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R J Guillory

Publications and source records attributed to R J Guillory.

At least 19 recordsLinked to original sources
PubMed1998

Papain proteolysis releases a soluble NADPH dependent diaphorase activity from bovine neutrophil membranes.

An NADPH dependent cytochrome c reductase has been purified from resting bovine neutrophil membranes. A high degree of purification, approaching homogeneity, is indicated by the presence of a single 75 kDa protein band on silver stained SDS-PAGE (10%). The purified protein catalyzes as well an NADPH dependent reduction of iodonitrotetrazolium violet (INT). Limited papain digestion of the purified preparation produces a 65 kDa product which retains both enzymatic activities. In a similar fashion papain digestion of the plasma membrane bound protein generates a fully active soluble NADPH dependent INT and cytochrome c reductase preparation (65 kDa). Proteolytic cleavage would appear to occur at a protein-membrane anchor remote from the proteins catalytic site. The cytochrome c reductase acts independently of the O2-generating cytochrome b558, a leukocyte plasma membrane protein which also catalyzes an NADPH dependent INT reduction.

Animals↗
PubMed1997

Purified leukocyte cytochrome b558 incorporated into liposomes catalyzes a cytosolic factor dependent diaphorase activity.

The leukocyte iodonitrotetrazolium violet (INT) reductase activity of disrupted bovine polymorphonuclear neutrophils is closely associated with the activation of the O2(-)-generating NADPH oxidase in a cell-free system. It is dependent upon NADPH, cytosolic factors, and amphiphiles (such as arachidonate), the same factors required for O2- generation. Both O2- generation and INT reductase activity are inhibited by phenylarsine oxide, an inhibitor of the activation of the NADPH oxidase [Li, J., & Guillory, R. J. (1997) J. Biochem. Mol. Biol. Biophys. (in press)]. In this report, the INT diaphorase activity of disrupted bovine polymorphonuclear neutrophils is shown to be resolved by DEAE-Sepharose chromatography into two fractions: an NADPH-cytochrome c reductase-containing fraction and a cytochrome b558-associated fraction. The diaphorase activity in the NADPH-cytochrome c reductase-containing portion is not dependent upon the presence of an amphiphile or phospholipid and is not associated with O2- generation. Upon incorporation into liposomes, the cytochrome b558-containing fraction demonstrates high O2- and INT reductase activities in the presence of cytosolic factors. Both O2- generation and INT reductase activities are SDS and FAD dependent and further stimulated by GTPgammaS. Phenylarsine oxide inhibits both O2- generation and INT reductase activities when added prior to activation by SDS. With the cytochrome b-containing liposomes, the Km values (O2- formation) for NADPH and NADH are 27.2 microM and 810 microM, and for INT reductase the Km values are 27.5 microM and 1017 microM, respectively. Under anaerobic conditions and thus in the absence of O2- formation, the NADPH-dependent INT reductase activity does not change, indicating that the dye reduction is not due to its direct reduction by O2 anion but is an intrinsic property of the superoxide-generating NADPH oxidase. Cytochrome b558 is the essential component of the NADPH oxidase and contains all the redox centers necessary for electron flow between NADPH and oxygen. The correlation of the activation and inhibition patterns for O2- generation and INT reduction by cytochrome b558 incorporated into artificial liposomes strongly indicates that the two activities are associated with the same membrane protein, cytochrome b558.

Anaerobiosis↗
PubMed1994

NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.

Polymorphonuclear neutrophil membranes, activated in a cell-free system, contain all of the essential components required for superoxide formation including the NADPH-binding component. Arylazido-beta-alanyl- [32P]NADPH--3'-O-(3-[N-(4-azido-2-nitrophenyl)amino] propionyl)-[32P]NADPH--an NADPH analogue and photoaffinity probe, has been used to identify the specific NADPH binding component of the oxidase in activated membranes. A protein of about 52 kDa was photodependently labeled in the activated membranes by arylazido-beta-alanyl-[32P]NADPH. Specificity of labeling was indicated by the absence of such labeling in nonactivated membranes. The 52-kDa-labeled protein was the only isotopically labeled protein extracted from the labeled membranes with the chaotrope sodium perchlorate. Sodium perchlorate extraction of the 52-kDa protein from activated membranes correlates with the loss of the membranes' superoxide-generating capability. Reconstitution of the lost activity for sodium perchlorate-extracted membranes was accomplished by reincubating the extracted membranes with cytosol. It is proposed that the arylazido- beta-alanyl-[32P]NADPH-labeled protein of 52- to 57-kDa present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase.

