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This chapter presents an overview of the biomedical significance of Pyrroloquinoline quinone (PQQ). It was first isolated from cultures of methylotrophic bacteria as an acetone adduct and its structure determined by X-ray diffraction and confirmed by organic synthesis. PQQ captures electrons from primary amines, catechols, catecholamines, ascorbic acid, and NAD(P)H to form PQQ-semiquinone and/or PQQ-hydroquinone. In a collaborative study with Dr. Manfred Karnovsky, an expert in respiratory burst biochemistry, they have recently shown that superoxide may be derived from a reaction involving the dioxygen oxidation of a reduced ortho-quinom, probably PQQ, which is present in phorbol myristate acetate-activated guinea pig peritoneal exudate neutrophils. The recent reports on the anti-inflammatory and antioxidant actions of PQQ are discussed. Rats given a variety of hepatotoxins capable of destroying the liver through the induction of oxidative stress injury are protected by PQQ in a dose-dependent manner. PQQ is a potent antioxidant and anti-inflammatory agent, which could be used pharmaceutically.

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.

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.

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 occured 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/CuA(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 α- maminoadipic-δ-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.

PQQ (methoxatin), a bis(quinone) tricarboxylic acid, is an organic cofactor in a variety of biological redox processes. It is effectively inhibited on a micromolar scale by alkynyl and aryl monoiodonium salts, whereas bis-(iodonium)triflates 5 and 8 are PQQ inhibitors at nanomolar levels

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.

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 [1]. Because spectroscopie data were found to be inconsistent [2] 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.

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.

Hemoglobin A1c, the most abundant minor hemoglobin component in human erythrocytes, is formed by the condensation of glucose with the N-terminal amino groups of the beta-chains of Hb A. The biosynthesis of this glycosylated hemoglobin was studied in vitro by incubating suspensions of reticulocytes and bone marrow cells with [3H]leucine or 59Fe-bound transferrin. In all experiments, the specific activity of Hb A1c was significantly lower than that of Hb A, suggesting that the formation of Hb A1c is a posttranslational modification. The formation of Hb A1c in vivo was determined in two individuals who were given an infusion of 59Fe-labeled transferrin. As expected, the specific activity of Hb A rose promptly to a maximum during the 1st week and remained nearly constant thereafter. In contrast, the specific activity of Hb A1c and also of Hbs A1a and A1b rose slowly, reaching that of Hb A by about day 60. These results indicate that Hb A1c is slowly formed during the 120-day life-span of the erythrocyte, probably by a nonenzymatic process. Patients with shortened erythrocyte life-span due to hemolysis had markedly decreased levels of Hb A1c.

The content of osteocalcin and protein bound gamma-carboxyglutamic acid (Gla) was studied as a function of bone maturation and mineralization in normal and vitamin D-deficient, rachitic chickens. The Gla/Ca2+ ratio was elevated in rachitic bone, particularly in the most undermineralized regions. For example, there is a 10- to 20-fold elevation in Gla/Ca2+ in the newly synthesized, least mineralized rachitic bone fraction, which progressively decreases to a 1.5-fold elevation in the most highly mineralized areas of rachitic tissue. Osteocalcin, which is the principal Gla-containing protein of mature bone, was quantitated by radioimmunoassay using specific antiserum to the 5670-dalton chicken protein. Surprisingly, the osteocalcin concentration is decreased 50% in vitamin D-deficient bone. From this we infer that accumulated Gla-containing protein in vitamin D-deficient and poorly mineralized bone may possibly represent a precursor of osteocalcin.

The activation of prolyl hydroxylase and lysyl hydroxylase by ascorbate was studied in young and senescent WI-38 fibroblast cultures using a tritium-release assay. Prolyl hydroxylase activity could be increased 3–4 fold in young cultures but remained unchanged in senescent cultures when these cultures underwent a two-hour preincubation in medium containing 0.2mM sodium ascorbate. Lysyl hydroxylase levels were unaffected both in young and senescent cultures. In another series of experiments, ascorbate was replaced with several other compounds in the tritium-release assay demonstrating that this reducing agent is not a specific cofactor of the partially purified enzymes from WI-38 cultures.

