Pyrroloquinoline quinone: a novel vitamin?

Pyrroloquinoline quinone (PQQ), otherwise known as methoxatin, is a water-soluble, redox-cycling orthoquinone that was initially isolated from cultures of methylotropic bacteria. It has been found to be a cofactor of some bacterial alcohol dehydrogenases, and is present in many animal tissues. It may be a novel vitamin because it has been shown to be essential for normal growth and development. The redox-cycling ability of PQQ enables it to scavenge or generate superoxide. When fed to animals as a supplement, PQQ prevents oxidative changes that would ordinarily occur. It has been reported to inhibit glutamate decarboxylase activity and protect against N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in the brain. It appears that in the whole animal, however, PQQ does not cross the blood-brain barrier. Furthermore, it increases nerve growth factor (NGF) synthesis in mouse astroglial cells, but has to be bound to glycine to penetrate and exert this effect in whole brain. It may therefore be regarded as a "Janus faced" molecule, with its potential for a therapeutic role in the brain still in question.

The effects of pyrroloquinoline quinone on heme-regulated eIF-2alpha kinase and eIF-2B activities in eukaryotic protein synthesis.

Pyrroloquinoline quinone (PQQ), a novel cofactor of biological redox processes, is ubiquitous in animal cells. We have examined the effects of PQQ on protein synthesis. PQQ inhibits protein synthesis in hemin-supplemented rabbit reticulocyte lysates. This inhibition is characterized by increased phosphorylation of eIF-2alpha and by diminished guanine nucleotide exchange activity of eIF-2B. The increased eIF-2alpha phosphorylation is the result of activation by PQQ of the heme-regulated eIF-2alpha kinase (HRI). The addition of 10 microM PQQ completely inhibits the increase in protein synthesis that occurs on the addition of hemin (20 microM) to heme-deficient lysates, whereas a lower concentration of PQQ (100 nM) causes a very slight stimulation of protein synthesis. The increased eIF-2alpha phosphorylation that occurs at high concentrations of PQQ inhibits eIF-2B activity, presumably due to formation of a 15S complex [eIF-2(alphaP).eIF-2B] in which eIF-2B becomes non-functional. Low concentrations of PQQ (0.1-1 microM) do not affect eIF-2alpha phosphorylation, but rather enhance the guanine nucleotide exchange activity of eIF-2B in reticulocyte lysates. In Chinese hamster ovary cell extract which is devoid of significant eIF-2alpha kinase activity, addition of both low and high concentrations of PQQ results in an increase in eIF-2B activity. The addition of PQQ to reticulocyte lysates activates HRI whereas addition of PQQ to purified HRI in vitro inhibits the autokinase and eIF-2alpha kinase activity of the HRI; the inhibition of purified HRI by PQQ is observed both in the presence and absence of hemin. These findings suggest that PQQ inhibits purified HRI by acting as an oxidant whereas in lysates in which PQQ is readily reduced, the PQQ acts as a reductant and increases the activities of both HRI and eIF-2B.

The catalysis of redox cycling by pyrroloquinoline quinone (PQQ), PQQ derivatives, and isomers and the specificity of inhibitors.

Pyrroloquinoline quinone (PQQ) is a widely distributed redox-active cofactor and essential nutrient. For its detection in protein-free ultrafiltrates or dialysates, a highly sensitive amplification assay was developed on the basis of PQQ's ability to catalyze redox cycling at pH 10 in the presence of excess glycine, oxygen, and nitro blue tetrazolium. Herein, we examine the propensities of PQQ, PQQ triester, and its various isomers, and certain PQQ triester derivatives, to catalyze glycine-fueled redox cycling and show that PQQ is the most capable of catalyzing redox cycling. Furthermore, PQQ has a unique pattern of inhibition induced by a series of PQQ antagonists of different potencies. The data indicate that putative PQQ from a biological sample, separated by HPLC and detected by the glycine-fueled redox-cycling assay, can be further identified as PQQ based on the profile of inhibition it displays with the antagonists such as those employed in this study. The methodology presented here should facilitate the specific detection of PQQ in biological samples.

