Inhibition of embryonic retinoic acid synthesis by aldehydes of lipid peroxidation and prevention of inhibition by reduced glutathione and glutathione S-transferases

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.     

Molecular cloning, characterization, and potential roles of cytosolic and mitochondrial aldehyde dehydrogenases in ethanol metabolism in Saccharomyces cerevisiae

The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K+ ions. In contrast, the activity of ALDH5 increased in the presence of K+ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption delta ald1 and delta ald2 strains on ethanol was marginally slower than that of the parent strain. The delta ald1 delta ald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.     

Stereospecificity of hydrogen transfer by the NADP-linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii

Class A and class B NAD(H)/NADP(H) coenzyme-dependent dehydrogenases distinguish between the diastereotopic hydrogens pro-R and pro-S at position 4 of the cofactor. We investigated the stereochemistry of hydride transfer in reactions catalyzed by an unusual thermophilic, zinc-containing, NADP-linked enzyme Thermoanaerobium brockii alcohol dehydrogenase (TBAD). Using proton NMR spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers the pro-R hydrogen from the pyridine 4 position of the reduced coenzyme. This stereospecificity is stable over (a) a broad range of temperatures up to 70 degrees C, (b) different concentrations of the coenzyme (catalytic or stoichiometric) and (c) a wide scope of substrates. Although NAD+ is not an effective coenzyme for TBAD, NADP+ and its synthetic analogs, 3-acetylpyridine-ADP+ and thio-NADP+, can be used successfully.     

Involvement of conserved glycine residues, 229 and 234, of Vibrio harveyi aldehyde dehydrogenase in activity and nucleotide binding

The involvement of two conserved glycine residues (Gly229 and Gly234) in activity and nucleotide binding in Vibrio harveyi aldehyde dehydrogenase (ALDH) have been investigated. Each of the glycine residues has been mutated to alanine and the mutant ALDHs have been expressed in Escherichia coli and specifically labelled with [35S]methionine. The G229A mutant was inactive with either NADP+ or NAD+ as coenzyme and did not bind to 2',5'-ADP Sepharose, indicating a complete loss of nucleotide affinity. In contrast, the G234A mutant showed a high affinity for 2',5'-ADP Sepharose. Purified G234A mutant showed similar kinetic properties to the native enzyme including a pre-steady-state burst of NADPH; however, the Michaelis constants for NAD+ and NADP+ were increased by 3- to 9-fold, showing that the mutation had an effect on saturation of the enzyme with NAD(P)+. These data are consistent with the structure for the nucleotide binding domain of Vh.ALDH being similar to that of class 3 or class 2 mammalian ALDHs which differ from the classical nucleotide binding domain found in most dehydrogenases.     

Mitochondrial malic enzymes. An association between NAD(P)+-dependent malic enzyme and cell renewal in Sprague-Dawley rat tissues

The activities of the mitochondrial NAD(P)+- and NADP+-dependent malic enzymes were measured in 11 tissues of the male Sprague-Dawley rat. The NAD(P)+-dependent malic enzyme was present in small intestinal mucosa, spleen, thymus, lung, and testis. Each of these tissues contain cells that are undergoing active rates of renewal. The NADP+-dependent malic enzyme was not confined to tissues undergoing cell renewal, and was present in mitochondria from brain, skeletal, and heart muscle, kidney, and lung and testis. Both enzymes were absent or at a low activity in normal and regenerating liver. The results support, and extent to nonneoplastic tissues, our proposal that cells which show active and sustained rates of renewal contain the NAD(P)+-dependent malic enzyme and have a unique intramitochondrial pathway for malate oxidation (Sauer, L.A., Dauchy, R.T., Nagel, W.O., and Morris, H.P. (1980) J. Biol. Chem. 255, 3844-3848).     

11 beta-hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described. 11 beta-HSD-1 is NADP(+)-dependent and has a Km in the micromolar range and bidirectional activity. 11 beta-HSD-2 is NAD(+)-dependent, has a Km in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11 beta-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD(+)-dependent 11 beta-HSD-2. We found that the Km for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD(+)-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, Km = 40 nM, NADP(+)-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11 beta-HSD-1 nor the 11 beta-HSD-2 and have named it 11 beta-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5 beta-dihydroprogesterone, and 5 alpha-dihydroprogesterone, but not bile acids.     

