Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylarnide gels

被引：14

作者：

Farkas, V ^{[1
]}

Ait-Mohand, F ^{[1
]}

Stratilová, E ^{[1
]}

机构：

[1] Slovak Acad Sci, Inst Chem, SK-84538 Bratislava, Slovakia

来源：

PLANT PHYSIOLOGY AND BIOCHEMISTRY | 2005年 / 43卷 / 05期

关键词：

isoelectric focusing; sulphorhodamine; transglycosylation; xyloglucan; xyloglucan endotransglycosylase; hydrolase; XET; XTH; zymogram;

D O I：

10.1016/j.plaphy.2005.03.006

中图分类号：

Q94 [植物学];

学科分类号：

071001 ;

摘要：

The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M-r polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure. (c) 2005 Elsevier SAS. All rights reserved.

引用

页码：431 / 435

页数：5

共 29 条

[21] Release of complexed xyloglucan endotransglycosylase (XET) from plant cell walls by a transglycosylation reaction with xyloglucan-derived oligosaccharides [J].

Sulová, Z ;

Baran, R ;

Farkas, V .

PLANT PHYSIOLOGY AND BIOCHEMISTRY, 2001, 39 (11) :927-932

[22] A COLORIMETRIC ASSAY FOR XYLOGLUCAN-ENDOTRANSGLYCOSYLASE FROM GERMINATING-SEEDS [J].

SULOVA, Z ;

LEDNICKA, M ;

FARKAS, V .

ANALYTICAL BIOCHEMISTRY, 1995, 229 (01) :80-85

[23]

Thompson JE, 1997, BIOCHEM J, V327, P699

[24] Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells [J].

Thompson, JE ;

Fry, SC .

PLANT JOURNAL, 2001, 26 (01) :23-34

[25] In vivo colocalization of xyloglucan endotransglycosylase activity and its donor substrate in the elongation zone of arabidopsis roots [J].

Vissenberg, K ;

Martinez-Vilchez, IM ;

Verbelen, JP ;

Miller, JG ;

Fry, SC .

PLANT CELL, 2000, 12 (07) :1229-1237

[26] Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays [J].

Vissenberg, K ;

Van Sandt, V ;

Fry, SC ;

Verbelen, JP .

JOURNAL OF EXPERIMENTAL BOTANY, 2003, 54 (381) :335-344

[27] A comprehensive expression analysis of all members of a gene family encoding cell-wall enzymes allowed us to predict cis-regulatory regions involved in cell-wall construction in specific organs of arabidopsis [J].

Yokoyama, R ;

Nishitani, K .

PLANT AND CELL PHYSIOLOGY, 2001, 42 (10) :1025-1033

[28] A surprising diversity and abundance of xyloglucan endotransglucosylase/hydrolases in rice. Classification and expression analysis [J].

Yokoyama, R ;

Rose, JKC ;

Nishitani, K .

PLANT PHYSIOLOGY, 2004, 134 (03) :1088-1099

[29] Functional diversity of xyloglucan-related proteins and its implications in the cell wall dynamics in plants [J].

Yokoyama, R ;

Nishitani, K .

PLANT BIOLOGY, 2000, 2 (06) :598-604

← 1 2 3 →