An NADPH oxidase superoxide-generating system in the rabbit aorta.

Superoxide anion can modulate vascular smooth muscle tone and potentially affect the growth response in vascular disease. The present studies were undertaken to characterize the source of superoxide in rabbit aorta. Rings of aorta (5 mm) were incubated in physiological salt solution (PSS) for 30 min at 37 degrees C in the presence of 10 mM diethyldithiocarbamate (DDC) with or without inhibitors of superoxide-generating systems. Rings were then placed in PSS containing 250 microM lucigenin at 37 degrees C in the presence or absence of inhibitors, and changes in amounts of superoxide were determined by measuring chemiluminescence (units). The inhibitors of xanthine oxidase, oxypurinol (300 microM), and of mitochondrial NADH dehydrogenase, rotenone (50 microM), had no significant effect on superoxide levels. An inhibitor of NADPH oxidase, iodonium thiophen, caused a concentration-dependent inhibition of superoxide anion (12.49 +/- 1.48 vs 5.27 +/- 1.81 and 2.30 +/- 0.36 units, control vs 7 microM and 70 microM iodonium thiopen, respectively). A structurally related iodonium compound, diphenyleneiodonium (20 microM), caused a 78% reduction in basal and DDC-evoked superoxide levels. In the presence or absence of DDC, exogenous administration of NADPH (10 microM-1 mM), but not NADP (1 mM), elicited a concentration-dependent rise in superoxide levels that was inhibited by iodonium thiophen. Particulate fractions of whole aortic tissue exhibited NADPH-dependent superoxide production that was inhibited by 1 microM diphenyleneiodonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of redox cycling of methoxatin (PQQ), and of superoxide release by phagocytic white cells.

The iodonium compounds diphenyleneiodonium and diphenyliodonium, and the amine compounds, 4,5-dimethyl phenylene diamine, N,N-dimethyl 1,4-phenylene diamine, 1,2-diamino-4,5-methyleneoxybenzene, and aminomalononitrile inhibit methoxatin's (PQQ's) redox activity in vitro, that is, the methoxatin-coupled oxidation of glycine and reduction of nitroblue tetrazolium to formazan. The compounds mentioned above also inhibit phorbol myristate acetate (PMA) stimulated superoxide release by phagocytic white cells--determined mainly as the superoxide dismutase sensitive reduction of ferricytochrome C. Related compounds, 3,4-diaminopyridine and 4-dimethylamino-benzylamine, did not inhibit redox activity of PQQ in vitro, nor did they inhibit PMA stimulated superoxide production in monocytes or neutrophils. Thus, there is a correlation between an agent's ability to inhibit PQQ redox cycling and its ability to inhibit superoxide release by phagocytes. The findings are a further indication that PQQ is involved in the respiratory burst of phagocytic cells.

Aspects of the release of superoxide by leukocytes, and a means by which this is switched off.

Although great progress has been made in understanding the respiratory burst of leukocytes that produce superoxide (O2-), it is possible that a component or components, might have been overlooked. Furthermore, O2- production and its sequels, though cardinal in bactericidal action, might ultimately be damaging to the host's own cells. It is important, therefore, that a biologic mechanism exist to turn off O2- production by stimulated leukocytes. This article offers evidence that methoxatin (PQQ), a redox-cycling orthoquinone, might be involved in O2- production by leukocytes. This is based on the fact that inhibitors of O2- production, such as diphenylene iodonium (DPI) and 4,5-dimethylphenylene diamine (DIMPDA), were shown to sequester PQQ in leukocytes, i.e., to form adducts with that substance. Addition of PQQ to cells blocked with the inhibitors partially restored O2- release. With respect to turning off cellular O2- release, a factor was observed to be released to the medium by old macrophages (14 days old, but not by those less than 7 days old). Such conditioned medium, when added to stimulated neutrophils or macrophages, blocked O2- release. This factor was sensitive to proteases, exhibited molecular sizes of 3 and 11 kDa, and its action was independent of the nature of the stimulus applied to the leukocytes. It was partially purified by column (sizing) chromatography and HPLC. It seems to be a general modulator of the release of reactive oxygen species by phagocytes and is irrespective of phagocytic cellular type, or species from which the cells were derived.