Affinity Labels↗
PubMed1994

Influences of lovastatin administration on the respiratory burst of leukocytes and the phosphorylation potential of mitochondria in guinea pigs.

Lovastatin, a cholesterol-lowering drug, decreased plasma cholesterol and cardiac tissue coenzyme Q10 levels in guinea pigs given 20 mg per kg body weight twice a day. Plasma cholesterol levels were reduced 40% in animals 2 to 4 months of age and 61% in animals 2 years of age. Coenzyme Q10 values in cardiac muscle and cardiac mitochondria of the treated, older group were decreased 31% and 37%, respectively. A significant decrease was not observed in coenzyme Q10 levels of the younger animal group. The potential to phosphorylate ADP to ATP driven by pyruvate-malate and succinate oxidation was decreased 43% and 45%, respectively, for cardiac mitochondria from the treated, 2-year-old animals. A decrease in phosphorylation potential was not observed for the younger group. The respiratory burst of leukocytes isolated from the intraperitoneal cavities of the treated, older animals was decreased 67%, while leukocytes isolated directly from their blood was decreased 76% (Diebold, B., Bhagavan, N. and Guillory, R. (1991) FASEB J. 5, A1203). In contrast to the intact leukocytes, the superoxide production of the cell-free systems prepared from leukocytes isolated from treated and untreated animals did not differ significantly. These observations suggest that in vivo lovastatin may not directly affect the leukocyte superoxide generating system, but may influence it indirectly possibly by modifying the lipid content of the membrane.

Animals↗
PubMed1994

Identification of the NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.

A cell-free system prepared from polymorphonuclear neutrophils is capable of NADPH-dependent generation of superoxide anion, but requires the simultaneous presence of plasma membranes, cytosol, arachidonate and guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The isolated membranes from such a preparation are able to catalyse NADPH-dependent superoxide formation independently of added cytosol and activators. Such activated membranes, activated in the cell-free system, must consequently contain all of the essential components required by the oxidase for superoxide formation, including the NADPH-binding component. Arylazido-beta-alanyl-[32P]NADPH (3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino] propionyl)-[32P]NADPH), an NADPH analogue and photoaffinity probe, is shown to act in the dark as a substrate for the oxidase activity in the activated membranes and an irreversible photodependent inhibitor following photoirradiation. The photoaffinity probe has been used to identify the specific NADPH-binding component of the oxidase in the activated membranes. In contrast with the sensitivity of the activated membranes, photoirradiation of arylazido-beta-alanyl-[32P]-NADPH under identical conditions, but with non-activated membranes, did not prevent subsequent activation of the treated membranes by cytosol, arachidonate and GTP[S]. However, photoirradiation of the cytosolic fraction in the presence of arylazido-beta-alanyl-[32P]NADPH resulted in an inhibition of the cytosol's ability to activate superoxide generation upon subsequent incubation with plasma membranes in the presence of arachidonate and GTP[S]. These observations are taken as a strong indication that the NADPH-binding protein of the oxidase is a cytosolic factor which associates with the plasma membrane upon activation. The superoxide-generating activity of the activated membranes was inhibited irreversibly in a concentration- and photo-dependent manner by arylazido-beta-alanyl-NADPH. The arylazido-beta-alanyl-NADPH-photodependent inhibition of superoxide generation in the activated membranes correlated with the photodependent labelling of a protein of 55.6 kDa by the arylazido-beta-alanyl-NADPH. Specificity of labelling was indicated by a lack of labelling of the 55.6 kDa region in the non-activated membranes and protection of the photodependent inhibition and labelling of the 55.6 kDa protein by NADPH. It is proposed that the arylazido-beta-alanyl-NADPH-labelled 55.6 kDa protein present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase.