Methods for quantitative analysis of the carboxylated amino acids, aminomalonic acid, β-carboxyaspartic acid, and γ-carboxyglutamic acid, are presented. These substances are acid labile and thus can be measured only after alkaline hydrolysis of proteins and peptides. Half-times for decarboxylation in 1 m HCl at 100°C are: aminomalonic acid (1.2 min); β-carboxyaspartic acid (1.7 min); and γ-carboxyglutamic acid (8.6 min). This property is useful for unequivocal identification in complex hydrolysates.

Osteocalcin is a vitamin K-dependent protein, synthesized in bone, which can be detected in serum. We have measured circulating osteocalcin levels in 10 patients with x-linked hypophosphatemia (XLH) and in 6 patients with autosomal recessive vitamin D dependence (ARVDD) who started 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] therapy. Patients with XLH were studied before and after 7-12 months of therapy that included 1,25-(OH)2D3 (10-72 ng/kg x day) and oral phosphate. Serum osteocalcin rose from 28 +/- 12 to 52 +/- 12 ng/ml (mean +/- SE; P less than 0.01) in concert with improvements in biochemical status and bone mineralization. Vitamin D therapy was withdrawn for 2 weeks from patients with ARVDD. The vitamin D-deplete status was evidenced by low 1,25-(OH)2D3 levels (12 +/- 2 pg/ml; n = 6). After 1 week of therapy with 1,25-(OH)2D3, serum calcium rose from 9.03 +/- 0.21 to 9.67 +/- 0.25 mg/dl (P less than 0.002), while serum phosphorus and alkaline phosphatase remained unchanged. Serum osteocalcin rose from 35 +/- 7 to 83 +/- 32 ng/ml (P less than 0.05). At 3 weeks, serum calcium remained elevated (9.63 +/- 0.18 ng/dl) over control levels (P less than 0.01); phosphorus and alkaline phosphatase were still unchanged. Serum osteocalcin rose to 114 +/- 42 ng/ml, significantly greater than values at 1 week (P less than 0.05). Thus, serum osteocalcin increases after 1,25-(OH)2D3 therapy in both ARVDD and XLH.

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.

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.

Acid-treatment facilitates PQQ detection by electron ionization mass spectroscopy with a molecular ion atM/e 330 and a base ion formed by triple decar☐ylation atM/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 car☐yl group, presumably the 9-car☐yl, 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 atM/e 332 with a base ion atM/e 200. There is compelling evidence for a dehydrogenated lactone, PQQ(−2H)L), atM/e 328 with a base ion atM/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 D 2 O, PQQ shows deuterated products with ions atM/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 atM/e 450, 452 and 454 and decar☐ylation-derived strong (base) ions atM/e 318, 320 and 322. Esterification of PQQ-DAN acid-products with triethyloxonium tetrafluoroborate gives triethyl esters derived from the 452 and 454 dalton compounds with very strong molecular ions atM/e 536 and 538, respectively. Ions derived from diethyl esterification of the 450 dalton compound atM/e 506 identify the 450 compound as the dehydrogenated monolactone of PQQ-DAN. Acid-hydrolyzed PQQ placed in neutral buffers yields PQQ(2H) as the main product with some PQQ also present. Both catalyze redox cycling and amplified formazan formation with glycinate and nitroblue tetrazolium. Therefore, acid hydrolysis of quinoproteins releases PQQ-products, detected well by redox-cycling. These products are not detected efficiently with carbonyl reagents in acid since acid-treatment of PQQ leads to 1) carbonyl group reduction and PQQ(2H) formation and 2) carbonyl masking of remaining PQQ by tautomeric lactonization.

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.

We have measured serum osteocalcin by radioimmunoassay, and urinary gamma-carboxyglutamic acid (Gla) by Dowex-1 chromatography in healthy adults and children. Circulating osteocalcin is 6.8 +/- 0.5 and 5.8 +/- 0.5 ng/ml in adult males (n = 25) and females (n = 26), respectively. Values are higher in children (n = 26), ranging from 25-30 ng/ml in a 1-year-old and declining to the adult level at puberty. The ratio of urinary Gla/creatinine as a function of age parallels the pattern of serum osteocalcin. Free Gla ranges from a high of 150 +/- 20 mumol Gla/g creatinine in infants (n = 17), decreasing until puberty at which time the excretion stabilizes at 44 +/- 4 mumol Gla/g creatinine (n = 27). The greater amounts of circulating osteocalcin and urinary Gla most likely reflect synthesis of osteocalcin during growth. With epiphyseal closure, levels decline and, in the adult, probably represent normal bone remodelling.

