Effect of oxygen on activation state of complex I and lack of oxaloacetate inhibition of complex II in Langendorff perfused rat heart

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.     

Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells.

A new method was developed for isolation of intracellular forms of simian virus 40 (SV40) nucleoprotein complexes from SV40-infected CV-1 cells late in the infectious cycle. In contrast to the Triton extraction method, which yields only a 60-70S complex, this new procedure yielded three forms of SV40 nucleoprotein complexes: complex I, complex II, and the nature virion (V). The three nucleoprotein complexes differed in physical as well as biochemical properties. Complex I, which is only a small portion of the total SV42 nucleoprotein complexes late during infection, was active in synthesizing both SV40-specific DNA and RNA. Pulse-labeling experiments suggest the following metabolic pathway: I leads to II leads to V. Conversion of complex I to II occurred shortly after the completion of SV40 DNA replication and resulted in the inactivation of the biosynthetic activities of I.     

Characterization of a camptothecin-resistant human DNA topoisomerase I

Topoisomerase I purified from a camptothecin-resistant human leukemia cell line and from the parental, camptothecin-sensitive line were compared in vitro. Relaxation of supercoiled DNA by the wild type enzyme was inhibited in the presence of camptothecin, while the mutant enzyme was unimpaired. Camptothecin altered the cleavage pattern of the wild type but not of the mutant enzyme. The stability of cleavable complexes was studied at a preferred topoisomerase I-binding sequence recognized by both enzymes. Camptothecin greatly enhanced the kinetic stability of the cleavable complex formed by the wild type enzyme, whereas that of the mutant enzyme was only marginally affected. In the absence of camptothecin, the cleavable complex formed by the mutant enzyme was stabilized relative to that of the wild type by several criteria. Thus, the mutant enzyme cleaved the topoisomerase I recognition sequence with 2-fold higher efficiency than the wild type enzyme. The mutant cleavable complex had a higher kinetic stability and was less sensitive to salt dissociation than the wild type complex. Furthermore, the mutant enzyme formed cleavable complexes in the absence of divalent cations, which were required for complex formation by the wild type enzyme.     

Microanalysis of the amino-acid sequence of monomeric beta-lactoglobulin I from donkey (Equus asinus) milk. The primary structure and its homology with a superfamily of hydrophobic molecule transporters

The complete primary structure of donkey beta-lactoglobulin I was determined by pulsed-liquid phase microsequencing of tryptic peptides. The protein has been isolated in monomeric form and it corresponds to monomeric beta-lactoglobulin of type I. With the inclusion of donkey beta-lactoglobulin I there are 13% common residues amongst the members of the beta-lactoglobulin family. Donkey beta-lactoglobulin I is homologous to the retinol-binding protein, bilin-binding protein and five other proteins belonging to the new superfamily of hydrophobic molecule transporters. A rapid method for peptide isolation and the strategy for microsequencing of this protein have been described.     

Effect of anoxia/reperfusion on the reversible active/de-active transition of NADH-ubiquinone oxidoreductase (complex I) in rat heart

The multi-subunit mammalian NADH-ubiquinone oxidoreductase (complex I) is part of the mitochondrial electron transport chain and physiologically serves to reduce ubiquinone with NADH as the electron donor. The three-dimensional structure of this enzyme complex remains to be elucidated and also little is known about the physiological regulation of complex I. The enzyme complex in vitro is known to exist as a mixture of active (A) and de-active (D) forms [Biochim. Biophys. Acta 1364 (1998) 169]. Studies are reported here examining the effect of anoxia and reperfusion on the A/D-equilibrium of complex I in rat hearts ex vivo. Complex I from the freshly isolated rat heart or after prolonged (1 h) normoxic perfusion exists in almost fully active form (87±2%). Either 30 min of nitrogen perfusion or global ischemia decreases the portion of active form of complex I to 40±2%. Upon re-oxygenation of cardiac tissue, complex I is converted back predominantly to the active form (80-85%). Abrupt alternation of anoxic and normoxic perfusion allows cycling between the two states of the enzyme. The possible role in the physiological regulation of complex I activity is discussed.     

