Rapid and specific assay of collagen glucosyltransferase with a Sepharose-bound substrate

✍ Scribed by Richard Reynertson; John Ford; Lennart Roden

• Book ID: 102983545
• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 664 KB
• Volume: 125
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90008-2

✦ Synopsis

A simple and specific assay has been developed for collagen glucosyltransferase in which a solid-phase substrate, consisting of a mixture of Type II collagen peptides linked to CNBractivated Sepharose 4B, is used. Following incubation with enzyme, the substrate is simply washed, and its radioactivity is determined. The specificity of the procedure has been validated by the isolation of radiolabeled Glc-Gal-Hyl from the reaction products after incubation with a crude enzyme preparation from embryonic chick cartilage. Under the conditions described, product formation was linear for 6 h and was proportional to the concentration of enzyme protein up to 180 pg/ml. The K, was 33 pM for UDP-glucose and 48 pM for the gel-bound collagen substrate, expressed as Gal-Hyl. The I',,,,, for the Sepharose-bound substrate was 30% of the value observed for the identical substrate in soluble form, suggesting that the solid matrix exerted some sterical hindrance on the bound substrate.