A sensitive fluorometric method for the quantitation of hemoglobin glycosylation, based upon periodate oxidation of the carbohydrate moieties present on both the α- and ϵ-amino groups of globin is described. The formaldehyde product is measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the condensation of formaldehyde with acetylacetone and ammonia. This method is rigorously designed to assay glycosylated hemoglobin levels and to give a direct measure of the number of glycogroups per milligram of hemoglobin. It requires only 1 mg of protein and may also be used to determine the extent of the nonenzmatic glycosylation of other proteins.

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.

Glucose reacts nonenzymatically with the NH2-terminal amino acid of the beta chain of human hemoglobin by way of a ketoamine linkage, resulting in the formation of hemoglobin AIc. Other minor components appear to be adducts of glucose 6-phosphate and fructose 1,6-diphosphate. These hemoglobins are formed slowly and continuously throughout the 120-day life-span of the red cell. There is a two- to threefold increase in hemoglobin AIc in the red cells of patients with diabetes mellitus. By providing an integrated measurement of blood glucose, hemoglobin AIc is useful in assessing the degree of diabetic control. Furthermore, this hemoglobin is a useful model of nonenzymatic glycosylation of other proteins that may be involved in the long-term complications of the disease.

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.

The use of a reaction involving [14C]cyanide and ammonia to stabilize and identify aldehydic cross-links in elastin and collagen is described. The products resulting from acid hydrolysis are in the form of 14C-labeled amino acids. The reaction was first studied with periodate-treated collagens, used as a model of collagen with aldehyde side chains. In these collagens the α-aminoglutaryl γ-semialdehyde residues react with [14C]cyanide and ammonia almost quantitatively and the expected product, after acid hydrolysis, is predominantly [14C]α, δ-diaminoadipic acid. This product indeed was isolated and identified. In elastin three amino acids derived from cross-links were isolated and their structures established. They are α,e-diaminopimelic acid, 1,9-diamino-1,9-dicarboxy-4-glycino-non-4-ene (carboxy amino aldol), and 2-amino-6-lysinopimelic acid (carboxylysinonorleucine). These compounds arise, respectively, from modification of residues of α-amino adipic acid δ-semialdehyde,aldol condensate of α-aminoadlpic acid δ-semialdehyde, and dehydrolysinonorleucine, and are in full agreement with structures of such compounds previously deduced using established sodium borohydride reduction procedures. Aldimines are modified with cyanide directly, not requiring the presence of ammonia. Cross-links already in the reduced form in the native protein, and the pyridinium forms of the desmosines do not incorporate the label and can be directly observed and quantitated by established procedures. The contents of modified cross-links can be estimated when the specific activity of Na14CN is known.

Vitamin K-dependent formation of the Ca 2+ -binding amino acid γ -carboxyglutamate is an important post-translational modification of proteins found in mineralized tissues. These and other carboxylated proteins, such as the one in kidney cortex, may have regulatory functions in calcium metabolism.

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.

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.

γ-Carboxyglutamic acid (Gla) is identified in the proteins associated with several types of ectopic calcifications in which hydroxyapatite is the major mineral component. These included the calcified skin and subcutaneous plaques from a patient with dermatomyositis, the calcium containing material extruded from the skin of a patient with scleroderma, and the calcified, atheromatous plaques from aorta. Alkaline hydrolysis of biopsy material from these and from normal tissue revealed the presence of Gla only in the plaque specimens. Since a γ-carboxyglutamic acid-containing protein is normally present in bone and absent in unmineralized tissues, the presence of Gla in soft tissue calcifications is a potentially significant finding, especially in view of its known calcium and phospholipid binding properties.

