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Biomedical subjects

P M Gallop

Publications and source records attributed to P M Gallop.

At least 19 recordsLinked to original sources
PubMed1994

Further evidence that pyrroloquinoline quinone interacts with the N-methyl-D-aspartate receptor redox site in rat cortical neurons in vitro.

In this study, we show that the essential nutrient pyrroloquinoline quinone (PQQ; 50 microM) regulates N-methyl-D-aspartate (NMDA; 10 microM) receptor activity primarily by reversing the increase in the frequency of openings of the receptor-associated ion channel after chemical reduction with dithiothreitol (DTT; 1 mM). Similar to other redox-active agents, PQQ (50-200 microM) had no effect on the single-channel conductance or arithmetic mean open time of NMDA-activated events. In other experiments, we observed that inhibitory effects of PQQ (50 microM) on NMDA (30 microM)-induced whole-cell responses could be abolished by prior N-ethylmaleimide (500 microM) alkylation of the putative thiol residues that likely comprise the redox site of the receptor. These results demonstrate that PQQ modulates the NMDA receptor by directly oxidizing its redox modulatory site.

Animals↗
PubMed1992

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry.

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occurred at a 2:1 PQQ/Cu(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/Cu(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that alpha-aminoadipic-delta-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.

Anaerobiosis↗
PubMed1992

Interaction of the putative essential nutrient pyrroloquinoline quinone with the N-methyl-D-aspartate receptor redox modulatory site.

The putative essential nutrient pyrroloquinoline quinone (PQQ) can efficiently mediate reduction and oxidation reactions in a variety of systems. Therefore, we investigated whether this compound could alter the function of the NMDA receptor via a recently described redox modulatory site. In rat cortical neurons in vitro, 50 microM PQQ could reverse the enhancement of 30 microM NMDA-induced whole-cell ionic currents produced by the reducing agent dithiothreitol (DTT; 2-4 mM). PQQ also depressed native responses in a DTT-reversible fashion. In addition, 50-200 microM PQQ produced a significant degree of neuroprotection in an acute model of NMDA-mediated neurotoxicity in astrocyte-rich cultures of rat cerebral cortex. Under certain conditions, PQQ can lead to the formation of oxygen-derived free radicals, and we have previously observed that these reactive species can oxidize the NMDA receptor. Nevertheless, the enzymatic free radical scavengers superoxide dismutase and catalase (10 micrograms/ml each) did not abolish the actions of PQQ. This observation held true even in astrocyte-poor cortical cultures, where neuronal processes are directly exposed to the extracellular milieu. Therefore, under in vitro conditions in which PQQ is presented without an exogenous electron donor, it appears as if the entire neuroprotective effect of PQQ is attributable to a direct oxidation of the NMDA receptor redox site. These results suggest the possibility of a novel role for PQQ, PQQ-like substances, and quinone-containing proteins in the brain, and may represent a novel therapeutic approach for the amelioration of NMDA receptor-mediated neurotoxic injury.

Animals↗
PubMed1991

Specific detection of quinoproteins by redox-cycling staining.

Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.

Animals↗
PubMed1990

Soybean lipoxygenase-1 is not a quinoprotein.

Soybean lipoxygenase-1 was reinvestigated with respect to its quinoprotein nature. It has been reported previously that soybean lipoxygenase-1 contains pyrroloquinoline quinone as the organic cofactor. Because spectroscopic data were found to be inconsistent with the evidence presented in [1], we sought to reproduce the published data by carefully following the procedures described in [1] and supplementing them with new analytical results. The combined data lead us to conclude that soybean lipoxygenase-1 is not a quinoprotein.

Coenzymes↗
PubMed1989

Acid-promoted tautomeric lactonization and oxidation-reduction of pyrroloquinoline quinone (PQQ).

Acid-treatment facilitates PQQ detection by electron ionization mass spectroscopy with a molecular ion at M/e 330 and a base ion formed by triple decarboxylation at M/e 198. Other ions found probably arise through acid-catalyzed tautomeric lactonization of PQQ to PQQ-lactone (PQQL) with subsequent oxidation of PQQL and reduction of PQQ. We propose that a carboxyl group, presumably the 9-carboxyl, attacks a double bond in PQQ, reversibly converting the 4,5-orthoquinone into an 4,5-enediol and forming an isomeric lactone, PQQL, of 330 daltons. The masking of carbonyls may explain the low reactivity of PQQ with carbonyl reagents in acid. Acid-promoted tautomeric lactonization with carbonyl-masking is known to occur with fluoresceins, phenolphthalein and other compounds, but has not been recognized before with PQQ. Acid-treated PQQ demonstrates molecular and other ions derived from reduced PQQ (PQQ(2H] or its lactone at M/e 332 with a base ion at M/e 200. There is compelling evidence for a dehydrogenated lactone, PQQ(-2H)L), at M/e 328 with a base ion at M/e 196. We suggest that PQQ, in tautomeric equilibrium with PQQL, oxidizes PQQL to PQQ(-2H)L (328 daltons), with its concurrent reduction to PQQ(2H) (332 daltons). With acidified D2O, PQQ shows deuterated products with ions at M/e values consistent with lactonization and oxidation-reduction. An analytically useful quinoxaline adduct, formed from PQQ and 2,3-diaminonaphthalene (PQQ-DAN) of 452 daltons, also undergoes acid-tautomerization-lactonization and oxidation-reduction similar to PQQ showing molecular ions at M/e 450, 452 and 454 and decarboxylation-derived strong (base) ions at M/e 318, 320 and 322.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1989

