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Plant Physiol, April 2001, Vol. 125, pp. 1723-1731
Biochemical Characterization of Wild-Type and Mutant Isoamylases
of Chlamydomonas reinhardtii Supports a Function of the
Multimeric Enzyme Organization in Amylopectin
Maturation1
David
Dauvillée,
Christophe
Colleoni,
Gregory
Mouille,
Matthew K.
Morell,
Christophe
d'Hulst,
Fabrice
Wattebled,
Luc
Liénard,
David
Delvallé,
Jean-Philippe
Ral,
Alan M.
Myers, and
Steven G.
Ball*
Laboratoire de Chimie Biologique, Unité Mixte de Recherche du
Centre National de la Recherche Scientifique, No. 8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'ascq cedex, France (D.D., C.C., G.M., C.d.H, F.W., L.L.,
D.D., J.-P.R., S.G.B.); Commonwealth Scientific and Industrial Research
Organization, Division of Plant Industry, G.P.O. Box 1600, Canberra,
Australian Capital Territory 2601, Australia (M.K.M.); and Department
of Biochemistry, Biophysics and Molecular Biology, Iowa State
University, Ames, Iowa 50011 (A.M.M.)
Chlamydomonas reinhardtii mutants of the
STA8 gene produce reduced amounts of high amylose starch
and phytoglycogen. In contrast to the previously described
phytoglycogen-producing mutants of C. reinhardtii that
contain no residual isoamylase activity, the sta8
mutants still contained 35% of the normal amount of enzyme activity.
We have purified this residual isoamylase and compared it with the
wild-type C. reinhardtii enzyme. We have found that the
high-mass multimeric enzyme has reduced its average mass at least by
one-half. This coincides with the disappearance of two out of the three
activity bands that can be seen on zymogram gels. Wild-type and mutant
enzymes are shown to be located within the plastid. In addition, they
both act by cleaving off the outer branches of polysaccharides with no
consistent difference in enzyme specificity. Because the mutant enzyme
was demonstrated to digest phytoglycogen to completion in vitro, we
propose that its inability to do so in vivo supports a function of the
enzyme complex architecture in the processing of pre-amylopectin chains.
1
This work was supported by the Ministère
de l'Education Nationale, by the Centre National de la Recherche
Scientifique, by Biogemma UK, and by the U.S. Department of Agriculture.
*
Corresponding author; e-mail steven.ball{at}univ-lille1.fr; fax
33-3-20-43-65-55.
© 2001 American Society of Plant Physiologists
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