In vitro studies of enzymatic properties of starch synthases and interactions between starch synthase I and starch branching enzymes from rice
Introduction
Plant starch contains linear or slightly branched amylose and highly branched amylopectin. In the fine structure of amylopectin most of the side chains having a degree of polymerization (DP) ≥ 10 form double helices [1], [2]. Thus, the length of amylopectin chains is responsible for the hydrophobicity and crystallinity of starch granules. It is established that amylopectin chains are elongated by starch synthase (SS). Plants generally have three major types of SS isozymes, namely SSI, SSII, and SSIII, whereas higher plants have also SSIV and in addition some of them have the SSV type [3], [4], [5], [6], [7]. In maturing rice endosperm, the major SS activity is accounted for by SSI, SSIIa, and SSIIIa [5], [7]. Our previous studies have examined the functions of individual SS isozymes by using rice mutants and transformants in which these genes are modified. The results suggested that SSI, SSIIa, and SSIIIa play distinct roles in amylopectin biosynthesis by forming short cluster chains [8], intermediate cluster chains (A and B1 chains) and longer cluster chains (B1 chains) [9], and long B1 and cluster-linking chains (B2–B3 chains) [10], respectively.
This paper aims to determine the kinetic parameters of rice SS isozymes toward the glucan substrate e7-amylopectin, which was prepared from rice endosperm starch of an ssI-deficient mutant line e7 [9]. Because e7-amylopectin is enriched in short chains of DP6-8 compared with wild-type amylopectin [8], it is a suitable substrate for analyzing the chain-length preference of individual SS isozymes.
For attempt to clarify chain elongation characteristics of SS isozymes, it is of great use to analyze quantitatively the chain-length distribution of the debranched SS reaction products. In this study, a fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was labeled at the reducing end of each malto-oligosaccharides (MOS) liberated after the treatment of the product with a bacterial isoamylase. The fluorephore-assisted carbohydrate capillary electrophoresis (FACE) method [11] can precisely and reproducibly determine the molar concentrations of MOS derived from the SS reaction products and the substrate. The method is useful to analyze in detail the change in fine structure of α-glucans during the SS enzymatic reactions.
The present study also showed that the synthesis of glucan by SSI in the absence of added primer was significantly enhanced in the presence of BE isozymes. This interaction was totally dependent on citrate. The activation of SSI-mediated glucan synthesis by BE was consistent with the recent observation that in the omission of exogenous glucan primer rice plastidial phosphorylase (Pho1) can synthesize glucans when the enzyme co-exists with BE [12]. We proposed that Pho1 might be involved with BE in the initiation process of starch biosynthesis in rice endosperm by generating branched maltodextrin and glucan from glucose 1-phosphate (G1P) [13], [14], [15]. A physiological role of the SSI and BE interaction during starch synthesis in rice endosperm is also discussed.
Section snippets
Reagents
ADPglucose and cluster dextrin (CD) were purchased from Sigma and Ezaki Glico Co., Ltd. (Osaka, Japan), respectively. In this study CD was occasionally used as glucan primer because it mimics the fine structure of cereal amylopectin produced by the cyclic branching reaction by Bacillus stearothermophilus BE with maize amylopectin, and is more easily dissolved in aqueous solution compared with normal cereal amylopectin [16]. Pseudomonas amyloderamosa isoamylase (PaISA), glucoamylase from Rhizopus
Comparison of basic properties of rice SS isozymes
To characterize the SS isozymes, their kinetic properties were determined using their purified recombinant proteins expressed in E. coli. When oyster glycogen was used as primer, activities of SSI, SSIIa, and SSIIIa were 1.83, 2.33, and 1.86 μmol ADPglucose utilized/mg protein/min, respectively (Table 1). The S0.5 value of SSI for ADPglucose (0.86 mM) was significantly greater than that of SSIIa (0.22 mM) or SSIIIa (0.26 mM). These S0.5 values are in the same range as Km values reported for SS
Discussion
SS plays an essential role in the elongation of α-1,4-glucosidic chains of amylopectin for starch biosynthesis. Three major forms of SS isozymes (SSI, SSIIa, and SSIIIa) are expressed in cereal endosperm and contribute to the fine structure of endosperm amylopectin [3], [4], [5], [6], [7]. It is essential to understand how these isozymes play their roles in the synthesis of the cluster structure of amylopectin, and to determine the enzymatic properties of the SS isozymes. In this paper, we
Acknowledgments
This study was funded by grants to Y.N. from The Ministry of Education, Sports, Science and Technology, Grant-in-Aid for Scientific Research (A) (No. 20248002) and to N. F. from the program for the Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry. We thank Dr. H. Satoh at Kyushu University for providing us with rice mutant lines. The DNA sequence analysis was performed with the aid of the Biotechnology Center, Akita Prefectural University. We also thank Dr.
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