Structure of 3-isopropylmalate dehydrogenase in complex with NAD+: ligand-induced loop closing and mechanism for cofactor specificity

BACKGROUND: The leucine biosynthetic enzyme 3-isopropylmalate dehydrogenase (IMDH) belongs to a unique class of bifunctional decarboxylating dehydrogenases. The two best-known members of this family, IMDH and isocitrate dehydrogenase (IDH), share a common structural framework and catalytic mechanism but have different substrate and cofactor specificities. IMDH is NAD(+)-dependent, while IDHs occur in both NAD(+)-dependent and NADP(+)-dependent forms. RESULTS: We have co-crystallized Thermus thermophilus IMDH with NAD+ and have determined the structure at 2.5 A resolution. NAD+ binds in an extended conformation. Comparisons with the structure in the absence of cofactor show that binding induces structural changes of up to 2.5 A in the five loops which form the dinucleotide-binding site. The adenine and diphosphate moieties of NAD+ are bound via interactions which are also present in the NADP(+)-IDH complex. Amino acids which interact with the NADP+ 2'-phosphate in IDH are substituted or absent in IMDH. The adenosine ribose forms two hydrogen bonds with Asp278, and the nicotinamide and nicotinamide ribose interact with Glu87 and Asp78, all unique to IMDH. CONCLUSIONS: NAD+ binding induces a conformational transition in IMDH, resulting in a structure that is intermediate between the most 'open' and 'closed' decarboxylating dehydrogenase conformations. Physiological specificity of IMDH for NAD+ versus NADP+ can be explained by the unique interaction between Asp278 and the free 2'-hydroxyl of the NAD+ adenosine, discrimination against the presence of the 2'-phosphate by the negative charge on Asp278, and the absence of potential favorable interactions with the 2'-phosphate of NADP+.     

Altered cell shape is linked to increased p34cdc2 gene expression in fibroblasts expressing a mutant E2F-1 transcription factor

Transhydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.     

Re-face stereospecificity at C4 of NAD(P) for alcohol dehydrogenase from Methanogenium organophilum and for (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as determined by 1H-NMR spectroscopy

The two diastereotopic protons at C4 of NAD(P)H are seen separately in 1H-NMR spectra. This fact was used to determine the stereospecificity at C4 of NAD(P) for the NADP-dependent alcohol dehydrogenase from Methanogenium organophilum and for the NAD-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans. The reduction of NADP+ with [2H6]ethanol was found to yield (4R)-[4-2H1]NADPH and the oxidation of (4R)-[4-2H1]NADH with 2-oxoglutarate to yield unlabelled [4-1H]NAD+. These results indicate that both enzymes are Re-face stereospecific at C4 of the pyridine nucleotides.     

Oxidative processes in tissues of the nematode Mecistocirrus digitatus

The tissues of the nematode M. digitatus were used to obtain mitochondria which swell under the effect of several factors. Intensive oxidation of succinate, fumarate, malate, oxalacetate, alpha-ketoglutarate and less intensive one -- of citrate, isocitrate and pyruvate were observed in the mitochondria; cis-aconitate was not oxidized at all. NAD and NADP increased the intensity of substrate oxidation, whereas parachlormercuribenzoate, malonate and benzoate decreased the latter.     

Rhodopsin arginine-135 mutants are phosphorylated by rhodopsin kinase and bind arrestin in the absence of 11-cis-retinal

Arginine-135, located at the border between the third transmembrane domain and the second cytoplasmic loop of rhodopsin, is one of the most highly conserved amino acids in the family of G protein-coupled receptors. The effect of mutation at Arg-135 on the ability of rhodopsin to undergo desensitization was investigated. Four mutants, R135K, R135Q, R135A, and R135L, were examined for their ability to be phosphorylated by rhodopsin kinase, to bind arrestin, and to activate the rod cell G protein, transducin (Gt). All of the mutants were phosphorylated, bound arrestin, and were able to activate Gt when reconstituted with 11-cis-retinal. Surprisingly, several of the mutants could be phosphorylated by rhodopsin kinase and could bind arrestin in the absence of 11-cis-retinal but were not able to activate Gt. These observations represent the first demonstration of a mutant G protein-coupled receptor that assumes a conformation able to interact with its G protein-coupled receptor kinase and arrestin, but not with its G protein, in the absence of ligand.     

