Phosphatidylinositol Specific Phospholipase C of Plant Stems

Abstract

A phosphatidylinositol-specific phospholipase C of plant stems (EC 3.1A.10) assayed at pH 6.6 and at 30°C cleaved phosphatidylinositol such that more than 85% of the product was inositol-l-phosphate. Other phospholipids were cleaved 5 to 10% or less under these conditions. The phospholipase had both a soluble and a membrane-associated form. The soluble activity accounted for approximately 85 to 90% of the activity and 15% was associated with membranes. The membrane-associated activity was most concentrated in the plasma membranes of hypocotyl segments of both soybean (Glycine max) and bushbean (Phaseolus vulgaris). The plasma membrane location was verified by analysis of highly purified plasma membranes prepared both by aqueous two-phase partitioning and by preparative free-flow electrophoresis and from the quantitation of the activity in all maior cell fractions. Internal membranes also contained phospholipase C activity but at specific activity levels of about 0.1 those present in plasma membranes. Golgi apparatus-enriched fractions from which plasma membrane contaminants were removed by twophase partition contained the activity at specific activity levels 0.2 those of plasma membrane. Both the soluble and the membrane-associated activity was stimulated by calcium but not by calmodulin, either alone or in the presence of calcium.There have been periodic reports of soluble phospholipase C activities in plants (8,9,11). Additionally, breakdown of membrane-associated phosphatidylinositol has been demonstrated based on analyses using radiolabeled phosphatidylinositol (5,8,9,20). The recent interest in phospholipases has been stimulated by their importance in signal-response transduction mechanisms (3,18,22) and the occurrence of phosphoinositol phosphates in plants (4,7,21,25). The latter in carrot cells appear concentrated in plasma membranes (JJ Wheeler, WF Boss, personal communication). In this paper we have examined the subcellular distribution of a phosphatidylinositol-specific phospholipase C of etiolated hypocotyls of soybean and bushbean and the response of the membrane-associated and soluble forms of the activity to calcium and calmodulin. The findings show a clear plasma membrane location of the membrane-associated form of the enzyme and a stimulation ofboth the soluble and the membraneassociated forms of the enzyme with calcium but not with calmodulin. The calcium-stimulated activity appears to be present in very low levels throughout the system of internal membranes as well. ' Supported in part by a grant 8206222 from the National Science Foundation (to D.J.M.

Purification and Characterization of Phosphatidylinositol-specific Phospholipase C in Suspension-cultured Cells of Rice (Oryza sativaL.)

“…5). Irvine et al 33 ) have demonstrated that dialyzed soluble extracts of celery and some other plants could hydrolyze PI in the presence of Ca 2 + in the millimolar range and at neutral pH, with the release of phosphoinositol and 1,2-cyclic phosphoinositol in the ratios expected for cleavage by a phospholipase C. Pfaffmann et al 16 ) have reported that, of the total PI-PLC activity of the homogenates, approximately 85% was soluble and 15% was membrane-associated in the soybean hypocotyl, and both the soluble and the membraneassociated activities were stimulated by Ca 2 + in the same manner as total activity with an optimum of 0.5 mM. Recently, the membrane-associated PI-PLC have been characterized in various higher plants.…”

“…19) The plasma membrane-bound PI-PLC was stimulated by a micro molar range of Ca 2 + 17 -20) and preferentially hydrolyzed PIP and PIP 2 more than pI. 16,17,19) In the rice suspension-cultured cells, most of PI-PLC activities are in the soluble fraction, but, there was little information for the soluble PI-PLC in higher plants. In this communication, we report enzymatic properties of PI-PLC purified from the soluble fraction of rice cells.…”

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells

“…Specifically, (a) the polyphosphoinositides must be located in the plasma membrane, (b) the enzymes responsible for the synthesis and degradation of polyphosphoinositides must be associated with the plasma membrane, (c) the polyphosphoinositide levels must transiently change upon cellular stimulation, (d) the products from the polyphosphoinositide hydrolysis must transiently increase upon stimulation, (e) a transient increase of free Ca2" in the cytosol must follow the transient increase of IP3. To date, only the first and the second criteria have been documented for several different plant tissues (15,19,25,29). There are few reports in the literature concerning polyphosphoinositide turnover in plants in response to stimuli.…”

Diacylglycerol Kinase from Suspension Cultured Plant Cells

“…the existence of PI and PI-4-phosphate kinases (8,9,31,32) and phospholipase C (4,5,10,11,18,23,24,27,28) kinase during the growth cycle of suspension-cultured plant cells (9). In the present work the purification of DG-kinase from suspension-cultured Catharanthus roseus cells is reported and its properties are compared with enzymes described from E. coli (3,17,20,30,34) and from animal cells (14,15,19).…”

“…the existence of PI and PI-4-phosphate kinases (8,9,31,32) and phospholipase C (4,5,10,11,18,23,24,27,28), have been reported recently. We assayed the in situ activity of DG- 'This work has been supported by the Deutsche Forschungsgemeinschaft (Wa 91) and the Fonds der Chemischen Industrie.…”