35] Lysyl oxidase: Preparation and role in elastin biosynthesis

Abstract

This chapter describes purification and assay methods utilized for the study of aortic lysyl oxidase and its activity toward elastin substrates. Lysyl oxidase is mostly insoluble in neutral salt buffers in a variety of connective tissues, although enzyme activity is present in the growth medium of fibroblast cell cultures as a soluble protein. The enzyme can be solubilized from connective tissue sources by buffers containing 4–6 M urea. Most purification schemes that have evolved begin with the ureasolubilized fraction and generally share other purification steps as well. There are two assay methods for lysyl oxidase: tritium release assay and enzyme-coupled spectrophotometric assays. In tritium release assay chick embryo aortas are incubated in organ culture with [6- 3 H]lysine in the presence of BAPN. The salineinsoluble, elastin-rich fraction is isolated from the pulsed aortas after incubation in organ culture. Oxidative deamination of the E-carbon of radioactive peptidyl lysine in this insoluble substrate releases a tritium ion that forms tritiated water by exchange during the assay. Tritiated water is distilled in vacuo from assay mixtures and quantified by liquid scintillation spectrometry. In enzyme-coupled spectrophotometric assays the production of n-butyraldehyde by lysyl oxidase is coupled to the reduction of NAD + by aldehyde dehydrogenase, measuring the change in absorption at 340 nm.