Interpersonal stress, depression, and disease activity in rheumatoid arthritis and osteoarthritis patients

The relationships among interpersonal stressors, depression, coping inefficiency, hormones (prolactin, cortisol, and estradiol), and disease activity were examined. The sample comprised 33 women with rheumatoid arthritis (RAs; age 37-78) and 37 women with osteoarthritis (OAs; age 47-91), who served as controls. In a regression analysis, interpersonal conflict events accounted for more than twice as much variance in depression in RAs than in OAs. In the RA patients, the immune-stimulating hormones prolactin and estradiol were significantly positively correlated with interpersonal conflicts, depression, coping inefficacy, and clinician ratings of disease activity, suggesting that RAs are more reactive to interpersonal stressors than are OAs, both psychologically and physiologically.     

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel modulator with neuroprotective properties

Lactate dehydrogenase from the malarial parasite Plasmodium falciparum has many amino acid residues that are unique compared to any other known lactate dehydrogenase. This includes residues that define the substrate and cofactor binding sites. Nevertheless, parasite lactate dehydrogenase exhibits high specificity for pyruvic acid, even more restricted than the specificity of human lactate dehydrogenases M4 and H4. Parasite lactate dehydrogenase exhibits high catalytic efficiency in the reduction of pyruvate, kcat/Km = 9.0 x 10(8) min(-1) M(-1). Parasite lactate dehydrogenase also exhibits similar cofactor specificity to the human isoforms in the oxidation of L-lactate with NAD+ and with a series of NAD+ analogs, suggesting a similar cofactor binding environment in spite of the numerous amino acid differences. Parasite lactate dehydrogenase exhibits an enhanced kcat with the analog 3-acetylpyridine adenine dinucleotide (APAD+) whereas the human isoforms exhibit a lower kcat. This differential response to APAD+ provides the kinetic basis for the enzyme-based detection of malarial parasites. A series of inhibitors structurally related to the natural product gossypol were shown to be competitive inhibitors of the binding of NADH. Slight changes in structure produced marked changes in selectivity of inhibition of lactate dehydrogenase. 7-p-Trifluoromethylbenzyl-8-deoxyhemigossylic acid inhibited parasite lactate dehydrogenase, Ki = 0.2 microM, which was 65- and 400-fold tighter binding compared to the M4 and H4 isoforms of human lactate dehydrogenase. The results suggest that the cofactor site of parasite lactate dehydrogenase may be a potential target for structure-based drug design.     

Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. Characterization of the beta H91, beta D392, and beta K424 mutants

Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated. Out of 16 charged residues studied, 6 have not been previously investigated. The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424. beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH. This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH. These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91. The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site. The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction. However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site. Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding. beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type. Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site. The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered.     

A possible functional relationship between microsomal aromatic aldehyde-ketone reductase and hexose-6-phosphate dehydrogenase

Aromatic ketone reductase activity of microsomes showed a unique cofactor requirement: Addition of NADP and glucose-6-phosphate was as effective as that of an artificial NADPH generating system, whereas NADPH alone served as a cofactor less efficiently. Microsomal aromatic ketone reductase, purified partially from guinea pig liver microsomes after solubilization with Triton X-100, reduced 5 beta-dihydrotestosterone, aromatic aldehydes, and ketones with NADPH as a cofactor. However, addition of hexose-6-phosphate dehydrogenase, purified from the same source, as an NADPH generator produced about 2 times higher activity than that of yeast glucose-6-phosphate dehydrogenase or NADPH alone.     

Glutamate, glutamine, and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: Even though the utilization of substrates alternative to glucose may play an important role in the survival of brain cells under ischemic conditions, evidence on changes in substrate selection by the adult brain in vivo during ischemic episodes remains very limited. This study investigates the utilization of glutamate, glutamine, and GABA as fuel by the neuronal and glial tricarboxylic acid cycles of both cerebral hemispheres after partially reversible focal cerebral ischemia (FCI). METHODS: Right hemisphere infarct was induced in adult Long-Evans rats by permanent occlusion of the right middle cerebral artery and transitory occlusion of both common carotid arteries. (1,2-13C2) acetate was infused for 60 minutes in the right carotid artery immediately after carotid recirculation had been re-established (1-hour group) or 23 hours later (24-hour group). Extracts from both cerebral hemispheres were prepared and analyzed separately by 13C nuclear magnetic resonance and computer-assisted metabolic modeling. RESULTS: FCI decreased the oxidative metabolism of glucose in the brain in a time-dependent manner. Reduced glucose oxidation was compensated for by increased oxidations of (13C) glutamate and (13C) GABA in the astrocytes of the ipsilateral hemispheres of both groups. Increased oxidative metabolism of (13C) glutamine in the neurons was favored by increased activity of the neuronal pyruvate recycling system in the 24-hour group. CONCLUSIONS: Data were obtained consistent with time-dependent changes in the utilization of glutamate and GABA or glutamine as metabolic substrates for the glial or neuronal compartments of rat brain after FCI.     

