Amylose Is Synthesized in Vitro by Extension of and Cleavage from Amylopectin

• Published in: The Journal of biological chemistry 1998-08, Vol.273 (35), p.22232-22240
• Main Authors: van de Wal, Marion, D'Hulst, Christophe, Vincken, Jean-Paul, Buléon, Alain, Visser, Richard, Ball, Steven
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALMIDON; AMIDON; AMILOPECTINA; AMILOSA; AMYLOPECTIN; Amylopectin - chemistry; Amylopectin - isolation & purification; Amylopectin - metabolism; AMYLOPECTINE; AMYLOSE; Amylose - biosynthesis; Amylose - chemistry; Amylose - isolation & purification; Biochemistry; Biochemistry, Molecular Biology; CARBOHYDRATE METABOLISM; CHLAMYDOMONAS REINHARDTII; Crystallography, X-Ray; Electrophoresis, Gel, Pulsed-Field; ENZYMIC ACTIVITY; EPS; Freshwater; GLICOSILTRANSFERASAS; GLYCOSYLTRANSFERASE; GLYCOSYLTRANSFERASES; GRANULE; GRANULE BOUND STARCH SYNTHASE; GRANULES; GRANULOS; HEXOSYLTRANSFERASES; Hydrolysis; ISOAMILASA; ISOAMYLASE; Kinetics; Laboratorium voor Plantenveredeling; Laboratory of Plant Breeding; Life Sciences; MALTOOLIGOSACCHARIDES; METABOLISME DES GLUCIDES; METABOLISMO DE CARBOHIDRATOS; OLIGOSACARIDOS; OLIGOSACCHARIDE; OLIGOSACCHARIDES; Oligosaccharides - metabolism; Plant Breeding; Plants - metabolism; STARCH
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.35.22232

Amylose synthesis was obtained in vitro from purified Chlamydomonas reinhardtii starch granules. Labeling experiments clearly indicate that initially the major granule-bound starch synthase extends glucans available on amylopectin. Amylose synthesis occurs thereafter at rates approaching or exceeding those of net polysaccharide synthesis. Although these results suggested that amylose originates from cleavage of a pre-existing external amylopectin chain, such transfer of chains from amylopectin to amylose was directly evidenced from pulse-chase experiments. The structure of the in vitro synthesized amylose could not be distinguished from in vivo synthesized amylose by a variety of methods. Moreover high molecular mass branched amylose synthesis preceded that of the low molecular mass, suggesting that chain termination occurs consequently to glucan cleavage. Short pulses of synthesis followed by incubation in buffer with or without ADP-Glc prove that transfer requires the presence of the glucosyl-nucleotide. Taken together, these observations make a compelling case for amylopectin acting as the in vivoprimer for amylose synthesis. They further prove that extension is followed by cleavage. A model is presented that can explain the major features of amylose synthesis in plants. The consequences of intensive amylose synthesis on the crystal organization of amylopectin are reported through wide angle x-ray analysis of the in vitrosynthesized polysaccharides.