Methoxatin (PQQ) in guinea-pig neutrophils.

PQQ, also called methoxatin, has been isolated from guinea-pig neutrophils. The organic cations diphenyleneiodonium (DPI) and diphenyliodonium (BPI) and the aromatic o-diamine 4,5-dimethylphenylenediamine (DIMPDA) sequester synthetic PQQ and inhibit its redox-cycling activity in a model system. Standards were made of adducts of tritiated PQQ with unlabeled DIMPDA and of unlabeled PQQ with tritiated DPI or DIMPDA. PQQ adducts were isolated from guinea-pig neutrophils with each of the tritiated inhibitors. They were separated and defined by high-performance liquid chromatography (HPLC). Tiron, a disodium benzene disulphonic acid, broke the DPI-PQQ adduct isolated from neutrophils and released free PQQ. Both DPI and DIMPDA, as well as BPI, blocked O2.- release by stimulated neutrophils. The blockade exerted by these inhibitors was released by the addition of PQQ to the cell suspensions. The data demonstrate the presence of PQQ in guinea-pig neutrophils and suggest that it has a possible role, direct or indirect, in the O2.(-)-producing respiratory burst.

The putative essential nutrient pyrroloquinoline quinone is neuroprotective in a rodent model of hypoxic/ischemic brain injury.

Pyrroloquinoline quinone is a ubiquitous redox cofactor and putative essential nutrient in mammals. Pyrroloquinoline quinone has recently been demonstrated to depress N-methyl-D-asparate induced electrical responses and is neuroprotective in vitro. In addition, pyrroloquinoline quinone has been demonstrated to act as a free radical scavenger in mammalian tissues. In this study, we demonstrate a neuroprotective effect of pyrroloquinoline quinone in an in vivo cerebral hypoxia/ischemia model in the rodent. Significant reduction in infarct size resulted from pyrroloquinoline quinone pretreatment and also when pyrroloquinoline quinone was administered following induction of hypoxia/ischemia. The neuroprotective effect was not dependent on change in core or cranial temperatures, as there was no difference between temperature measurements in pyrroloquinoline quinone-treated and vehicle-treated controls. No changes in electroencephalographic activity were observed at neuroprotective doses. These findings suggest that pyrroloquinoline quinone may represent a novel class of quinoid reagents of potential use in the treatment of neurological disorders that involve excitotoxicity. This study demonstrates a protective effect of the novel essential nutrient pyrroloquinoline quinone on brain injury in a rodent model of cerebral hypoxia/ischemia. Pyrroloquinoline quinone was neuroprotective when administered before and even after the insult, and did not appear to have significant neurobehavioral side effects. Pyrroloquinoline quinone represents a new class of agents with potential use in the therapy of stroke.

Covalent protein crosslinks: general detection, quantitation, and characterization via modification with diphenylborinic acid.

Progressive crosslinking of proteins appears to be a general phenomenon in aging cells and tissues. Crosslinked proteins can form insoluble aggregates which become increasingly resistant to proteolysis as more crosslinks form. However, most evidence for progressive crosslinking with age is indirect, and little is known about the chemical mechanisms involved. We have therefore developed a method for detection and isolation of any type of stable covalent crosslink from protein hydrolysates which requires no prior knowledge of the molecular structure of whatever crosslink(s) may be present. It utilizes the specificity of the diphenylborinic acid reagent for alpha-amino acid groups and the chromatographic properties and uv absorbance of the crosslink derivatives. The method is demonstrated using eight different crosslinks from collagen and fibrin, and a general procedure is given for detection of any type of crosslink in a protein hydrolysate.

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.

Characterization of the glycine-dependent redox-cycling activity in animal fluids and tissues using specific inhibitors and activators: evidence for presence of PQQ.