Modification of erythrocyte membrane sialoglycoprotein(s) in isolated form, or when in membrane, is described using sequential sodium periodate oxidation and tritiated sodium borohydride reduction, essentially as suggested by Van Lenten and Ashwell ((1971) J. Biol. Chem. 246, 1889). Conditions of modification were investigated; under the optimal but nondestructive conditions selected, the modification appears specific for sialyl residues and leads to incorporation of tritium into the modified product, 5-acetamido-3,5-dideoxy-l-arabino-2-heptulosonic acid, identified by its susceptibility to neuraminidase and mild acid hydrolysis, and by its identity with the authentic compound in chromatographic behavior, several colorimetric assays, and extent of tritium incorporation. The selectivity of the modification for the sialoglycoprotein(s) is further demonstrated by carrying out the modification on solubilized membrane proteins; only the sialoglycoprotein(s) incorporates the label. In the modification of the intact membranes some lipid components also become radioactive. The modification of intact erythrocytes, the membranes, or the isolated sialoglycoprotein(s) results in loss of the M blood group activity, but the A and B blood group activities and the phytohemagglutinin binding sites are not affected. The modification offers a handle for isolation of membrane sialoglycoprotein(s) and study of their disposition, and possibly, for evaluation of the physiological role of the terminal polyhydroxy side chains of their sialyl residues.

A brief review of the status of current knowledge on the maturation of connective tissue proteins is presented and includes new information relating to collagen and elastin crosslinks. Some consideration is given to pertinent methodology, such as the NaBH4 reduction technique where tritium or deuterium is either in the reagent or the solvent or in both. The most recently developed methodology involving [14C] cyanide incorporation into various maturation compounds is also briefly considered. In addition, a general overview of the field and of some of the remaining problems is given.

Reduction of insoluble collagens from bone, skin, and tendon with NaB3H4 produced several radioactive substances which were isolated from acid hydrolysates of the protein. One of these substances, aldol-histidine, was most abundant in cow skin collagen and also appeared in reconstituted fibrils of purified cow skin tropocollagen. Another compound, histidino-hydroxymerodesmosine, was abundant in all of the collagens studied, and it appeared to be structurally related to aldol-histidine. The postulated structures of both compounds were obtained primarily by high and low resolution mass spectrometry and by high resolution nuclear magnetic resonance spectrometry. Ancillary data were provided by ultraviolet spectrometry and by colorimetric methods. Both compounds were present in partially purified, large peptides obtained from cyanogen bromide digests of insoluble collagen and of reconstituted collagen fibrils. These results suggest that aldol-histidine and histidino-hydroxymerodesmosine serve as cross-links in collagen, potentially uniting three or four polypeptide chains, respectively.

PRIMARY STRUCTURE OF THE CoLLAGENS. • . . . . . . . . . . . . . . . • . • . .. ...... ... .... 618 Collagens of chain composition (a1h a2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 619 Collagens of chain composition [a1(II)]a and [al(III)Ja. ... . . ......... . . . . . .. 627 Collagen of chain composition [aI, a2, a3] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 628 BIOSYNTHESIS OF CoLLAGEN MOLECULES...... .. . . • . . .. . .. . . .. .. . . . . . .. . ..... 630 HYDROXYLATION OF PROLINE AND LYSINE RESIDUES IN CoLLAGEN.... . . . . ....... 634 BASEMENT MEMBRANES AND THE GLYCOSYLATION OF CoLLAGENS.... .. . . . .. ..... 637 ELASTIN. . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 640 CROSSLINKS OF CoLLAGEN AND ELASTIN ...... ... ...... 652 Lysine (L) . . .. .. .... .. .. . . ... . ..... . .. . .. . . . ..... 653 o-Hydroxylysine (HL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 653 a-Amino adipic acid a-semialdehyde (AL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 653 a-Hydroxy a-amino adipic acid a-semialdehyde (HAL) . . . . . . . . . . . . . . . . . . . .. .. 654 Dehydrolysinonorleucine (deLNL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 655 Lysinonorleucine (LNL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. 655 "Aldor' (ALAL).... . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. 656 Dehydrohydroxylysinonorleucine (deHLNLa, deHLNLb) . . . . . . . . . . . . . . . . . . . . . 656 o-Hydroxylysinonorleucine (HLNL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 656 Dehydrohydroxylysinohydroxynorleucine (deHLHNL). . .... ...... 656 Hydroxylysinohydroxynorleucine (HLHNL) ... . ... " .. . . .. . . .. .. .. . . . . ..... 657 Syndesine (S). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 657 Dehydromerodesmosine (deM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 657 Merodesmosine (M). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 657 Dehydrohydroxymerodesmosine (deHM) . . . . . . .. . .. . .. . . .... .. .... ......... 657 Dihydrodesmosines (dihydroD) . . . . . . . . . . . . . . . . .. . . . . . . . " ... .. 658 Desmosines (D) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 658 Tetrahydrodesmosines (tetrahydroD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 658 Compound 285 . . . . . . . . . . . . . . . . ...... . . ....... . . . . . .... . . . . . .. . . . . . . . . , 659 Posthistidines . .. . . . . ....... . . . . . . . . .... " .. . . . . . . . . . . . .. .. 659 Compound 16 . . . . . . . . . . . . . . . . . ...... . ..... . . . . " .. .. .. .. .... . . . . ....... 659 COLLAGENASESAND ELASTASES . . • . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . 660 CoNCLUDING REMARKS 660 APPENDIX . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . 664

