CELL-FREE SYNTHESIS OF PECTIN - IDENTIFICATION AND PARTIAL CHARACTERIZATION OF POLYGALACTURONATE 4-ALPHA-GALACTURONOSYLTRANSFERASE AND ITS PRODUCTS FROM MEMBRANE PREPARATIONS OF TOBACCO CELL-SUSPENSION CULTURES

被引:44
作者
DOONG, RL
LILJEBJELKE, K
FRALISH, G
KUMAR, A
MOHNEN, D
机构
[1] UNIV GEORGIA,COMPLEX CARBOHYDRATE RES CTR,ATHENS,GA 30602
[2] UNIV GEORGIA,DEPT BIOCHEM & MOLEC BIOL,ATHENS,GA 30602
关键词
D O I
10.1104/pp.109.1.141
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Polygalacturonate 4-alpha-galacturonosyltransferase (EC 2.4.1.43) activity has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell-suspension cultures. Incubation of UDP-[C-14]galacturonic acid with tobacco membranes results in a time-dependent incorporation of [C-14]galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product. The optimal synthesis of product occurs at a pH of 7.8, 25 to 30 degrees C, an apparent K-m for UDP-D-galacturonic acid of approximately 8.9 mu M, and a V-max of approximately 150 pmol min(-1) mg(-1) protein. The product was characterized by scintillation counting, thin-layer chromatography, high-performance anion-exchange chromatography, and gel-filtration chromatography in combination with enzymatic and chemical treatments. The intact product has a molecular mass of approximately 105,000 D based on dextran molecular standards. The product was treated with base to hydrolyze ester linkages (e.g. methyl esters), digested with a homogeneous endopolygalacturonase (EPGase), or base and EPGase treated. Base and EPGase treatment results in cleavage of 34 to 89% of C-14-labeled product into components that co-chromatograph with mono-, di-, and trigalacturonic acid, indicating that a large portion of product contains contiguous 1,4-linked alpha-D-galactosyluronic acid residues. Optimal EPGase fragmentation of the product requires base treatment prior to enzymatic digestion, suggesting that 45 to 67% of the galacturonic acid residues in the synthesized homogalacturonan are esterified. At least 40% of the base-sensitive linkages were shown to be methyl esters by comparing the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase.
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页码:141 / 152
页数:12
相关论文
共 60 条
[41]  
Mohnen D, 1993, Semin Cell Biol, V4, P93, DOI 10.1006/scel.1993.1012
[42]  
MORRE JD, 1987, BIOL MEMBRANES PRACT, P37
[43]   DETERMINATION OF THE PATTERN OF METHYL ESTERIFICATION IN PECTIN - DISTRIBUTION OF CONTIGUOUS NONESTERIFIED RESIDUES [J].
MORT, AJ ;
QIU, F ;
MANESS, NO .
CARBOHYDRATE RESEARCH, 1993, 247 :21-35
[44]   PROBLEMS ENCOUNTERED DURING THE EXTRACTION, PURIFICATION, AND CHROMATOGRAPHY OF PECTIC FRAGMENTS, AND SOME SOLUTIONS TO THEM [J].
MORT, AJ ;
MOERSCHBACHER, BM ;
PIERCE, ML ;
MANESS, NO .
CARBOHYDRATE RESEARCH, 1991, 215 (01) :219-227
[45]  
NORTHCOTE D H, 1985, P177
[46]  
O'Neill M.A., 1990, METHODS PLANT BIOCH, V2, DOI DOI 10.1016/B978-0-12-461012-5.50018-5
[47]  
Rexova-Benkova L, 1976, Adv Carbohydr Chem Biochem, V33, P323, DOI 10.1016/S0065-2318(08)60285-1
[48]  
REXOVABENKOVA L, 1977, COLLECT CZECH CHEM C, V42, P3204
[49]   Structures at the plant cell surface [J].
Roberts, K. .
CURRENT OPINION IN CELL BIOLOGY, 1990, 2 (05) :920-928
[50]   PARTIAL-PURIFICATION OF GOLGI-BOUND ARABINOSYLTRANSFERASE AND 2 ISOFORMS OF XYLOSYLTRANSFERASE FROM FRENCH BEAN (PHASEOLUS-VULGARIS L) [J].
RODGERS, MW ;
BOLWELL, GP .
BIOCHEMICAL JOURNAL, 1992, 288 :817-822