Affinity Labels↗
PubMed1990

Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling.

Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin with N-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin, Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10(-8)M. Mojave toxin, caudoxin, notexin, Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while beta-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.

Affinity Labels↗
PubMed1985

The [4B-3H] NADH-H2O exchange reaction of the mitochondrial NADH dehydrogenase.

The purified mitochondrial NADH dehydrogenase enzyme has been shown to catalyze a rapid [4B-3H] NADH-H2O exchange reaction. When the enzyme is subjected to a single freeze-thaw cycle there is a complete loss of NADH dehydrogenation without a measurable decrease in the [4B-3H] NADH-H2O exchange. Complete loss of the [4B-3H] NADH-H2O exchange follows brief exposure to ultraviolet photoirradiation. The differential sensitivity of the water exchange reaction and the dehydrogenase activity suggests a direct involvement of the enzymes flavin cofactor in the catalysis of the [4B-3H] NADH-H2O exchange. Arylazido-beta-alanyl NAD+ (A3'-0-[3-[N-4-azido-2-nitrophenyl)amino] propionyl]NAD+) is shown to be a potent photodependent inhibitor of the [4B-3H] NADH-H2O exchange activity following photoirradiation with visible light. This is consistent with the observed photodependent inhibition of the dehydrogenase activity by this photoprobe (Chen, S. and Guillory, R.J. (1981) J. Biol. Chem. 256, 8318-8323).

Animals↗
PubMed1985

Interaction of [3H]arylazido aminopropionyl ATP ([3H]ANAPP3) with P2-purinergic receptors in the smooth muscle of the isolated guinea-pig vas deferens.

Following its photolysis in the presence of the isolated guinea-pig vas deferens, the ATP photoaffinity label ANAPP3 produces a specific antagonism of adenine nucleotide-induced contractile responses which are mediated by P2-purinergic receptors. To characterize the site of covalent photoincorporation of ANAPP3, intact vasa deferentia were treated with [3H]ANAPP3 and samples of homogenate, cytosol and a crude membrane fraction were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Photolysis of [3H]ANAPP3 (10(-5) M; 3.0 mu Ci/ml) resulted in the incorporation of radioactivity into cellular components with apparent molecular weights of 54-66 and 43-57 kilodaltons. The photoincorporation of [3H]ANAPP3 was associated with the crude membrane fraction and not the cytosol, was reduced in the presence of ATP in an ATP-concentration-dependent manner, was lessened following pretreatment of the tissues with photolyzed nonradiolabeled ANAPP3, and was unaffected by the nucleoside transport inhibitor, dipyridamole. In tension studies on the same tissues the presence of ATP resulted in a concentration-dependent reduction in the initial contractile response to [3H]ANAPP3 the response to 3H was antagonized in tissues which had been pretreated with nonradiolabeled ANAPP3, and dipyridamole had no effect on the contractile response to [3H]ANAPP3. According to several criteria these findings indicate that the antagonism by photolyzed ANAPP3 of adenine nucleotide-induced responses is a direct result of the covalent insertion at or near the recognition site of cell-surface P2-purinergic receptors.

Adenosine Triphosphate↗
PubMed1985

Studies of the ferricyanide reductase activities of the mitochondrial reduced nicotinamide adenine dinucleotide-ubiquinone reductase (complex I) utilizing arylazido-beta-alanyl NAD+ and arylazido-beta-alanyl NADP+.

