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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.

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.

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.

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.

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.

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.

The specialized calcium binding amino acid, γ-carboxyglutamic acid (Gla) is quantitated in developing atherosclerotic plaque relative to progression of the disease, and a Gla-containing protein isolated from calcified atherosclerotic plaque is partially characterized. Low levels of Gla are found in fatty streak and fibrous plaque lesions, and a marked increase in Gla content occurs in calcified plaque. A unique Gla-containing protein is purified from 0.5M EDTA (pH 8.0) extracts of calcified plaque, named atherocalcin. The protein containing 19 Gla residues/1000 amino acids is 80,000 molecular weight, with a pI of 4.16 – 4.3 and is uniquely different from other known Gla-containing proteins. The implications of this work for the further understanding of the pathogenesis and therapy of atherosclerosis are discussed.

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.

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 specific radioactive label was introduced into the sialoglycoproteins of the erythrocyte membrane by sequential sodium periodate oxidation and tritiated sodium borohydride reduction. This was achieved whether the sialoglycoproteins were isolated, present in situ within the intact erythrocyte, or the isolated erythrocyte membranes. The label is found in the oligosaccharide chains of the sialoglycoproteins predominantly in residues which were formerly those of bound sialic acid. The method appears selective for the sialoglycoproteins and certain, as yet unidentified lipid components.

Summary An ethanol fractionation procedure was developed for the isolation of glycoproteins from aqueous pyridine solubilized human erythrocyte membranes. The preparation resembles that obtained by extraction with phenol in its amino acid and carbohydrate composition and the presence of antigens. In addition to the glycoproteins the preparation contains glycolipid components.

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.