E, Cross-section of a broccoli stem through a leaf abscission zone. cortex of broccoli stems and in vascular tissue, especially in leaf traces. The -glucosidases Indotecan (EC 3.2.1.20, -d-glucoside glucohydrolase) are a widespread and diverse group of enzymes that serve a variety of functions, depending on the subcellular location and organism in which they are found. Despite their origins in several unrelated gene families, they all share an unusually wide and overlapping substrate specificity, making identification from biochemical properties alone difficult (for review, see Frandsen and Svensson, 1998). Various forms of -glucosidase can hydrolyze -1,1-; -1,2-; -1,3-; -1,4-; and -1,6-linked Glc from glycoproteins or from the nonreducing ends of carbohydrates ranging in size from disaccharides to starch. They can also catalyze -glucosyltransferase reactions (Yamasaki and Suzuki, 1980; Yamasaki and Konno, Indotecan 1985). Apoplastic forms of -glucosidase having acidic pH optima occur widely in plants (Klis, 1971; Parr and Edelman, 1975; Yamasaki and Konno, 1987, 1992; Beers et al., 1990), but their function remains elusive because of the apparent lack of appropriate substrates in the apoplast (Fry, 1995). A chloroplastic form of -glucosidase with a neutral pH optimum was isolated from pea and is thought to function in starch degradation, perhaps by removing rare -1,2 or -1,3 linkages that, if present, could block the action of the more abundant amylases and phosphorylases (Beers et al., 1990; Sun et al., 1995). Two different ER forms of -glucosidase are also known: glucosidase I and II, which sequentially remove -1,2- and -1,3-linked Glc, respectively, from nascent glycoproteins as part of the quality control system that functions during glycoprotein folding (Hebert et al., 1995). We began characterizing the -glucosidases of Arabidopsis by Indotecan searching the Arabidopsis EST collection for genes homologous to Family 31 -glucosidases, which include mammalian lysosomal -glucosidase (Hoefsloot et al., 1988) and intestinal sucrase/isomaltase (Chantret et al., 1992) and several fungal -glucosidases (Dohman et al., 1990; Sugimoto and Suzuki, 1996; Nakamura et al., 1997a). The ER glucosidase II is usually a distant relative of this family (Trombetta et al., 1996; Arendt and Ostergaard, 1997). ESTs from several different Arabidopsis genes were identified, and one, 38A2T7 (accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”T04333″,”term_id”:”315493″,”term_text”:”T04333″T04333), was used as a probe to clone the gene (Monroe et al., 1997). The deduced amino acid sequence of contains 902 amino acids with a predicted mass of 101 kD, including the putative signal sequence. The amino acid sequence of is usually 42% to 48% identical to those of the recently reported cDNA clones from barley (Tibbot and Skadsen, 1996), Mouse monoclonal to HAUSP spinach (Sugimoto et al., Indotecan 1997), and sugar beet (Matsui et al., 1997). Although it is not completely sequenced, a second Arabidopsis gene, and each of the other herb -glucosidases. The polypeptides of the -glucosidases all have deduced masses of just over 100 kD, but the products of the four sequenced genes are all subjected to posttranslational modification, including proteolysis and glycosylation, probably in the secretory pathway, since all of the genes contain putative signal sequences. In barley seeds antibodies against the cloned -glucosidase expressed in recognize polypeptides of 81 and 95 kD (Tibbot et al., 1998). Active forms of barley seed -glucosidase are also glycosylated (Sun and Henson, 1990). Four active forms of -glucosidase from spinach seeds were separated by Sugimoto et al. (1995) and found to be 78, 78, 82, and 82 kD. The smaller forms were much more active against soluble starch than were the larger forms, but peptide sequences from three of the forms were all found within a single deduced cDNA sequence (Sugimoto et al., 1997). The active product of the sugar beet -glucosidase is usually 91 kD (Chiba et al., 1978). As with spinach seeds, four forms of the sugar beet enzyme, varying both in affinity for the cell wall fraction and in substrate specificity, were separated from cultured cells (Yamasaki and Konno, 1989). Apparently, posttranslational modifications affect not only the size of the enzymes but also their substrate specificity. Other acidic -glucosidases for which sequence information is usually lacking are also glycosylated, including those from soybean (Yamasaki and Konno, 1985) and banana (Konishi et al., 1991). We conclude that most if not all acidic -glucosidases are glycosylated and proteolytically processed, but for no form of the enzyme is the extent of glycosylation or the specific proteolytic cleavage sites known. Apoplastic -glucosidases are well characterized biochemically, and some have been sequenced; however, little is known about their physiological function, especially in vegetative tissues. In this paper we describe the gene family from Arabidopsis and report around the isolation.