Peptidylglycine alpha-amidating monooxygenase activity in spinal cord, dorsal roots, and dorsal root ganglia of Macaca fascicularis.

Several peptides synthesized by primary sensory neurons are alpha-amidated at the C-terminal residue, including vasoactive intestinal polypeptide, neurokinin A and substance P, which is also abundant in spinal cord. In pituitary and other tissues, the C-terminal amidation is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM). In the present study, soluble PAM activity in spinal cord and in primary sensory neurons is quantified and characterized as to cofactor and cosubstrate requirements and substrate specificity.

Animals↗
PubMed1989

Separation and determination of alpha-amino acids by boroxazolidone formation.

Reaction of an alpha-amino acid (alpha-AA) with 1,1-diphenylborinic acid (DPBA) leads to the formation of a kinetically stable adduct at pH 2-5 in which both the alpha-amino and the alpha-carboxyl groups are bound to boron forming a cyclic mixed anhydride termed a boroxazolidone. In this adduct, the greater than N:B bond is coordinate, involving the free electron pair of nitrogen, thereby satisfying the octet rule for the second electron shell of boron (Group IIIA). Consequently, the alpha-amino function of the boroxazolidone can be primary, secondary, or tertiary, as demonstrated by boroxazolidone formation with glycine, N-methylglycine, and N,N-dimethylglycine. On reaction with DPBA, the alpha-AA moiety of N-terminal gamma-glutamyl peptides is also derivatized as demonstrated by the formation of a glutathione boroxazolidone. The 1,1-diphenylboroxazolidone adducts of alpha-AA may be separated by reversed-phase (RP)-HPLC (AA-DPBA/RP-HPLC) enabling the derivatization procedure to be used as a precolumn reaction for alpha-AA analysis. Under the conditions we describe here, DPBA is not stably reactive with the epsilon-amino group of lysine. Furthermore, it does not complex with amide bonds of the peptide backbone or to any side chains of the common amino acids. Reaction of an alpha-AA mixture with DPBA, followed by RP-HPLC (AA-DPBA/RP-HPLC) is then a simple method by which to analyze alpha-AA in a mixture with peptides and amines. Precolumn reaction with DPBA may be used to separate peptides from alpha-AA and from those peptides which contain an alpha-AA moiety. Unreacted peptides are bound only weakly to the HPLC column and thus are separated from reacted alpha-amino acids which are retained as 1,1-diphenylboroxazolidones until their selective elution. This method is particularly suited for the analysis of alpha-amino acids that are derived from post-translational modification of protein side chains.

Amino Acids↗
PubMed1989

PQQ, the elusive coenzyme.

The recently discovered redox coenzyme, PQQ (methoxatin), is widely distributed. Quantitation of protein-bound PQQ has been difficult, but unique redox cycling reactions, which reflect its striking biological properties, reveal trace amounts. PQQ is a potential target for drugs.

Animals↗
PubMed1989

Methoxatin (PQQ), coenzyme for copper-dependent amine and mixed-function oxidation in mammalian tissues.

The newly discovered coenzyme PQQ can now be measured at picomole levels with redox cycling methods developed in our laboratory. PQQ-peptides have been obtained from digests of the copper quinoenzymes, diamine oxidase, lysyl oxidase and dopamine-beta-hydroxylase. PQQ is present in egg yolk and milk, suggesting its immediate availability for developing embryos and newborn animals. We suggest that PQQ, when exposed in traumatized, ischemic, inflammed or pathological tissues, may catalyze the formation of large amounts of superoxide and should be considered as a source of oxidative stress when planning pharmacotherapeutic intervention. PQQ and quinoproteins play a role in the redox metabolism and structural integrity of cells and tissues.

Amines↗
PubMed1988

The amplified detection of free and bound methoxatin (PQQ) with nitroblue tetrazolium redox reactions: insights into the PQQ-locus.

Porcine kidney diamine oxidase, a PQQ-enzyme, can be directly measured by formazan production with putrescine and nitroblue tetrazolium. This cyclic reaction in air is unaffected by superoxide dimutase, suggesting a two electron transfer between substrate-reduced PQQ-locus and nitroblue tetrazolium, without intermediate formation of superoxide. With albumin-bound PQQ and detergent-exposed PQQ-loci, glycine can be oxidized by PQQ and electrons repetitively transferred through PQQ-sites to nitroblue tetrazolium, the rate of formazan production detecting picomoles of exposed PQQ-locus. Exposed PQQ-loci are also reducible with NaCNBH3. Nitroblue tetrazolium, reoxidizes the reduced PQQ-locus with formazan production. These experiments suggest that the PQQ-locus of quinoproteins contains a [ketone-ketoimine in equilibrium with ketoamine] redox center.

