Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Characterization of the enzymatic polymerization of 2,6-linked fructan by leaf extracts from timothy grass (Phleum pratense)
Autore:
Cairns, AJ; Nash, R; De Carvalho, MAM; Sims, IM;
Indirizzi:
Inst Grassland & Environm Res, Dept Cell Biol, Aberystwyth SY23 3EB, Dyfed, Inst Grassland & Environm Res Aberystwyth Dyfed Wales SY23 3EB EB, Dyfed, Inst Bot S Fisiol & Bioquim Plantas, BR-01061970 Sao Paulo, Brazil Inst Bot S Fisiol & Bioquim Plantas Sao Paulo Brazil BR-01061970 BCrazil Univstraliarne, Sch Bot, CRC Ind Plant Biopolymers, Parkville, Vic 3052, Au Univ Melbourne Parkville Vic Australia 3052 mers, Parkville, Vic 3052, Au
Titolo Testata:
NEW PHYTOLOGIST
fascicolo: 1, volume: 142, anno: 1999,
pagine: 79 - 91
SICI:
0028-646X(199904)142:1<79:COTEPO>2.0.ZU;2-W
Fonte:
ISI
Lingua:
ENG
Soggetto:
LOLIUM-TEMULENTUM L; EXCISED LEAVES; BARLEY LEAVES; KEY ENZYME; FRUCTOSYLTRANSFERASE ACTIVITY; CICHORIUM-INTYBUS; DENOVO SYNTHESIS; BIOSYNTHESIS; SUCROSE; PLANTS;
Keywords:
invertase; glycosyl-linkage analysis; oligosaccharide; polymerase; polysaccharide; sucrose; vacuole;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Citazioni:
34
Recensione:
Indirizzi per estratti:
Indirizzo: Cairns, AJ InstSY23ssland & Environm Res, Dept Cell Biol, Plas Gogerddan, Aberystwyth Inst Grassland & Environm Res Plas Gogerddan Aberystwyth Dyfed Wales SY23 3EB
Citazione:
A.J. Cairns et al., "Characterization of the enzymatic polymerization of 2,6-linked fructan by leaf extracts from timothy grass (Phleum pratense)", NEW PHYTOL, 142(1), 1999, pp. 79-91

Abstract

A fructan polymerase activity was partially purified and concentrated by sequential acid and salt precipitation from extracts of excised, illuminatedleaves of timothy grass (Phleum pratense). The polymerase catalysed the denovo synthesis of oligo- and polyfructan from sucrose as sole substrate atnear-physiological rates (0.5-0.9 mg g(-1) fresh mass h(-1); 0.9-1.5 nkat g(-1)). Rates of in vitro polymerisation were high, at up to 4.1 mg cm(-3) h(-1) (7.1 nkat cm(-3)) of total products of degree of polymerization greater than 2 (DP > 2). The trisaccharides 1-kestose and 6-kestose together with oligosaccharides of up to DP = c. 10 were synthesized in under 2 h at 30 degrees C. In longer incubations, ethanol-precipitable polymers of DP = c. 10-35 (1.6-5.7 kDa) were detected by anion-exchange chromatography and pulsed amperometry. When this polymeric product was used as a primer and re-incubated with fresh enzyme and sucrose, abundant polymers of up to DP = 50 (8.1 kDa) were formed. The structure of the polymeric enzyme product was compared with native fructan from timothy leaves and with standard inulin, using glycosyl-linkage analysis followed by identification of partially methylated alditol acetate derivatives by GC-MS. The deduced structure was a linear (unbranched) 2,6-linked fructose chain terminated with glucose and fructose. The linkage structures of the native and enzyme-generated polymers wereidentical, increasing confidence in the physiological relevance of the activity. After ultracentrifugation of tissue homogenates at 265 000 g(av), the polymerase remained in the supernatant, demonstrating no tight association with particulate components. The polymerizing reaction was dependent on enzyme concentration, requiring at least 3 g fresh mass equivalent cm(-3) (c. 2.7 nkat cm(-3)) for the efficient in vitro generation of fructans of DP > 3. In common with other trisaccharide-synthesizing and oligofructan-glycosylating enzymes from grasses, the polymerase reaction exhibited both a maximal velocity at pH 5.0-5.5 and a low affinity for sucrose. The polymerization reaction did not saturate fully even at 1.5 M sucrose, and the concentration causing half maximal velocity (apparent K-m) was c. 560 mM. The preparation contained substantial invertase activity (1.8 mg sucrose g(-1) freshmass h(-1) = 1.5 nkat g(-1) fresh mass) with a K-m for sucrose hydrolysis of 5 mM. A single peak of polymerase activity with an M-r of 51 kDa was recovered from size-exclusion chromatography (SEC). Invertases of M-r 51 and 110 kDa were identified in the preparation. The 110-kDa invertase isoform exhibited no polymerase activity, but synthesized trisaccharide (mainly 1-kestose) from sucrose. The 51-kDa isoform co-eluted with the polymerase. The trisaccharide fraction produced by this isoform contained abundant 1- and 6-kestose. After SEC, the purification of the polymerase was 41-fold relativeto the original tissue homogenate. The properties of enzymatic polymerization of fructan are discussed with respect to the physiology of accumulationin grass leaves and other systems.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/11/20 alle ore 12:06:56