A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase.
A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.
D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D002855:Chromatography, Thin Layer; D003094:Collagen / Q000138:chemical synthesis* / Q000378:metabolism; D004535:Ehlers-Danlos Syndrome / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007553:Isotope Labeling; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012867:Skin / Q000201:enzymology; D014316:Tritium
D010446:Peptide Fragments; D014316:Tritium; D003094:Collagen; C045653:collagen I, alpha chain (98-110); D006899:Mixed Function Oxygenases; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase