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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.

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.

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.

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.

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-peformance liquid-perfomance 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.

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 metalic cations and heterocyclic aromatic cations, like the N-methyl phenazonium cation and aryl-iodonium compounds, strongly and specifically inhibit this redox-cycling 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.

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.

Hypusine (N(-)-(4-amino-2-hydroxybutyl) lysine), a spermidine-dependent post-translational protein modification, is synthesized by various mammalian cells in culture. Experiments described in this paper demonstrated a relationship between rates of cellular growth and the synthesis of hypusine. Cells that divide at fast rates have a high rate of hypusine synthesis. In kinetic experiments, a positive relationship is evident between the rates of protein, DNA and hypusine synthesis. Cells seeded at high density, growing non-exponentially, synthesized less hypusine than logarithmically growing cells seeded at low density. Slowing the growth rate of cells by modification of the external milieu also results in a decreased rate of hypusine synthesis. These results provide additional evidence of the association of hypusine with cell proliferation in cultured cell lines and suggest a possible role for this unusual post-translational modification in the complex macromolecular events leading to cellular growth.

The pentapeptide Phe-Leu-Glu-Glu-Leu, tritiated at the gamma carbon of each Glu residue, has been synthesized. In a system using microsomal preparations derived from rat liver, vitamin K-dependent tritium release from the L-Glu residues of this substrate can occur without the concurrent gamma-carboxylation of Glu. This tritium release reaction, which indicates cleavage of the gamma C-H bond, although easily uncoupled from CO2-dependent gamma C carboxylation, does require the reduced (hydroquinone) form of vitamin K and oxygen. The data argue against a concerted mechanism for the cleavage of the gamma C-H bond and carboxylation and against a mechanism in which the vitamin functions solely to transfer or activate CO2. Although the tritium release is related clearly to the oxidation of vitamin KH2, it is not yet established how the subsequent carboxylation proceeds. However, two carboxylation mechanisms compatible with the results are discussed.

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.

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.

An unknown amino acid was purified from bovine ligament elastin. The compound was shown by proton NMR to have a pyridinium ring structure similar to isodesmosine, yet containing an olefinic double bond on one additional side chain which was not attached to the pyridinium ring. Mass spectral analysis confirmed the NMR data and indicated a parent compound with a mass of 653. A structure is proposed that is derived from the condensation of five lysine residues. The trivial name of pentasine is proposed for this compound.

Hypusine-containing protein identified as eukaryote initiationtranslation factor 4D was labeled with [ 14 C]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.

Mass spectral evidence is presented that the elastin of bovine ligamentum nuchae contains, in addition to desmosine and isodesmosine, larger levels of their respective cyclic dehydrodesmopiperidine precursors. In addition to being precursors of the desmosines, such compounds are most likely cross-links in themselves. In fact, the occurrence of these compounds, now isolated as their reduced derivatives, may clarify the discrepancies in the stoichiometry of the conversion of lysine residues to cross-linking components as noted by several workers (1,2,3). With this study the occurrence of all of the tetrafunctional amino acid cyclic structures related to the desmosines is now confimed by mass spectrometry.

A new trifunctional crosslink, 2,10-diamino-5-hydroxymethyl-6-(N-1-histidyl)-undecandioic acid, termed aldol-histidine, was isolated from borohydride reduced cow skin insoluble collagen. The compound was characterized by pmr spectroscopy and mass spectrometry of several volatile derivatives. The crosslink may be derived from Michael addition of a histidine residue to the known aldol crosslink, 2,10-diamino-5-formyl-5-undecandioic acid. This is the first example of a histidine containing crosslink found in structural protein.

Summary Chromatographic fractionation of acid hydrolysates of N aBH4-reduced reconstituded collagen fibrils was carried out. Two compounds were isolated and shown to be hydroxylysinonorleucine and its lactone. The lactone underwent dehydration under certain circumstances yielding two new compounds which by mass spectrometry were both found to be Δ3,4-dehydrolysinonorleucine. The two compounds are probably the cis, trans isomers of Δ3,4-dehydrolysinonorleucine.

Previous studies have identified gamma-carboxyglutamic acid as a constituent of one or more protein(s) synthesized by rat and chicken kidney microsomes in vitro in a vitamin K-dependent post-translational reaction [1]. Incubation of microsomes from a mouse kidney cell line (RAG) with [14C]NaHCO3 results in formation of protein-bound [14C]gamma-carboxylglutamic acid. Incorporation is stimulated threefold by addition of the active vitamin K compound 2-methyl, 3-farnesyl, 1,4-naphthoquinone. At least 90% of incorporated, nondialyzable [14C] is situated in the gamma-carboxyl group of gamma-carboxyglutamic acid residues.

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.

