Association of Latent Cellulase Activity with Plasma Membranes from Kidney Bean Abscission Zones

Abstract

Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10-to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which celiulase was associated had a peak equilibrium density of 1.16 to 1.17 g/ cm3 which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate celiulase was associated with the plasma membrane.Abscission zones from kidney bean seedlings contained cellulase which existed in two forms (9,14). An intracellular form with a p13 of 4.5 was present at all times, and an extracellular form with a pl of 9.5 increased during abscission or after treatment with ethylene (10). The 4.5-pI form was buffer-soluble whereas the 9.5-pI form was extracted from cell walls by high salt. The 9.5-pl form hydrolyzed cellulose (9) and appeared to cause breakdown of cell walls during abscission.The relationship between the forms of cellulase and their function during abscission is not known. One possibility is that the 4.5-pI form is transported out of the cell to give rise to the 9.5-pI form which functions in cell wall breakdown. As a first step to testing this hypothesis, we have determined that cellulase is associated with cell membranes and identified the membrane system with which it is associated. The results show that from 5 to 10% of the total cellulase activity in the tissue was associated with the plasma membrane whereas no significant activity was found in other membranes. MATERIALS AND METHODSPlant Material and Cell Fractionation. Seeds of Phaseolus vulgaris L., cv. Red Kidney, were germinated in a mixture of vermiculite and pea gravel (3:1) and grown for 10 to 12 days at 24 C and a light intensity of 2,000 ft-c. One-cm sections of petiole including the laminar abscission zone were excised into a chilled beaker and homogenized at a ratio of 3 ml buffer/I g fresh weight of tissue in a polytron (Brinkmann Instruments) at 2 C for 45 sec at a setting of 5. The homogenization buffer contained 20 mm tris-MES, pH 7.2, 0.3 M sucrose, 3 mM EDTA, and 0.1 % BSA. The homogenate was squeezed through a 50-,um mesh nylon cloth, and cell fractions were collected by centrifugation at 2,000g for 10 min and 13,000g for 15 min at 2 C. These pellets were washed by suspending in homogenization buffer and centrifuging at the original forces. An additional cell fraction was obtained by centrifuging the 13,000g supernatant at 80,000g for 45 min onto a cushion of 55% (w/w) sucrose.This fraction was washed by suspending in homogenization buffer without sucrose and pelleting again onto a sucrose cushion.Sucrose Density Gradients. Linear sucrose density gradients we...

Differential Ion Stimulation of Plasmalemma Adenosine Triphosphatase from Leaf Epidermis and Mesophyll of Nicotiana rustica L

“…DISCUSSION The isopycnic gradients from epidermis and mesophyll tissue both exhibited a plasmalemma ATPase with peak activity at 38% sucrose. The position of the peak ATPase activity agrees well with plasma membrane isolated from other plant tissues (8,11,16,20) and the enzyme markers cellulase and glucan synthetase support its identity as plasmalemma. This fraction is also apparently relatively free of contaminating membranes, particularly chloroplast fragments which comprise a lighter fraction immediately above the plasmalemma.…”

“…1). The ATPase peak at 38% sucrose appears to be plasmalemma since glucan synthetase at high substrate levels and cellulase activity, both markers for plasmalemma (11,20), also exhibit peak activity at 38% sucrose (Fig. 1).…”

“…A number of workers have characterized this enzyme from root and stem preparations but very few investigations have been carried out with photosynthetic tissue (1,11) in spite of strong evidence suggesting its involvement with the regulation of stomatal opening (8,19). Virtually all articles to date have dealt with homogenized organs (see the recent article by Leonard and Hotchkiss [16] for an exception).…”

β-Glucosidase Activity in Corn Roots

“…The first washing removed the greatest amount of enzyme activity (36%) as well as the greatest amount of protein (16%) when compared to the unwashed control (Table II). The first wash removed soluble extraneous protein (7). Successive washings removed considerably less of the total j8-glucosidase activity and less protein with a concomitant small decrease in specific activity (successive washing removed some membranes due to the change in sedimentation path length).…”

“…Soluble enzymes migrate further into sucrose gradients upon increased centrifugation time (rate sedimentation [13]). If membrane-bound, the (3-glucosidase activity should equilibrate at the isopycnic density of the associated membrane and not change in density with prolonged centrifugation time (7). Figure 2A shows the 'particulate' (3-glucosidase activity associated with the 1,000 to 120,000g membrane pellet was actually a soluble enzyme which moved further into the sucrose gradient with longer centrifugation time.…”