An ultrafiltration assay for lysyl oxidase.
A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.
Shackleton
D R
DR
Department of Biochemistry, University of Edinburgh Medical School, United Kingdom.
Hulmes
D J
DJ
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000629:Aminopropionitrile / Q000378:metabolism; D000818:Animals; D002642:Chick Embryo; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004549:Elastin / Q000378:metabolism; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism*; D013552:Swine; D014316:Tritium; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods; D014508:Urea / Q000494:pharmacology
D014316:Tritium; D000629:Aminopropionitrile; D014508:Urea; D004549:Elastin; D000594:Amino Acid Oxidoreductases; D008249:Protein-Lysine 6-Oxidase