The Involvement of Glycosidases in the Cell Wall Metabolism of Suspension-cultured Acer pseudoplatanus Cells

Abstract

Several glycosidases have been isolated from suspensioncultured sycamore (Acer pseudoplatanus) cells. These include an a-galactosidase, an a-mannosidase, a f-N-acetylglucosaminidase, a ,B-glucosidase, and two ,3-galactosidases.The pH optimum of each of these enzymes was determined. Glycosidases have been reported in a variety of bacteria, fungi, animals, and higher plants. In plants, glycosidases have long been known to be present in seeds and storage material (1,2,4,8,9,15,17,(24)(25)(26)(27). Recently, there has been considerable interest in these glycosidases. One of the major reasons for the increased interest in these enzymes has been their usefulness as tools for studying the structure of oligo-and polysaccharides. Li has studied an a-mannosidase from jack bean meal and has used it to study a-mannosides present in glycoproteins (15-17). Agrawal and Bahl have studied the glycosidases from the cotyledons of Phaseolus vulgaris (1, 4). They have used these enzymes to study the structure of the saccharide portion of a glycoprotein hormone (3). An a-galactosidase from coffee beans has been used to study various galactomannans (7).Further interest in these enzymes stems from the possibility that they may be involved in growth of microbial and plant cells. ,-1 , 3-Glucanases have been reported to be widespread in higher plants (6). Among these are two reports of ,B-1 ,3-glucanase activity in suspension-cultured plant cells (10,18 growth (19,20,28,29). Cell wall-degrading enzymes have been reported to be present in crude preparations of cell walls (12)(13)(14). These findings suggest that glycosidases partially degrade cell walls and, in so doing, facilitate cell growth.In this report we describe a number of glycosidases of sycamore cells (Acer pseudoplatanus) grown in suspension culture. Some of the glycosidases are found associated with the cell walls. The activity of some of these hydrolytic enzymes is greater during log phase growth than prior to or after the growth period of these suspension-cultured cells.MATERIAL AND METHODS Sycamore cells (A. pseudoplatanus) were grown on M-6 media as described earlier (5) except that 20 g/liter of sucrose was used rather than 40 g/liter.For the growth experiments, cells were grown in 2800-ml Fernbach flasks with a three-way stopcock attached near the bottom. The stopcock was sterilized by passing 70% ethanol through it before and after each use. Cells were withdrawn while the culture was agitated by a magnetic stirrer. Representative samples of the cell population could be obtained in this manner.The cells were collected on a coarse scintered glass funnel. The collected cells were washed with 5 volumes of 0.5 M potassium phosphate, pH 7.0, and the wash solution was saved. The washed cells were suspended in additional phosphate buffer (1.0 ml of buffer for each g of cells) and disrupted by forcing the suspension through a French pressure cell at 2,000 p.s.i. The resulting preparation was centrifuged for 60 min at 40,000g.The enzymes in the 0.5 M wash from the whole ...

Distribution of α-Galactosidase in Cucurbita pepo

“…leaves (3,8) where an involvement in galactolipid metabolism of organelle membranes was suggested, and it has also been reported in walls of epicotyl tissue (12) and in plant cell and tissue cultures (10,13). Our interest in the distribution of a-galactosidase in Cucurbita pepo resulted from our discovery that immature importing leaves possess an extremely active ability to hydrolyze raffimose, stachyose, and verbascose entering their tissue from the phloem and are incapable of any detectable synthesis or accumulation of these sugars.…”

Host-Pathogen Interactions

“…The elicitor, as a foreign molecule, must approach its site of action in the soybean cell from outside the cell. Previous studies have shown that glycosidases bound to the walls of intact plant cells will hydrolyze exogenously added substrates (14,17).…”

Host-Pathogen Interactions

“…This culture was also grown in modified B5 medium according to published methods (6,14). Cell suspension cultures of sycamore (Acer pseudoplatanus) were grown in modified M-6 medium with 20 g/l sucrose (19).…”