A direct approach has been developed for quantitative identification of the calcium-binding amino acid, gamma-carboxyglutamate, in proteins. This should be advantageous for the study of numerous systems where specific roles for the binding of calcium or other divalent cations are suspected. Investigation of mineralized tissue, where calcium-binding proteins are implicated in the mineralization process, revealed that gamma-carboxyglutamate was present in proteins solubilized from chicken bone with neutral aqueous ethylenediamine tetraacetic acid. This was established by direct isolation of the amino acid from alkaline hydrolysates and its quantitative conversion to glutamic acid by decarboxylation in 0.05 M HCl at 100 degrees. The kinetics of decarboxylation and chromatographic behavior are identical to those of gamma-carboxyglutamate from human prothrombin. After resolution of the soluble bone proteins by phosphate gradient elution from hydroxyapatite, gamma-carboxyglutamate was found to be concentrated primarily in one BaSO4-adsorbable anionic protein species; bone collagen was devoid of the amino acid. In view of the recently discovered requirement of vitamin K for generation of calcium binding sites (gamma-carboxyglutamate) by gamma-carboxylation of specific glutamic acid residues in prothrombin, our findings may implicate vitamin K metabolism in normal bone development and suggest a role for the gamma-carboxyglutamate-rich protein in regulation of calcium salt deposition in mineralized tissues.

A new process has been developed which is called "Boradeption" to signify boronic acid--dependent phase transfer of water-insoluble agents. Highly fluorescent boronic acid dervatives, FluoroBoras, are solubilized with a physiologically compatible carrier buffer containing a receptor group for boronate adduct formation. The system can be used to stain living cells. In another variation of the Boradeption concept, an insoluble reporter molecule containing a boronate receptor is solubilized with a carrier buffer containing a boronic acid functional group. The boronate-receptor complexes, which are in dynamic equilibrium, can be designed as vital stains and reagents for a variety of biological and medical applications.

Elastin from anatomically defined regions of young calf lung and dog aorta was isolated and purified by a procedure which sequentially removed lipids, collagen, structural glycoproteins, and the microfibrillar proteins without apparent damage to the cross-linking residues, which have been shown to be sensitive to autoclaving and hot alkali treatment. One of the methods described was effective in obtaining pure elastin from lung parenchyma. Visceral pleura was found to be the richest source (25% dry weight) of elastin in the lung tissues examined. The amino acid compositions of the elastins purified by different methods were compared for purity and for the detection of possible damage to cross-linking compounds. Cross-linking profiles were obtained by column chromatography either after reduction with 3[H]NaBH4 or after reaction with 14[C]NaCN and NH3. The 3[H]NaBH4 method, under carefully controlled conditions, proved not to be quantitatively reproducible. The reaction of elastin with 14[C]NaCN and NH3 appeared preferable due to its reproducibility; this procedure required one type of hydrolysis for the analysis of all the cross-linking compounds. Examination of the cross-linking profiles of the elastins from various tissue regions revealed differences in the type, distribution, and quality of cross-links.

A new trifunctional crosslink, termed hydroxyaldol-histidine, was isolated from cow skin collagen. This compound was not reducible by sodium borohydride; it was characterized by PMR spectroscopy and by low and high resolution mass spectroscopy of volatile derivatives. This crosslink is identical to an unknown amino acid previously detected in pure collagen-derived peptides. We postulate that it arises by condensation of peptidyl allysine, hydroxyallysine and histidine. This is the first example of a non-borohydride reducible crosslink found in collagen.

Several peptides synthesized by primary sensory neurons are α-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 α-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.

Logarithmically growing Chinese hamster ovary cells, cultured in the presence of [1, 4- 14 C] putrescine, synthesize a protein(s) containing the unusual amino acid hypusine [ N e-(4-amino-2-hydroxybutyl)lysine]. This protein was separated and identified by two-dimensional gel electrophoresis and fluorography. The labeled hypusine isolated from an acid hydrolysate of the cell protein by ion exchange chromatography was identified by oxidative degradation and analyses of the products. Hydralazine, one of the most frequently prescribed drugs for the treatment of moderate to severe hypertension, added to the culture, resulted in the accumulation of a protein(s) containing the precursor amino acid deoxyhypusine [ N e-(4-aminobutyl)lysine]. Demonstration of this intermediate and its subsequent conversion to hypusine suggests that the synthesis occurs in several steps, one of these involving a hydroxylation reaction which can be inhibited by hydralazine.

Summary A four-fold difference in the number of valyl-proline sequences recovered from bovine ligamentum nuchae elastin and ear cartilage elastin indicates that there are significant primary sequence differences between the two types of elastin. These results support our previous suggestion that the elastin associated with elastic cartilage is a different genetic type of elastin.

