Mercedes A. Paz

Collagen synthesized and modified by aging fibroblasts in culture.

SummaryCollagen is produced by WI-38 diploid human fibroblast cultures throughout their life cycle. It is examined by a sensitive method based on the analysis of specific peptides obtained after digestion with bacterial collagenase. The production and hydroxylation of the collagen is strongly dependent upon the age (population doublings) of the culture and the presence of ascorbic acid. Young cultures (passage 26) produce large amounts of collagen in the absence of ascorbic acid, and this collagen is about 50% hydroxylated compared to that produced by young cultures in the presence of ascorbic acid. Ascorbic acid reduces to about one-half the amount of collagen produced by these young cultures. The young confluent cultures also depend strongly on ascorbic acid for hydroxylation of proline. The dependence declines rapidly with the age of the culture. The collagen produced by young cultures supplied with ascorbic acid is very similar to the type I collagen produced by normal individuals and has about the same degree of hydroxylation of its prolyl residues. The amount of collagen produced by “older” cultures is unaffected by ascorbic acid, but the degree of hydroxylation is normal only if ascorbic acid is present, and is decreased to about 60 to 70% in the absence of the vitamin. “Senescent” cultures showed little, if any, dependency on ascorbic acid, and the collagen produced, with and without the vitamin, is about 80% hydroxylated.The prolyl hydroxylation system of the WI-38 cells and the various controls on the system are age-dependent.

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

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

PQQ, the elusive coenzyme

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

Cellular proliferation and hypusine synthesis

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.

Separation and determination of α-amino acids by boroxazolidone formation

Abstract Reaction of an α-amino acid (α-AA) with 1,1-diphenylborinic acid (DPBA) leads to the formation of a kinetically stable adduct at pH 2–5 in which both the α-amino and the α-carboxyl groups are bound to boron forming a cyclic mixed anhydride termed a boroxazolidone. In this adduct, the > 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 α-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 α-AA moiety of N-terminal γ-glutamyl peptides is also derivatized as demonstrated by the formation of a glutathione boroxazolidone. The 1,1-diphenylboroxazolidone adducts of α-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 α-AA analysis. Under the conditions we describe here, DPBA is not stably reactive with the ϵ-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 α-AA mixture with DPBA, followed by RP-HPLC (AA-DPBA/RP-HPLC) is then a simple method by which to analyze α-AA in a mixture with peptides and amines. Precolumn reaction with DPBA may be used to separate peptides from α-AA and from those peptides which contain an α-AA moiety. Unreacted peptides are bound only weakly to the HPLC column and thus are separated from reacted α-amino acids which are retained as 1,1-diphenylboroxazolidones until their selective elution. This method is particularly suited for the analysis of α-amino acids that are derived from post-translational modification of protein side chains.

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. Ruckers 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. PQQs 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.

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.

[31] Elastn isolation and cross-linking

Publisher Summary This chapter presents the procedure for isolation and purification of elastin from histologically defined regions of calf lung, other bovine tissues, and dog aorta and to measure the nature and distribution of cross-links. The tissues are separated as far as technically possible into anatomically defined regions. Representative samples were taken for histological examination to confirm that the tissues were normal and that adequate isolation of the defined areas had occurred. A modified Verhoeffs iron hematoxylin stain is used for this purpose and, in addition, standard electronmicroscopy methods are employed to examine the elastin preparations. The procedure for analysis of elastin cross-links allows a specific cleavage of iso(desmosine) and may be useful in sequence studies of cross-linked (pyridinium)-containing peptide. However, to reduce the size of the peptide, an additional doublebond cleavage is necessary if it is assumed that two peptides are cross-linked by the pyridinium compound.

Differences in valyl-proline sequence content in elastins from various bovine tissues.

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.

Collagen prolyl hydroxylation in WI-38 fibroblast cultures: Action of hydralazine

SummaryThe action of hydralazine on collagen prolyl hydroxylation was studied in a cell culture system using WI-38 fibroblasts. The prolyl hydroxylation level was determined by a method involving the digestion of collagen by bacterial collagenase and the examination of specific peptides. The presence of low concentrations of hydralazine (0.2 mM) in both “young” and “old” fibroblast cultures strongly inhibited collagen prolyl hydroxylation. The degree of inhibition was greater in serum-deficient cultures. No significant improvement in the degree of hydroxylation was observed by increasing either ascorbate or iron levels in the hydralazine-containing cultures in which hydroxylation was inhibited. Some of the reported side effects of hydralazine seen in patients might be related to its inhibitory effects on mixed function oxidative (MFO) hydroxylation systems. While the ascorbate dependence of the prolyl hydroxylase system of WI-38 decreased with the “age” of the culture, hydralazine inhibition of hydroxylation was dramatic with cultures of all “ages”.