Scientia

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Comparative Biochemistry and Physiology, Part B 142 (2005) 56 – 66 www.elsevier.com/locate/cbpb

Two forms of a-amylase in mantle tissue of Mytilus galloprovincialis: Purification and molecular properties of form II M. Lombran˜a, P. Sua´rez, F. San Juan * Departamento de Bioquı´mica, Gene´tica e Inmunolgı´a, Facultad de Ciencias del Mar, Universidad de Vigo, Lagoas-Marcosende s/n, 36200 Vigo, Spain Received 18 February 2005; received in revised form 27 May 2005; accepted 30 May 2005 Available online 11 July 2005

Abstract a-amylase activity has been shown for the first time in a non-digestive tissue from Mytilus galloprovincialis. a-amylase from mussel mantle tissue has been purified by affinity chromatography on insoluble starch, followed by gel-filtration chromatography on Superdex-200. The chromatographic and electrophoretic behaviour of M. galloprovincialis a-amylase and stability characteristics suggest two forms of this enzyme: one form forming stable aggregates (form I) and a monomeric form (form II) that is more abundant, active and unstable. Both forms show an inverse quantitative variation. Purified form II was highly unstable and the molecular mass was estimated to be 66 kDa by sodium dodecyl sulphate (SDS)-gel electrophoresis. Maximum activity was noted at pH 6.5 and 35 -C. D 2005 Elsevier Inc. All rights reserved. Keywords: Affinity chromatography; a-amylase; Bivalves; Glycogen metabolism; Mantle tissue; Molecular characterization; Mytilus; Purification

1. Introduction Culture of molluscs, more specifically of Mytilus galloprovincialis, is one of the most important sources of richness in the Galician community due to the special environmental characteristics of its estuaries (Figueras, 1990; CaceresMartinez and Figueras, 1997; Sanchez-Mata and Mora, 2000; Keldany, 2002). The main aspects of mollusc culture are growth and reproduction and both are closely linked. Indepth studies of biochemical and metabolic levels would lead to a greater knowledge of bivalves reproduction and a better control and optimization of their production. The mantle tissue of Mytilus has two interrelated physiological functions: accumulation of reserve substances and development of the gonad that invades the mantle, proliferating at the expense of the reserve tissue (Lubet, 1957). This basically comprises two main cellular types: vesicular cells (VC) that mainly store glycogen and adipogranular cells (AGC) that store lipids, proteins and a

* Corresponding author. Tel.: +34 9 86 812574; fax: +34 9 86 812556. E-mail address: fsanjuan@uvigo.es (F. San Juan). 1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.05.017

small amount of glycogen (Lubet et al., 1978; Pipe, 1987; Crespo and Espinosa, 1990; Freites et al., 2002, 2003). In Mytilus, the glycogen of the mantle tissue is the main energy source for sustaining gonadal development and a clear relationship exists between its degradation and gametogenic development (Bayne et al., 1982; Gabbott, 1983; Gabbott and Peek, 1991; Sua´rez et al., 2005). Despite this, few studies deal with the alternative metabolic pathways of glycogen metabolism in these organisms, as well as the enzymes involved (Alemany and Rosell-Pe´rez, 1973; Va´zquez-Baanante and Rossel-Pe´rez, 1979; Zaba, 1981; San Juan Serrano et al., 1991; Ibarguren and Ramos-Martı´nez, 1991; Fernandez et al., 1994). There are two known pathways to degrade this polysaccharide: hydrolytic and phosphorolytic pathways. The main enzyme activities in both pathways (glycosidases and glycogen phosphorylase) are found in this tissue (Gabbott and Peek, 1991; Zaba, 1981; Peek and Gabbott, 1990; San Juan Serrano et al., 1991), but a-amylase activity and relative contribution of amylase to hydrolytic pathway in glycogen utilization for gametogenic development is still unknown. a-amylase (1,4-a-d-glucan glucanohydrolase EC. 3.2. 1.1) catalyzes the endohydrolysis of 1,4-a-d-glucosidic


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