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Radiochemical Estimation of Polyamine Oxidase

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Polyamine oxidases, defined in this chapter as amine oxidases capable of catalyzing the oxidation of spermidine or spermine (1 ,2 ) (see Chapter 1 ), are present in plants, bacteria, fungi, protozoa, worms, and all mammalian cells (2 ,3 ) (see references in Chapter 1 ). In mammalian cells spermine and spermidine can be converted back to putrescine by the pathways shown in Chapter 1 , Fig. 5 . The first step is the acetylation of an aminopropyl group to give the N 1 -acetyl derivative. This is cleaved by polyamine oxidase to form an aldehyde, 3-acetamidopropanal and either spermidine or putrescine (see Chapter 1 , Fig. 6 ). However, acetylation is not a prerequisite for oxidation, and nonacetylated spermidine or spermine can also act as substrates, but less efficiently. Oxidation of spermine or spermidine by some tissue enzymes is stimulated if benzaldehyde is added to the assay mixture (Table 1 ). This stimulation results in a lowering of the K m value for the substrate, probably as a consequence of Schiff base formation between the aldehyde and the primary amino groups of spermine or spermidine, and appears to mimic the effects of in vivo acetylation. Use of benzaldehyde in this assay substitutes for the nonavailability of radiolabeled N 1 -acetylspermine or N 1 -acetylspermidine, the preferred substrates of tissue polyamine oxidase. The effect of benzaldehyde addition on the particular polyamine oxidase being assayed should always be examined before routinely including it in the assay mixture.
Table 1  The Effect of Benzaldehyde on K m Values of Rat Liver Polyamine Oxidase for Spermine, and Spermidine ( 12 14 )
 

Km (�M )

−Benzaldehyde

+ Benzaldehyde

Spermidine

50

15

N 1 -Acetylspermidine

14

Spermine

20

5

N 1 -Acetylspermine

0.6

N 1 ,N 12 -Diacetylspermine

50

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