizes NADH to provide electrons via cytochrome b5 (cyt b5) to ER-associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b5R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi et al., 1999, Biochem. J. 338, 499-505). Maize b5R isoforms are produced by a small multi-gene family. The NFR cDNAs were shown to encode active b5Rs by heterologous expression in yeast. Both reductases, in addition to Fe3+-chelates, efficiently reduced Cu2+-chelates. Using a polyclonal antibody able to recognize both NFR I and NFR II isoforms, no ER or mitochondrial localization could be detected in maize roots. Unexpectedly, maize b5Rs were found to be targeted to the tonoplast. Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activity is associated with tonoplast-enriched maize root fractions. Tonoplast targeting is not consistent with a role in desaturase reactions or with the other functions ascribed to date to plant b5R. This indicates that alternative ER-associated electron donors for desaturases need to be sought, and that plant b5Rs may have previously unexpected functions.     

Identification, expression pattern, and subcellular location of human RIP isoforms

XRIP alpha was identified as an adapter protein involved in RAP nuclear import. Several homologs were reported in mammal EST analysis, but the expression pattern and genomic organization of hRIP isoforms were not clarified yet. We isolated nine isoforms of hRIP from a premade human fetal brain library. hRIP alpha is the longest isoform with 219 residues, containing a N-terminal arginine-rich basic region, followed by an acidic region and two C-terminal Zn finger-like structures. hRIP beta deletes one Zn-finger-like structure. Three hRIP alpha isoforms and four hRIP sigma isoforms express truncated proteins due to frame shift. hRIP gamma isoforms lost the C-terminal Zn-finger-like structure. hRIP delta isoforms only contain the N-terminal arginine-rich basic region and the core sequence of the acidic region. The genomic organization of hRIP was identified by bioinformatic analysis. hRIP, containing seven exons, is located at human chromosome 17p13. hRIP was expressed in all 16 detected human tissues with a similar pattern. All EGFP-hRIP fusion proteins were located at the nucleus in the HEK293 cell. The two-polar molecular structure of hRIP might be involved in the basic cell function, and plays a role in the alternative nuclear ingress.     

Auxin biogenesis: subcellular compartmentation of indoleacetaldehyde reductases in cucumber seedlings

Subcellular fractionation of cucumber (Cucumis sativus L.) seedlings was achieved, and two of the enzymes in the auxin biosynthetic pathway were localized. NADH-specific indoleacetaldehyde reductase activity was observed only in the cytosol fractions obtained from separated hypocotyl and cotyledon tissue. In contrast, a portion of the NADPH-specific indoleacetaldehyde reductase activity was associated with a microsomal fraction derived from these tissues. The NADPH-specific indoleacetaldehyde reductase was consistently found to be more firmly associated with the microsomal fraction derived from hypocotyls than with that from the cotyledons. These results indicate a division of the terminal steps of auxin biogenesis into at least two subcellular compartments.     

Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases

Background  

Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments.     

Aldose and aldehyde reductases in human tissues

Immunochemical characterizations of aldose reductase and aldehyde reductases I and II, partially purified by DEAE-cellulose (DE-52) column chromatography from human tissues, were carried out by immunotitration, using antisera raised against the homogenous preparations of human and bovine lens aldose reductase and human placenta aldehyde reductase I and aldehyde reductase II. Anti-aldose reductase antiserum cross-reacted with aldehyde reductase I, anti-aldehyde reductase I antiserum cross-reacted with aldose reductase and anti-aldehyde reductase II antiserum precipitated aldehyde reductase II, but did not cross-react with aldose reductase or aldehyde reductase I from all the tissues examined. DE-52 elution profiles, substrate specificity and immunochemical characterization indicate that aldose reductase is present in human aorta, brain, erythrocyte and muscle; aldehyde reductase I is present in human kidney, liver and placenta; and aldehyde reductase II is present in human brain, erythrocyte, kidney, liver, lung and placenta. Monospecific anti-α and anti-β antisera were purified from placenta anti-aldehyde reductase I antiserum, using immunoaffinity techniques. Anti-α antiserum precipitated both aldehyde reductase I and aldose reductase, whereas anti-β antibodies cross-reacted with only aldehyde reductase I. Based on these studies, a three gene loci model is proposed to explain the genetic interrelationships among these enzymes. Aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α and β subunits and aldehyde reductase II is a monomer of δ subunits.     

Function and subcellular location of Ro52beta

Autoantigen Ro52alpha was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52beta, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52beta in contrast to Ro52alpha. Our biochemical assays revealed that both Ro52alpha and Ro52beta function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52alpha and Ro52beta are ubiquitinated when overexpressed with ubiquitin in HEK293T cells, suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52alpha mainly localizes to the cytoplasmic rod-like structures, whereas Ro52beta diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52alpha, the dimerization might be required for the localization of Ro52alpha to the rod-like structures. On the basis of these results, Ro52alpha and Ro52beta appear to ubiquitinate their particular substrates at different locations.