Effect of anoxia/reperfusion on the reversible active/de-active transition of NADH-ubiquinone oxidoreductase (complex I) in rat heart

The multi-subunit mammalian NADH-ubiquinone oxidoreductase (complex I) is part of the mitochondrial electron transport chain and physiologically serves to reduce ubiquinone with NADH as the electron donor. The three-dimensional structure of this enzyme complex remains to be elucidated and also little is known about the physiological regulation of complex I. The enzyme complex in vitro is known to exist as a mixture of active (A) and de-active (D) forms [Biochim. Biophys. Acta 1364 (1998) 169]. Studies are reported here examining the effect of anoxia and reperfusion on the A/D-equilibrium of complex I in rat hearts ex vivo. Complex I from the freshly isolated rat heart or after prolonged (1 h) normoxic perfusion exists in almost fully active form (87+/-2%). Either 30 min of nitrogen perfusion or global ischemia decreases the portion of active form of complex I to 40+/-2%. Upon re-oxygenation of cardiac tissue, complex I is converted back predominantly to the active form (80-85%). Abrupt alternation of anoxic and normoxic perfusion allows cycling between the two states of the enzyme. The possible role in the physiological regulation of complex I activity is discussed.     

Carbonic Anhydrase Inhibitors: Metal Complexes of a Sulfanilamide Derived Schiff base and their Interaction with Isozymes I,II and IV

Metal complexes of aromatic/heterocyclic sulfonamides act as stronger inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to the uncomplexed sulfonamides from which they are derived. Here we report the synthesis and inhibition studies against the physiologically relevant isozymes CA I, CA II and CA IV, of a series of metal complexes (Co(II), Ni(II) and Cu(II) derivatives) of a Schiff-base ligand, obtained from sulfanilamide and salicylaldehyde. The best activity was observed for the Cu(II) and Co(II) complexes, against CA II and CA IV, for which inhibition constants in the range of 15-39 and 72-108 nM, respectively, were seen. The enhanced efficacy in inhibiting the enzyme may be due to a dual mechanism of action of the metal complexes, which interact with CA both by means of the sulfonamide moieties as well as the metal ions present in their molecule.     

Carbonic anhydrase inhibitors: metal complexes of a sulfanilamide derived Schiff base and their interaction with isozymes I,II and IV

Metal complexes of aromatic/heterocyclic sulfonamides act as stronger inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to the uncomplexed sulfonamides from which they are derived. Here we report the synthesis and inhibition studies against the physiologically relevant isozymes CA I, CA II and CA IV, of a series of metal complexes (Co(II), Ni(II) and Cu(II) derivatives) of a Schiff-base ligand, obtained from sulfanilamide and salicylaldehyde. The best activity was observed for the Cu(II) and Co(II) complexes, against CA II and CA IV, for which inhibition constants in the range of 15-39 and 72-108nM, respectively, were seen. The enhanced efficacy in inhibiting the enzyme may be due to a dual mechanism of action of the metal complexes, which interact with CA both by means of the sulfonamide moieties as well as the metal ions present in their molecule.     

Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides.

A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.     

Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 x 10(4). On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 x 10(5)) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 X 10(5), and a ganglioside/protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides GD1a and GD1b, which, like GT1b, are potential substrates for the enzyme and GM1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. GT1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with GD1a and GD1b.     

Nitrosyl-heme complexes are formed in the ischemic heart: evidence of nitrite-derived nitric oxide formation, storage, and signaling in post-ischemic tissues