Pyrroloquinoline quinone, a redox cofactor first isolated from bacteria, efficiently catalyzes the nonenzymatic oxidation of glycine in the presence of nitroblue tetrazolium. We report that certain metallic cations and heterocyclic aromatic cations, like the N-methyl phenazonium cation and aryl-iodonium compounds, strongly and specifically inhibit this redoxcycling activity. The inhibition by metal cations is reversed by Tiron and that of the aromatic cations by Tiron and thyroxine. These inhibitors and activators affect authentic PQQ and the redox-activity of putative PQQ isolated from biological sources in a similar manner. This indicates that pyrroloquinoline quinone occurs naturally in animal tissues and fluids.

Reaction of lysyl oxidase with trans-2-phenylcyclopropylamine.

trans-2-Phenylcyclopropylamine hydrochloride (tranylcypromine; TCP) was found to be both an inhibitor and a substrate of lysyl oxidase, the enzyme which oxidizes peptidyl lysine in elastin and collagen to initiate cross-linking in these proteins. The reaction of TCP with this enzyme was further characterized in view of the potential interference that chronic administration of this antidepressant compound may exert on the development and repair of connective tissues. In contrast to the irreversible and/or competitive inhibitors of lysyl oxidase previously described, TCP noncompetitively and reversibly inhibited the oxidation of both alkylamine and elastin substrates with Ki values of 386 and 375 microM, respectively. The noncompetitive mode of interaction affected the accessibility of the active site to productive amine substrates since the reductive trapping of n-hexylamine to lysyl oxidase was largely prevented by the presence of TCP. It was of additional interest that lysyl oxidase catalyzed a limited degree of conversion of TCP to cinnamaldehyde accompanied by the production of hydrogen peroxide. The lack of significant incorporation of protein-bound tritium accompanying reduction of the enzyme-TCP complex with [3H]NaBH4 argued against the formation of a Schiff base between the enzyme and the cinnamaldehyde product as the basis of the inhibitory effect. Spectral evidence was also obtained for an additional interaction between TCP and lysyl oxidase that was independent of the inhibitory effect of TCP. Cyclopropylamine, lacking the benzene moiety of TCP, inhibited lysyl oxidase irreversibly and competitively, and was not a substrate, pointing toward a defining role for the benzene moiety in the interaction of TCP with lysyl oxidase.

Is the antioxidant, anti-inflammatory putative new vitamin, PQQ, involved with nitric oxide in bone metabolism?

Our laboratory recently isolated free PQQ (2,7,9-tricarboxy-pyrroloquinoline quinone, methoxatin), a bacterial redox cofactor, from red cells, neutrophils, serum and milk and found free PQQ in CSF, synovial fluid and bile. The metabolism and functions of PQQ and ascorbate may be coupled. Physiologically, free PQQ catalyzes dioxygen-superoxide interconversion, and participates in both superoxide generation (respiratory burst) and scavenging (cell protection). Using a labeled aromatic o-diamine, superoxide formation by activated neutrophils was inhibited and the labeled phenazine adduct of PQQ could be isolated from the inhibited cells (Karnovsky et al., 1992). PQQ may convert xanthine oxidase to xanthine dehydrogenase (XD) and could be the physiological coenzyme of XD. PQQ plus copper, form a potent amine-oxidizing system. Shah et al., 1992 found that PQQ-Cu2+ catalyzes the oxidation of epsilon-amino groups in collagen and elastin. Rucker's lab (Smidt et al., 1991) has found that PQQ may be a vitamin for mouse pups. Watanabe et al., 1988 and Nishigori et al., 1989, showed that injected PQQ protects animals against oxidative stress injury. PQQ's in vivo antioxidant action, spares reduced glutathione. PQQ, as an actively transported organic anion, concentrates in cells. In other experiments (Aizenman et al., 1992), PQQ protected neurons against the neurotoxin action of the glutamate-receptor against NMDA. We shall consider possible roles for PQQ in the biosynthesis of nitric oxide (NO, endothelium-derived relaxing factor, EDRF) from L-arginine and in NO removal by superoxide. NO has now been linked to the inhibition of osteoclastic bone resorption.

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.

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.

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.

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.

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)