Serological differences between the multiple amine oxidases of yeasts and comparison of the specificities of the purified enzymes from Candida utilis and Pichia pastoris

Abstract

1. Antiserum to purified methylamine oxidase of Candida boidinii formed precipitin lines (with spurs) in double-diffusion tests with crude extracts of methylamine-grown cells of the following yeast species: Candida nagoyaensis, Candida nemodendra, Hansenula minuta, Hansenula polymorpha and Pichia pinus. No cross-reaction was observed with extracts of Candida lipolytica, Candida steatolytica, Candida tropicalis, Candida utilis, Pichia pastoris, Sporobolomyces albo-rubescens, Sporopachydermia cereana or Trigonopsis variabilis. Quantitative enzyme assays enabled the relative titre of antiserum against the various methylamine oxidases to be determined. 2. The amine oxidases from two non-cross-reacting species, C. utilis and P. pastoris, were purified to near homogeneity. 3. The methylamine oxidases, despite their serological non-similarity, showed very similar catalytic properties to methylamine oxidase from C. boidinii. Their heat-stability, pH optima, molecular weights, substrate specificities and sensitivity to inhibitors are reported. 4. The benzylamine oxidases of C. utilis and P. pastoris both oxidized putrescine, and the latter enzyme failed to show any cross-reaction with antibody to C. boidinii methylamine oxidase. Benzylamine oxidase from C. boidinii itself also did not cross-react with antibody to methylamine oxidase. The heat-stability, molecular weights, substrate specificities and sensitivity to inhibitors of the benzylamine/putrescine oxidases are reported. 5. The benzylamine/putrescine oxidase of C. utilis differed only slightly from that of C. boidinii. 6. Benzylamine/putrescine oxidase from P. pastoris differed from the Candida enzymes in heat-stability, subunit molecular weight and substrate specificity. In particular it catalysed the oxidation of the primary amino groups of spermine, spermidine, lysine, ornithine and 1,2-diaminoethane, which are not substrates for either of the Candida benzylamine oxidases that have been purified. 7. Spermine and spermidine were oxidized at both primary amino groups; in the case of spermidine this is a different specificity from that of plasma amine oxidase. 8. Under appropriate conditions, P. pastoris benzylamine/putrescine oxidase (which is very easy to purify) can be a useful analytical tool in measuring polyamines.