In addition to the generation from specific nitric-oxide (NO) synthases, NO formation from nitrite occurs in ischemic tissues, such as the heart. Although NO binding to heme-centers is the basis for NO-mediated signaling as occurs through guanylate cyclase, it is not known if this process is triggered with physiologically relevant periods of sublethal ischemia and if nitrite serves as a critical substrate. Therefore electron paramagnetic resonance studies were performed to measure nitrosylheme formation during the time course of myocardial ischemia and reperfusion and the role of nitrite in this process. Rat hearts were either partially nitrite-depleted by nitrite-free buffer perfusion or nitrite-enriched by preinfusion with 50 microm nitrite. Ischemic hearts loaded with nitrite showed prominent spectra of six-coordinate nitrosyl-heme complexes, primarily NO-myoglobin, that increased as a function of ischemic duration, whereas in nonischemic-controls these signals were not seen. Total nitrosyl-heme concentrations within the heart were 6.6 +/- 0.7 microm after 30 min of ischemia. Nitrite-depleted hearts also gave rise to NO-heme signals during ischemia, but levels were 8-fold lower. Nitrite-mediated NO-heme complex formation during ischemia was associated with activation of guanylate cyclase. Upon reperfusion, the levels of NO-heme complexes decreased 3-fold by the first 15 min but remained elevated for over 45 min. The decrease in NO-heme complex levels was paralleled by the formation of nitrate, suggesting the oxidation of heme-bound NO upon reperfusion. Thus, nitrite-mediated NO-heme formation occurs progressively during ischemia, with these complexes serving as a store of NO with concordant activation of NO signaling pathways.     

Reaction of fluorescein bimercuric acetate with a molybdenum center of xanthine oxidase from milk

Inhibition of milk xanthine oxidase by fluorescein bimercuriacetate (FMA) allows for the classification of S-containing groups according to their localization and role in the catalytic activity of the enzyme. The enzyme (E) complexes with FMA (E--FMA I and E--FMA II) differing in their activity, stoichiometry and spectral properties were studied at various experimental conditions, reaction time and FMA concentrations. The enzyme molecule contains 5 groups that are reactive towards FMA (E--FMA I) and are localized outside the active center. That these groups have no concern with activity and are subjected to modification irrespective of whether or not the xanthine oxidase molecule has an intact Mo-center. The formation of an inactive E--FMA II complex is associated with an additional (in comparison with E--FMA I) binding of two FMA molecules per molecule of the active enzyme. The stoichiometry of the E--FMA II complex was determined by the X-ray fluorescent method from the amount of the Hg in enzyme. A kinetic scheme of xanthine oxidase inhibition by FMA is proposed, according to which the inhibition is a result of modification of two groups in the enzyme active center, of which only one is essential for the enzyme activity. This scheme also postulates the role of reversible E--FMA complexes in the course of irreversible inhibition. Xanthine oxidase is protected against FMA by the substrate (xanthine), competitive inhibitors (azaxanthine and allopurinol) and acceptor (2,6-dichlorophenolindophenol), i. e., compounds which interact with the Mo-center of the enzyme. The EPR spectra of the dithionite-reduced E--FMA II complex were found to contain a "slow" signal, Mo(V), typical of the Mo-center devoid of labile sulphur. It was assumed that the essential group interacting with FMA in the active center of xanthine oxidase as a terminal sulphur which is a component of the coordination region of Mo.     

Isolation and properties of noncovalent complex of transketolase with RNA

A method for isolation of homogenous transketolase from baker's yeast using immunoaffinity chromatography was significantly simplified. It was demonstrated that transketolase could be isolated from fresh yeast in the form of a complex with a high molecular weight RNA. Storage of yeast led to the dissociation of the complex to a low molecular weight complex and then to the free enzyme. Conditions were chosen for complex dissociation and free enzyme isolation. In comparison to the free enzyme, the specific activities of the high and low molecular weight complexes were decreased 20-25- and 3-5.5-fold, respectively. The affinity to the cofactor thiamine diphosphate and to xylulose-5-phosphate (donor substrate) did not change for the low molecular weight complex, while the time of binding to calcium increased. The latter was necessary for the complete manifestation of the enzymatic activity. Changes in the circular dichroism spectrum between 300 and 360 nm after the addition of thiamine diphosphate, which characterize the formation of the catalytically active holoenzyme, were significantly lower for the low molecular weight complex than for the free enzyme.     