Nicotinate adenine dinucleotide phosphate (NAADP) triggers a specific calcium release system in sea urchin eggs

Transient fluxes of intracellular ionized calcium (Ca2+) from intracellular stores are integral components of regulatory signaling pathways operating in numerous biological regulations, including in early stages of egg fertilization. Therefore, we explored whether NADP, which is rapidly generated by phosphorylation of NAD upon fertilization may, directly or indirectly, exert a regulatory role as a trigger of Ca2+ release from intracellular stores in sea urchin eggs. NADP had no effect, but we found that the deamidated derivative of NADP, nicotinate adenine dinucleotide phosphate (beta-NAADP), is a potent and specific stimulus (ED50 16 nM) for Ca2+ release in sea urchin egg homogenates. NAADP triggers the Ca2+ release via a mechanism which is distinct from the well-known Ca2+ release systems triggered either by inositol-1,4,5-triphosphate (IP3) or by cyclic adenosine diphospho-ribose (cADPR). The NAADP-induced release of Ca2+ is not blocked by heparin, an antagonist of IP3, or by procaine or ruthenium red, antagonists of cADPR. However, it is selectively blocked by thionicotinamide-NADP which does not inhibit the actions of IP3 or cADPR. NAADP produced by heating of NADP in alkaline (pH = 12) medium or synthetized enzymatically by nicotinic acid-NADP reaction catalyzed by NAD glycohydrolase have identical properties. The results presented herein thus describe a novel endocellular Ca(2+)-releasing system controlled by NAADP as a specific stimulus. The NAADP-controlled Ca2+ release system may be an integral component of multiple intracellular regulations occurring in fertilized sea urchin eggs, which are mediated by intracellular Ca2+ release, and may also have similar role(s) in other tissues.     

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I). Effect of substrates on the fragmentation of subunits by trypsin

It has been shown that treatment of bovine mitochondrial complex I (NADH-ubiquinone oxidoreductase) with NADH or NADPH, but not with NAD or NADP, increases the susceptibility of a number of subunits to tryptic degradation. This increased susceptibility involved subunits that contain electron carriers, such as FMN and iron-sulfur clusters, as well as subunits that lack electron carriers. Results shown elsewhere on changes in the cross-linking pattern of complex I subunits when the enzyme was pretreated with NADH or NADPH (Belogrudov, G., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576) also indicated that complex I undergoes extensive conformation changes when reduced by substrate. Furthermore, we had previously shown that in submitochondrial particles the affinity of complex I for NAD increases by >/=20-fold in electron transfer from succinate to NAD when the particles are energized by ATP hydrolysis. Together, these results suggest that energy coupling in complex I may involve protein conformation changes as a key step. In addition, it has been shown here that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kDa subunit a 33-kDa degradation product that resisted further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bound to a NADP-agarose affinity column, and could be eluted with a buffer containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved in intramitochondrial fatty acid synthesis.     

The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury

Nicotinamide nucleotide transhydrogenase constitutes a proton pump which links the NAD(H) and NADP(H) pools in the cell by catalyzing a reversible reduction of NADP+ by NADH. The recent cloning and characterization of several proton-pumping transhydrogenases show that they share a number of features. They are composed of three domains, i.e., the hydrophilic domains I and III containing the NAD(H)- and NADP(H)-binding sites, respectively, and domain II containing the transmembrane and proton-conducting region. When expressed separately, the two hydrophilic domains interact directly and catalyze hydride transfer reactions similar to those catalyzed by the wild-type enzyme. An extensive mutagenesis program has established several amino acid residues as important for both catalysis and proton pumping. Conformational changes mediating the redox-driven proton pumping by the enzyme are being characterized. With the cloned, well-characterized and easily accessible transhydrogenases from E. coli and Rhodospirillum rubrum at hand, the overall aim of the transhydrogenase research, the understanding of the conformationally driven proton pumping mechanism, is within reach.