Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans1(PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content -effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.1 The abbreviations used are: proteoglycans (PG); keratan sulfate (KS); heparan sulfate (HS); dermatan sulfate (DS); chondroitin sulfate (CS); glycosaminoglycan (GAG); affinity coelectrophoresis (ACE); circular dichroism (CD); advanced glycation end products (AGE); cyanogen bromide (CB); 3-(N-morpholino)-2hydroxypropanesulfaonate (MOPSO) *Address correspondence to: James D. San Antonio, Department of Medicine and the Cardeza Foundation for Hematologic Research, Thomas Jefferson University, 809 Curtis, 1015 Walnut St., Philadelphia, PA 19107-5099, Ph: 215-955-6121, Fax: 215-923-3836 [Linsenmayer, 1991;Prockop and Kivirikko, 1995] for review). It is synthesized as a soluble, procollagen form containing two α1, and one α2 chains, each of about 1000 amino acids. Upon secretion from the cell the propeptides are cleaved, and the collagen monomer is assembled into the fibril, proposed to consist of aggregates of microfibrils, or 5-mer bundles of overlapping monomers. Type I collagen fibrils contribute to the integrity and function of many tissues via interactions with other collagens, PGs, and growth and differentiation factors [DiLullo et al., 2002]. In aging, type I collagen b...

The RecA protein of Escherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

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The distribution of rat liver epoxide hydrolase in various subcellular fractions was investigated by immuno-electron-microscopy. Ferritin-linked monospecific anti-(epoxide hydrolase) immunoglobulins bound specifically to the cytoplasmic surfaces of total microsomal preparations and smooth and rough microsomal fractions as well as the nuclear envelope. Specific binding was not observed when the ferritin conjugates were incubated with peroxisomes, lysosomes and mitochondria. The average specific ferritin load of the individual subcellular fractions correlated well with the measured epoxide hydrolase activities. This correlation was observed with fractions prepared from control, phenobarbitone-treated and 2-acetamidofluorene-treated rats.