Cell Walls of Phaseolus vulgaris Leaves Contain the Azocoll-Digesting Proteinase

Abstract

The leaves of Phaseolus vulgaris L. cv Greensleeves contain an endopeptidase with a pH optimum of 9.0 and an isoelectric point between 10.0 and 10.5. This endopeptidase is the only abundant Azocoll-digesting proteinase in the leaves. The activity of this enzyme is highest in immature leaves and declines as the leaf matures and senesces. Enzymically isolated protoplasts contain very little of this proteinase. The proteinase can be recovered readily from the extracellular fluid obtained by gentle centrifugation of leaf strips vacuum-infiltrated with a buffered solution. These experiments indicate that the Azocoll digesting proteinase is located in the periplasmic space and/or the cell wall.Our present view of the structure of the cell wall is one in which cellulose fibrils are embedded in a three-dimensional matrix of hemicelluloses, pectic polysaccharides, and glycoproteins with hydroxyproline-rich domains. 3 To whom reprint requests should be addressed at Department of Biology, C-0 16, UCSD, La Jolla, CA 92093. described (3).Enzyme Extraction. Immediately after the leaves were taken from the plants, they were weighed, cut into small pieces, rinsed with distilled H20 and cooled to OC. The leaves were then homogenized in 4 volumes of 50 mm K-phosphate (pH 7.4) containing 1% insoluble PVP (Sigma). Homogenization was for 3 min at 0C with a Polytron homogenizer (Kinematica, Luzern, Switzerland). The homogenate was strained through four layers of cheesecloth and centrifuged at 25,000g for 15 min. The supernatant was used as a source of enzymes.Column Chromatography. Extract from 10 g of leaves were loaded on a DEAE-cellulose (Whatman, DE 32) column (30 x 1 cm), equilibrated with 50 mm K-phosphate (pH 7.4). The column was washed with equilibration buffer till the nonabsorbed protein was rinsed from the column and then eluted with equilibration buffer containing 1.0 M NaCl. Fractions were collected and assayed for enzyme activity. Azocollase activity was not absorbed and was recovered in the void volume of the column. The azocollase containing fractions were pooled, dialyzed against 1 mm K-phosphate (pH 7.4), and subjected to preparative isoelectrofocusing using a L. K. B. (Bromma, Sweden) 8001 column with a 1% ampholine (L. K. B.) solution in the pH 3.5 to 10.0 range. The column was run at 1 w and 4C for 40 h. Protoplast Isolation. Leaves were cut in 4-mm2 pieces, vacuum infiltrated, and incubated in a solution of 0.37% Cellulysin (Calbiochem) and 0.1% polycillin in mannitol medium (0.6 M mannitol in 10 mM Mes-NaOH buffer, pH 5.5). After 16 h of incubation at 27C with gentle shaking, the macerated tissue was resuspended in this medium and carefully strained through two layers of cheesecloth. The filtrate was centrifuged at 22g for 20 min. The pellet, which contained the intact protoplasts, was resuspended in mannitol medium containing 25% Ficoll and overlayered with a solution composed of 15% Ficoll in mannitol medium. The gradient was centrifuged at 40g for 15 min and the protoplasts formed a layer on top of t...

Nucellain, a Barley Homolog of the Dicot Vacuolar-Processing Protease, Is Localized in Nucellar Cell Walls

“…One example of this is peroxidases, which are believed to cause reduction of cell wall extensibility by forming bridges between phenolic residues on neighboring cell wall proteins or polysaccharides (Kim et al, 1989). Similarly, proteinases have also been suggested to modify cell wall polypeptides during growth (Van Der Wilden et al, 1983;Varner and Lin, 1989). Therefore, we propose that nucellain plays a role in processing and/or turnover of cell wall proteins.…”

Purification and Developmental Analysis of a Metalloendoproteinase from the Leaves of Glycine max

“…Calcium has also been shown to be an absolute requirement for the calpains, a class of proteinases that have been observed in animal and fungal systems. These enzymes have yet to be described in higher plants (17 Van der Wilden et al (25) speculated that the bean proteinase may be localized in cell walls and may possibly be involved in the metabolism of cell wall proteins. We find this speculation appealing in light of the growing list of proteins, such as hydroxyproline-rich proteins, proline-rich proteins, glycinerich proteins, pathogenesis-related proteins, peroxidases, phosphatases, and glycosidases, residing outside the plasma membrane (4,6,11,12,26).…”

“…Those proteinases involved with the metabolism of seed storage proteins and senescence have received the most attention (8,22). With the exception of a few reports (3,19,25), the literature contains little information on the presence of the metalloproteinase class of enzymes in plants. Indeed, to date, there have been no definitive reports of metalloproteinases in plants.…”

“…Those proteinases involved with the metabolism of seed storage proteins and senescence have received the most attention (8,22). With the exception of a few reports (3,19,25) the metallo and cysteine classes of proteinases, respectively. Their findings have aided us in further characterization of the Azocollase A activity, including the purification to homogeneity of this enzyme.…”

Association of a 33-Kilodalton Cysteine Proteinase Found in Corn Callus with the Inhibition of Fall Armyworm Larval Growth

“…The released hydrolases may be the first line of defense against pathogen or herbivore attack (Boller, 1986). Some proteinases such as the one found in Phaseolus vulgaris leaves (Van der Wilden et al, 1983) are present in the cell wall and thus provide another site of defense. Feeding by Spodoptera littoralis induces Leu aminopeptidase in tomato (Pautot et al, 1993).…”