Tài liệu Báo cáo Y học: Purification, characterization, immunolocalization and structural analysis of

Purification, characterization, immunolocalization and structural

analysis of the abundant cytoplasmic b-amylase from Calystegia

sepium (hedge bindweed) rhizomes

Els J. M. Van Damme1

, Jialiang Hu1

, Annick Barre2

, Bettina Hause3

, Geert Baggerman4

, Pierre Rouge´

2 and

Willy J. Peumans1

1

Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Leuven, Belgium; 2

Institut de Pharmacologie et

Biologie Structurale, Unite´ Mixte de Recherche Centre National de la Recherche Scientifique 5089, Toulouse, France; 3

Institute of Plant

Biochemistry, Halle, Germany; 4

Laboratory of Developmental Physiology and Molecular Biology, Katholieke Universiteit Leuven, Leuven,

Belgium

An abundant catalytically active b-amylase (EC 3.2.1.2)

was isolated from resting rhizomes of hedge bindweed

(Calystegia sepium ). Biochemical analysis of the purified

protein, molecular modeling, and cloning of the correspond￾ing gene indicated that this enzyme resembles previously

characterized plant b-amylases with regard to its amino-acid

sequence, molecular structure and catalytic activities.

Immunolocalization demonstrated that the b-amylase is

exclusively located in the cytoplasm. It is suggested that the

hedge bindweed rhizome b-amylase is a cytoplasmic

vegetative storage protein.

Keywords: b-amylase; Calystegia sepium; hedge bindweed;

immunolocalization; vegetative storage protein.

Exo-hydrolases catalyzing the release of b-maltose from the

nonreducing ends of a-1,4-linked oligo- and polyglucans

(also so-called b- or exo-amylases) (EC 3.2.1.2) have been

studied for several decades because they are possibly

involved in starch metabolism in plants, and play an

important role in biotechnological processes whereby starch

is converted into simple sugars. In the past, research on

b-amylases has been focussed on the abundant b-amylases

found in the endosperm of barley (Hordeum vulgare ) and

some other cereals [1], soybean (Glycine max ) seeds [2] and

sweet potato (Ipomoea batatas) tubers [3]. During the last

decade, evidence has accumulated that b-amylases are

ubiquitous in flowering plants. Cereals such as barley, wheat

(Triticum aestivum ), rye (Secale cereale ) and maize (Zea

mays) also contain, besides the classical abundant and

highly active endosperm b-amylases, low levels of another

so-called ‘tissue-ubiquitous’ form in leaves and roots [1].

b-Amylases have also been identified in roots of alfalfa

(Medicago sativa ) and several other forage legumes

including sweetclover (Melilotus officinalis), red clover

(Trifolium pratense ), birdsfoot trefoil (Lotus corniculatus)

[4], and in pea (Pisum sativum ) epicotyls [5]. In addition,

b-amylases have been identified in species of the families

Solanaceae (potato, Solanum tuberosum ) [6] and Brassica￾ceae (Arabidopsis thaliana and Streptanthus tortuosus)

[7,8].

Extensive enzymatic studies of several b-amylases

unambiguously demonstrated that these enzymes exclu￾sively catalyze the release of b-maltose from the

nonreducing ends of a-1,4-linked oligo- and polyglucans.

Accordingly, b-amylases are believed to be involved in the

degradation of starch in the plant and/or a-1,4-linked

oligoglucans. Though this presumed role might hold true for

some b-amylases, it certainly cannot be extrapolated to all

plant b-amylases because (a) some b-amylases occur in

tissues that are devoid of starch, (b) many plant b-amylases

are spatially separated from their presumed substrate (i.e.

starch), and (c) inbred lines of rye lacking the abundant

endosperm b-amylase germinate normally [9]. This implies

that some b-amylases are not required and even not involved

in starch degradation but fulfil another role [10]. It has been

proposed, for example, that the abundant b-amylases from

cereal endosperm and alfalfa taproots function as seed

storage proteins and vegetative storage proteins (VSPs),

respectively [1,4]. A major difficulty in confirming the role

of b-amylases is the lack of insight in their subcellular

location. According to some reports, b-amylase is an

extrachloroplastic protein restricted to the cytoplasm of

spinach cells [11] and A. thaliana leaves [7], which implies

that the enzyme does not contribute to the amylolytic

activity of the chloroplast. Others, however, presented

evidence for a vacuolar location (e.g. in pea and wheat leaf

protoplasts) [12]. Indirect evidence based on the absence of

a signal peptide from the deduced sequence of all

b-amylases cloned thus far suggests that the enzyme is

located in the cytoplasm [10]. Although there is evidence

that in A. thaliana leaves one particular b-amylase is

Correspondence to E. J. M. Van Damme, Katholieke Universiteit

Leuven, Laboratory for Phytopathology and Plant Protection, Willem

de Croylaan 42, 3001 Leuven, Belgium. Fax: þ 32 16 322976,

Tel.: þ 32 16 322379, E-mail: [email protected]

Enzyme: b-amylase (EC 3.2.1.2).

Note: the nucleotide sequence reported in this paper has been submitted

to the GenBanke/EMBL Data library under the accession number

AF284857.

(Received 6 July 2001, revised 5 October 2001, accepted 8 October

2001)

Abbreviations: CalsepRRP, Calystegia sepium RNase-related protein;

HCA, hydrophobic cluster analysis; VSP, vegetative storage protein;

Calsepa, C. sepium agglutinin.

Eur. J. Biochem. 268, 6263–6273 (2001) q FEBS 2001