Amine Oxidase (Copper-Containing)↗
PubMed1988

The interaction of aminogroups with pyrroloquinoline quinone as detected by the reduction of nitroblue tetrazolium.

The interaction of pyrroloquinoline quinone (PQQ) with amino groups was followed by measuring the capacity of adducts to reduce nitroblue tetrazolium (NBT). Of the natural amino acids only glycine, ornithine, and lysine interacted strongly with PQQ. The reducing activity of other less reactive amino acids, but not of lysine, was increased by ammonia, primary or secondary amines. Divalent cations, in contrast inhibited development of NBT-reducing activity. PQQ also developed NBT-reactivity in the presence of serotonin and albumin. A reaction scheme is proposed which explains these findings. It is suggested that the NBT-reducing activity of plasma which is not caused by glycation of plasma proteins, arises from PQQ adducts inherent to plasma. This NBT-reducing activity corresponds to approximately 10 micrograms PQQ/ml plasma.

Alanine↗
PubMed1987

Effect of hydroxyorganoboranes on synthesis, transport and N-linked glycosylation of plasma proteins.

Utilizing a recently developed method (Boradeption) for transferring water-insoluble hydroxyorganoborane compounds into the cells, we observed inhibition of protein synthesis by three of these compounds and inhibition of secretion of plasma proteins by four of them in human hepatoma HepG2 cells. These effects were specific in that the cell viability was not affected and an increase in protein catabolism was not observed. Three compounds caused a compound-specific alterations in the electrophoretic mobility of secreted glycoproteins due to underlying changes in the N-linked carbohydrate moieties. Results presented suggest a potential new source of cellular probes.

Blood Proteins↗
PubMed1987

The formation and stability of the hypusine containing protein in Chinese hamster ovary cells.

Hypusine-containing protein identified as eukaryote initiation-translation factor 4D was labeled with [14C]spermidine in logarithmically growing Chinese hamster ovary cells. Radioautography of the cellular proteins separated by polyacrylamide gel electrophoresis showed the label in a single protein of 18000 Mr. Time course analysis showed that this protein remained undegraded for up to 72 hours after its synthesis. Radioactivity present in the amino acid hypusine, isolated after acid hydrolysis, remained constant during the same period of time. These results indicate that the hypusine-containing protein has a long half-life.

Animals↗
PubMed1986

"Glycated" osteocalcin in human and bovine bone. The effect of age.

Purified osteocalcin from cow and calf bone was analyzed for nonenzymatic glycosylation (glycation) by sodium [3H]borohydride reduction. Calf bone was found to be approximately 5% glycated, while bone from mature cows was 10% glycated. These results were confirmed by a second method which utilizes periodate oxidation followed by formaldehyde fluorescence. Osteocalcin in human bone was also found to be glycated. The content of glycated osteocalcin from the bones of 47 nondiabetic individuals, aged 0.6-97, was dependent upon age. The extent of glycation was lowest in children, was constant through the adult years, and increased linearly in bone taken from individuals aged 60-97. Glycated osteocalcin was purified by boronate affinity chromatography and subjected to one-step Edman degradation. It was established that the site of glycation was the amino-terminal tyrosine. Increases in the amount of glycated osteocalcin in the bones of older individuals may play a role in the pathogenesis of senile osteoporosis and in the osteopenia which may accompany diabetes mellitus.

Aging↗
PubMed1985

Determination of osteocalcin in human serum: results with two kits compared with those by a well-characterized assay.

Osteocalcin, the vitamin K-dependent protein in bone, can also be detected in serum and is receiving increased attention as a marker for bone turnover in evaluating patients with metabolic bone disease. We compared results for patients as determined with two commercial radioimmunoassay kits (Immuno Nuclear Corp. and Seragen Inc.) and with our in-house radioimmunoassay (Methods Enzymol 107: 517, 1984). Results by our assay correlated well (r greater than 0.9) with those by both kits, but the values by the Immuno Nuclear and Seragen methods were respectively 40% and 10% lower than by our radioimmunoassay. Within-run variation (CV) for the two kit methods was respectively 7.3% and 9.8%, run-to-run CV was 9.7% and 8.6%. The standard curve was linear from 1 to 25 micrograms/L for the Immuno Nuclear kit, from 4 to 100 micrograms/L for the Seragen equilibrium method, and from 1 to 25 micrograms/L for the Seragen nonequilibrium method. A second freeze-thaw cycle reduced the serum values by 20% to 40% for both kit methods. A third freeze-thaw cycle further reduced values and eliminated any correlation among methods.

Calcium-Binding Proteins↗