The NADH and NADPH ferricyanide reductase activities present in mitochondrial NADH-CoQ reductase preparations have been studied utilizing two photoaffinity pyridine nucleotide analogues: arylazido-beta-alanyl NAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]NAD+) and arylazido-beta-alanyl NADP+ (N3'-O-[3-[N-(4-azido-3-nitrophenyl)amino]propionyl]NADP+). For the NADH-K3Fe(CN)6 reductase activity, arylazido-beta-alanyl NAD+ was found to be, in the dark, a competitive inhibitor with respect to both NADH and K3Fe(CN)6 with Ki,app values of 9.7 and 15.5 microM, respectively. In comparison the NADP+ analogue exhibited weak noncompetitive inhibitor activity for this reaction against both substrates. Upon photoirradiation arylazido-beta-alanyl NAD+ inhibited NADH-K3Fe(CN)6 reductase up to 70% in the presence of a 25-fold molar excess of analogue over the enzyme concentration. This photodependent inhibition could be prevented by the presence, during irradiation, of the natural substrate NADH. In contrast complex kinetic results were obtained with studies of the effects of the pyridine nucleotide analogues of NADPH-K3Fe(CN)6 reductase activity in the dark. Photoirradiation of either analogue in the presence of the enzyme complex resulted in an activation of NADPH-dependent activity. The possibility that the NADPH-K3Fe(CN)6 reductase activity of complex I represents a summation of the combined ferricyanide reductase activity of the NADPH-NAD+ transhydrogenase and NADH oxidoreductase is suggested.

Animals↗
PubMed1984

The reaction mechanism of the mitochondrial pyridine nucleotide transhydrogenase. A study utilizing arylazido-pyridine nucleotide analogues.

The addition of a purified mitochondrial pyridine nucleotide transhydrogenase enzyme preparation to complex I (NADH-CoQ reductase) results in a significant increase in the NADPH-AcPyAD+ transhydrogenase activity of the complex without influencing the NADH-AcPyAD+ transhydrogenase activity. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of complex I, the purified transhydrogenase enzyme preparation was found to co-migrate with the Mr = 130,000 (130K) subunit of the NADH-CoQ reductase. Loss of the NADPH-NAD+ transhydrogenase activity of complex I following limited tryptic digestion was associated with a corresponding loss of the 130K subunit from the complex. These results suggest that the 130K subunit of complex I is the specific peptide responsible for the catalysis of the NADPH-NAD+ transhydrogenase activity observed in complex I. Studies have been carried out testing the influence of photoaffinity pyridine nucleotide probes on the NADPH-NAD+ transhydrogenase activity catalyzed at three levels of resolution, i.e. a homogeneous transhydrogenase preparation, a partially resolved membrane preparation (complex I), and an intact mitochondrial membrane preparation (EDTA particles). Such studies have revealed arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+) to be a potent inhibitor and an active site-directed reagent for NADPH-NAD+ transhydrogenation at all three levels of resolution. On the other hand, arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino]propionyl)NAD+ does not produce a significant degree of inhibition of NADPH-NAD+ transhydrogenase activities prior to or following photoirradiation. Nevertheless, the NAD+ analogue has been found to specifically label, covalently, the transhydrogenase protein following photoirradiation of an enzyme-analogue mixture. Arylazido-beta-alanyl NAD+ can as well function as a substrate during transhydrogenation by virtue of being able to accept a hydride ion from NADPH. An interpretation of the observed nucleotide photoprobe specificity for interaction at the active site for transhydrogenation is advanced. In this interpretation, an ordered binding of substrate involves an initial NADP(H) (or NADP+ photoprobe) interaction with a hydrophobic region at the transhydrogenation site. This initial reactivity is followed by a positioning of NAD(H) (or the NAD+ photoprobe analogue) above or periphery to the NADP(H) nucleotide present at the active site region. Supportive evidence for this model for transhydrogenation is presented and discussed.

Affinity Labels↗
PubMed1984

Identification of the NADH-NAD+ transhydrogenase peptide of the mitochondrial NADH-CoQ reductase (Complex I). A photodependent labeling study utilizing arylazido-beta-alanyl NAD+.

