Diamine oxidase of Lathyrus sativus seedlings. Purification and properties.
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Date
1979-06
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Abstract
Diamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated
seedlings of Lathyrus sativus by MnCl2 treatment, (NH4)2SO4 and acetone fractionations,
DEAE-Sephadex chromatography followed by gel filtration on Sephadex
G-200. A single step purification of the enzyme was achieved by using an immunoaffinity
column, wherein rabbit antibodies to the homogeneous diamine oxidase
were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous
by electrophoretic, immunological and ultracentrifugal criteria. It had an
Mr of 148,000 (6·46S) and was a dimer with similar sub-units (Mr 75,000). Amino
acid analysis showed the absence of cysteine residues although it contained five
disulphide bonds. The enzyme had copper (2·7 g atom/mol enzyme) but was not
a glycoprotein. No absorption maximum in the visible region was detectable.
Ethylenediamine 1,3-diaminopropane and histamine were potent competitive
inhibitors for the substrate putrescine. The addition of monospecific antibodies
to the enzyme increased the Km for benzyl amine without any change in the Vmax.
Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity
with the antibodies to the L. sativus enzyme.
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Keywords
Diamine oxidase, Lathyrus sativus, purification, properties, immunoaffinity procedure, sub-units, copper content
Citation
Suresh M R, Adiga P R. Diamine oxidase of Lathyrus sativus seedlings. Purification and properties. Journal of Biosciences. 1979 Jun; 1(2): 109-124.