Kinetic analysis was made of the dialysis of digests of ichthyocol gelatin prepared with collagenase. It was found that most, if not all, of the peptides in such digests are dialyzable, but at greatly different rates. First, there is a class of slowly dialyzable peptides that contain most of the tyrosine, ester groups, and carbohydrate moieties of the parent protein molecule. Second, there is a class of slowly dialyzable peptides, which are rich in the polar amino acids and appear to originate from the polar or “noncrystalline” regions of the protein molecule. Third, there is a class of rapidly dialyzable peptides that are rich in proline and hydroxyproline and appear to originate from the “crystalline” regions of the collagen molecule. After 48 hr., the most slowly dialyzable peptides remain in an aggregate fraction which, operationally, has been designated a “non-dialyzable” fraction. It has been established that the aggregate peptides have, relative to original gelatin, increased contents of glutamic acid, aspartic acid, and lysine, and contain major portions of the carbohydrate, tyrosine, and hydroxylamine-sensitive (“ester-like”) bonds of gelatin. However, they have relatively decreased contents of proline, hydroxyproline, arginine, and leucine. Their Contents of glycine and alanine are relatively unchanged. Dinitrophenylation studies indicated that the average peptide length of the “nondialyzable” peptides was 11–15 residues. On the basis of these and other studies, a representation is made of the polar and nonpolar regions of the collagen molecule.

1. An alternative simplified procedure is described for the isolation of a 30-residue peptide obtained by digestion with collagenase of carp swim bladder tropocollagen (ichthyocol). The peptide contains an aldehydic component as a 2,4-dinitrophenylhydrazone. The aldehyde dinitrophenylhydrazone is present in stoichiometric proportion to the amino acids in the peptide. 2. This peptide is shown to be devoid of free terminal amino groups by lack of reactivity with ninhydrin or phenylisothiocyanate. Since it is prepared by treatment with collagenase and yet devoid of any terminal α-amino group including that of a glycine residue, the peptide most likely arises from a blocked amino-terminal position of a tropocollagen chain. 3. The aldehyde-bearing component most likely is not bound to any of the following amino acid side chains for the reasons given: (a) to side chain carboxyl groups of glutamyl or aspartyl residues in ester linkage, since treatment with aqueous hydrazine does not release the dinitrophenylhydrazone from the peptide; (b) to the hydroxyl group of a serine residue by means of a glycosidic-like attachment, since mild treatment with NaOH does not promote β elimination in the serine; (c) to side chains of either threonine, hydroxylysine, hydroxyproline, lysine, arginine, or histidine, since residues of these are not present in the peptide; (d) to the hydroxyl side chain of a tyrosine residue, since this amino acid, while present in the aldehyde-dinitrophenylhydrazone-peptide obtained following digestion with collagenase, is absent from the aldehyde-dinitrophenylhydrazone-peptide obtained by further digestion with Pronase. 4. The peptide obtained with collagenase is cleaved further into at least seven peptides which have been partially characterized. A yellow heptapeptide containing the aldehydic component as a 2,4-dinitrophenylhydrazine derivative is also without free amino groups, and this locates the aldehyde-bearing component within 7 residues of a blocked amino terminus of a tropocollagen chain. 5. Ultraviolet absorption spectra of derivatives of the aldehyde component, and its tendency to undergo acid-catalyzed α,β-dehydration to α,β-unsaturated aldehyde derivatives, suggest that its first 3 carbon atoms are involved in a β-hydroxyaldehyde structure. The aldehyde-bearing component must contain an additional functional group with which it makes covalent attachment to the protein. 6. It is suggested that the aldehyde component, by virtue of its chemical nature, reactivity, stoichiometry, and location in the protein, may be involved in the maturation and cross-linking of soluble tropocollagen to form insoluble mature tissue collagen.