Incubation of Complex I (NADH-CoQ reductase) of ox heart mitochondria at 4 degrees C in the presence of 0.5 M NaClO4 followed by ammonium sulfate fractionation of the solubilized proteins results in the isolation of a resolved preparation still capable of catalyzing NADH-NAD+ transhydrogenation but having only low levels of NADH dehydrogenase activity. A number of NAD(H) analogues, including the photoaffinity probes, arylazido-beta-alanyl NAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]NAD+ and arylazido-beta-alanyl AcPyAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]AcPyAD+ can be utilized as substrates for transhydrogenation in this preparation. A further incubation (10 min) of the resolved NADH-NAD+ transhydrogenase in the presence of 0.5 M NaClO4, but now at 30 degrees C, results in the complete loss of this transhydrogenase activity. Photoaffinity labeling experiments utilizing arylazido-[3-3H]beta-alanyl NAD+ and arylazido-[3-3H]beta-alanyl AcPyAD+ with the resolved NADH-NAD+ transhydrogenase preparation prior to and following NaClO4 (30 degrees C) treatment indicates that the 42,000 molecular weight component of Complex I is the pyridine nucleotide binding site responsible for the major NADH-NAD+ (DD) transhydrogenase activity of Complex I.

Animals↗
PubMed1983

Photoaffinity labeling: theory and practice.

The potential for generating a reactive reagent in situ by photoexcitation and for labeling sites not containing reactive nucleophilic residues provides photoaffinity labeling with a special advantage in the study of ligand-binding sites. There are, however, problems in the application of the photoaffinity technique. A number of concerns about this approach are examined by using as a background studies with arylazido-beta-alanyl nucleotide probes developed in this laboratory. It is concluded that photoaffinity labeling is likely to emerge as one of the major techniques for elucidating structure-function relationships within biological membranes. In the future the design of photoprobes with shorter activation half-lives may make it possible to investigate time-dependent (i.e., conformational) changes occurring at the active site of enzymes and within complex ligand-binding sites.

Adenosine Triphosphate↗
PubMed1981

Studies on the interaction of arylazido-beta-alanyl NAD+ with the mitochondrial NADH dehydrogenase.

Arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) NAD+) is a potent competitive inhibitor with respect to NADH (apparent Ki, 1.7-2.7 microM) for the purified mitochondrial NADH dehydrogenase (EC 1.6.99.3). Upon photoirradiation of the dehydrogenase in the presence of arylazido-beta-[3-3H]-alanyl NAD+, radioactivity was found to be associated with the Mr = 57,000 subunit without significant labeling of what are considered to be the enzyme's two smaller subunits. This labeling could be prevented by the presence of NADH during photoirradiation. In contrast to arylazido-beta-alanyl NAD+, arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]-propionyl) NADP+) did not inhibit dehydrogenase activity nor did photoirradiation of the enzyme in the presence of the radiolabeled analogue (arylazido-beta-[3-3H]alanyl NADP+) result in radioactivity being incorporated into the enzyme. It is concluded that the Mr -- 57,000 subunit contains the pyridine nucleotide-binding site of the mitochondrial NADH electron transport system.

Affinity Labels↗
PubMed1981

Photodependent incorporation of arylazido-beta-alanyl-NAD+ into the coenzyme binding site of yeast glyceraldehyde-3-phosphate dehydrogenase.

Yeast glyceraldehyde-3-phosphate dehydrogenase was labeled in a photodependent reaction by the arylazido-beta-alanyl derivative of NAD+. This analogue was bound covalently to the enzyme and could be reduced in situ by the substrate glyceraldehyde-3-phosphate. That this derivative was bound to the active site in the proper orientation was shown by fluorescence experiments, from the retention of the enzymatic activity when the photolysis of the enzyme-analogue binary complex was carried out in the presence of NAD+. In the dark a non-photodependent competitive inhibition corresponding to a KI-value of 150 microM was observed. Thiol groups of the enzyme were not modified in the photolabeling reaction. Of the various arylazido-beta-alanyl nucleotide derivatives studied, the NADP+ derivative influenced the enzymatic activity to the greatest extent; this is probably due to an ionic bond between enzyme and nucleotide, in addition to the covalent bond of the photolytic reaction.

Affinity Labels↗