A new trifunctional crosslink, isolated from NaB3H4-reduced calf skin insoluble collagen, is described. The postulated structure of this compound, hydroxymerodesmosine, is based upon the analysis and comparison of its mass spectral fragmentation pattern with other crosslinks obtained from collagen and elastin.

Treatment of calf skin insoluble collagen with NaB 3 H 4 followed by acid hydrolysis and ion-exchange chromatography yields a new compound which is prominent in the chromatograms of several collagens. We now describe this compound as reduced N ϵ -hexosylhydroxylysine. It appears to arise by reduction of the Schiff base between the carbonyl moiety of a hexose and the ϵ-amino group of hydroxylysine; the postulated structure was derived from both high- and low-resolution mass spectrometry and by comparison with synthetic N ϵ -galactosylhydroxylysine. Conceivably, the unreduced compound may be a crosslink, uniting collagen and glycoproteins or proteoglycans in the connective tissue.

A summary of results and ideas concerning special features of the covalent structure of collagen is presented. Our recent data on the nature of aspartyl ester pairs in collagen cross-linking and speculations about how these may function in fiber maturation are discussed. An important new aldehydic component associated with the cross-link sites has been detected and is under investigation. A model of collagen based upon 4 subunits of 25,000 to 30,000 per alpha component has been developed. These subunits appear to be held together in a linear array by three pairs of ester bonds. A more detailed picture of the distribution of "crystalline" and "amorphous" regions along the tropocollagen molecule has been proposed primarily based on the results of analyses of collagen peptide fractions obtained with collagenase. Other topics such as gamma-glutamyl bonds in collagen and results with carbonyl group-detecting reagents which demonstrate the presence of small amounts of alpha-keto acid groups are briefly considered.

Following reduction with sodium borotritide, bone and dentine collagens are found to contain only a few labelled substances, in contrast with the soft tissue collagens. The reduced aldehydes, ϵ-hydroxynorleucine (HNL) and σ, ϵ-dihydroxynorleucine (DHNL) are abundant in the mineralized collagens and their relative proportions differ in fetal and mature tissues. The reduced crosslinks, σ-hydroxylysinonor leucine (HLNL) and σ, σ-dihydroxylysinonorleucine (DHLNL) together account for a major portion of the radioactivity and their relative abundance varies with age. In contrast with the results of others, we find that DHLNL is the major reducible crosslink of mineralized collagens, comprising up to 60% of the total radioactivity. Moreover, reduction with NaBD4 shows that a portion of the Schiff base precursor of DHLNL becomes reduced in vivo. The marked insolubility of bone and dentine collagens may, in part, be attributed to such in vivo reduction.

Summary The crosslinks in elastin of bovine ligamentum nuchae and of the intimal-medial and adventitial segments of rat thoracic aorta were evaluated comparatively. Reaction with [14C] -sodium cyanide and ammonia was used to stabilize and identify aldehydes and certain aldehyde-derived crosslinks. The derivatives obtained demonstrated the presence in the aortic elastins of α-amino adipic acid σ-semialdehyde, dehydrolysinonorleucine and the aldol condensate of αamino adipic acid σ-semialdehyde. In addition to lysinonorleucine and the desmosines, other, but as yet uncharacterized compounds were found to be present.

Summary Elastin of bovine ligamentum nuchae was prepared without the use of hot alkali and subsequently reduced either with 3[H]NaBH4 or 3[H]NaBD4. Reduction with NaBD4 permitted the measurement of the ratio of merodesmosine to dehydromerodesmosine in natural elastin, and in this case was found to be approximately one. Thus in ligamentum elastin, untreated with hot alkali and/or borohydride, two kinds of Schiff base crosslinks, dehydrolysinonorleucine and dehydromerodesmosine, have been shown to occur as such. They also occur naturally in the respective forms of their reduced derivatives, lysinonorleucine and merodesmosine.

THE clear-cut demonstration by Piez1 that the 3 α-chains of cod-fish skin tropocollagen are unique, and the likelihood that a similar condition is true for all species of tropocollagen, have led me to consider various simple arrays of sub-units within the chains which could explain their known compositional features. I have arrived at a model in which an α-chain consists of 6 sub-units, each of approximately 17,000 molecular weight, and falling into 3 different compositional types that I call A, B and C, respectively. A tropocollagen unit, then, in this model would consist of a total of 18 sub-units: 6A, 6B, 6C. Of all the possible combinations of 6 sub-units of 3 types in an α-chain, one which fits the known analyses of the amino-acid compositions of the several types of α-chains in the various tropocollagens is the following: α1 = 3A + 2B + C; α2 = 3C + 2A + B; α3 = 3B + 2C + A For purposes of reference we designate this particular model as the 3,2,1 : A,B,C model, indicating that each different α-chain in the tropocollagen unit consists of a different array of the sub-units, but similar in that it has 3 of one kind of sub-unit, 2 of a second, and 1 of the third.