Perfusion chromatography—a new procedure for very rapid isolation of integral photosynthetic membrane proteins

The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.Abbreviations Chl chlorophyll - LDAO N,N dimethyldodecylamine-N-oxide - LHC light-harvesting complex - PS photosystem - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Docking multiple conformations of a flexible ligand into a protein binding site using NMR restraints

A method is described for docking a large, flexible ligand using intra-ligand conformational restraints from exchange-transferred NOE (etNOE) data. Numerous conformations of the ligand are generated in isolation, and a subset of representative conformations is selected. A crude model of the protein-ligand complex is used as a template for overlaying the selected ligand structures, and each complex is conformationally relaxed by molecular mechanics to optimize the interaction. Finally, the complexes were assessed for structural quality. Alternative approaches are described for the three steps of the method: generation of the initial docking template; selection of a subset of ligand conformations; and conformational sampling of the complex. The template is generated either by manual docking using interactive graphics or by a computational grid-based search of the binding site. A subset of conformations from the total number of peptides calculated in isolation is selected based on either low energy and satisfaction of the etNOE restraints, or a cluster analysis of the full set. To optimize the interactions in the complex, either a restrained Monte Carlo-energy minimization (MCM) protocol or a restrained simulated annealing (SA) protocol were used. This work produced 53 initial complexes of which 8 were assessed in detail. With the etNOE conformational restraints, all of the approaches provide reasonable models. The grid-based approach to generate an initial docking template allows a large volume to be sampled, and as a result, two distinct binding modes were identified for a fifteen-residue peptide binding to an enzyme active site.     

Generation of reactive oxygen species by the mitochondrial electron transport chain

Generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain (ETC), which is composed of four multiprotein complexes named complex I-IV, is believed to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Previous studies have identified the ubiquinone of complex III and an unknown component of complex I as the major sites of ROS generation. Here we show that the physiologically relevant ROS generation supported by the complex II substrate succinate occurs at the flavin mononucleotide group (FMN) of complex I through reversed electron transfer, not at the ubiquinone of complex III as commonly believed. Indirect evidence indicates that the unknown ROS-generating site within complex I is also likely to be the FMN group. It is therefore suggested that the major physiologically and pathologically relevant ROS-generating site in mitochondria is limited to the FMN group of complex I. These new insights clarify an elusive target for intervening mitochondrial ROS-related processes or diseases.     

Enzyme inhibition by iminosugars: Analysis and insight into the glycosidase–iminosugar dependency of pH

Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme–inhibitor complexes with three (EH₃I), two (EH₂I), one (EHI), or no protons (EI), are possible. In the present work an analysis method is presented that from pH-inhibition data allows one to distinguish between the different complexes and determine which protonation state is preferred. It is also possible to determine the pH-independent binding constants of the inhibitor. Analysis of pH data for imino- and azasugar inhibition of β-glucosidases revealed that basic glycosidase inhibitors bind as the monoprotonated (EHI) complex. Three neutral inhibitors were also studied and two of these were also bound exclusively as the EHI complex while a third bound both as a EHI and a EH₂I complex.     

Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase

Human retinol dehydrogenase 10 (RDH10) was implicated in the oxidation of all-trans-retinol for biosynthesis of all-trans-retinoic acid, however, initial assays suggested that RDH10 prefers NADP(+) as a cofactor, undermining its role as an oxidative enzyme. Here, we present evidence that RDH10 is, in fact, a strictly NAD(+)-dependent enzyme with multisubstrate specificity that recognizes cis-retinols as well as all-trans-retinol as substrates. RDH10 has a relatively high apparent K(m) value for NAD(+) (~100 microm) but the lowest apparent K(m) value for all-trans-retinol (~0.035 microm) among all NAD(+)-dependent retinoid oxidoreductases. Due to its high affinity for all-trans-retinol, RDH10 exhibits a greater rate of retinol oxidation in the presence of cellular retinol-binding protein type I (CRBPI) than human microsomal RoDH4, but like RoDH4, RDH10 does not recognize retinol bound to CRBPI as a substrate. Consistent with its preference for NAD(+), RDH10 functions exclusively in the oxidative direction in the cells, increasing the levels of retinaldehyde and retinoic acid. Targeted small interfering RNA-mediated silencing of endogenous RDH10 or RoDH4 expression in human cells results in a significant decrease in retinoic acid production from retinol, identifying both human enzymes as physiologically relevant retinol dehydrogenases. The dual cis/trans substrate specificity suggests a dual physiological role for RDH10: in the biosynthesis of 11-cis-retinaldehyde for vision as well as the biosynthesis of all-trans-retinoic acid for differentiation and development.     

Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.     

Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method.

A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.