STRICTOSIDINE SYNTHASE FROM CATHARANTHUS-ROSEUS - PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS

被引:39
作者
DEWAAL, A
MEIJER, AH
VERPOORTE, R
机构
[1] LEIDEN UNIV, GORLAEUS LABS, DIV PHARMACOGNOSY, 2300 RA LEIDEN, NETHERLANDS
[2] LEIDEN UNIV, GORLAEUS LABS, AMSTERDAM CTR DRUG RES, 2300 RA LEIDEN, NETHERLANDS
关键词
D O I
10.1042/bj3060571
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell suspension cultures were purified and characterized. A purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing. Four of six isoforms were purified to apparent homogeneity, whereas two others were nearly homogeneous. All strictosidine synthase isoforms were found to be glycoproteins. The isoforms were also found in leaves and roots of the plant, in seedlings and in hairy root cultures. The ratio of the different isoforms differed slightly between these sources. The kinetic parameters of the isoforms showed no significant differences. The maximal velocity (300-400 nkat/mg of protein) is the highest reported so far. It was demonstrated that the apparent Michaelis constant for tryptamine (approx. 9 mu M) is much lower than values reported previously. The presence of weak product inhibition (K-p approx. 35 times K-m) was established, whereas substrate inhibition was not detected.
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页码:571 / 580
页数:10
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