Multiple phosphoinositide‐specific phospholipases C in oat roots: characterization and partial purification

Abstract

SummaryPhosphoinositide-specific phospholipases C are critical enzymes in the transduction of hormonal and environmental signals into animal cells. Several different isozymes of phospholipese C are known in mammalian systems which differ in their expression and regulation. Elucidation of the regulation of these phospholipases C has greatly advanced our understanding of the control of mammalian cell growth and development. Plant cells, too, contain phospholipeses C specific for phosphoinositides, and there is evidence that they may be involved in plant cell responses to environmental stimuli. This paper reports that there are at least four variants of phosphoinositidespecific phospholipase C in the roots of oat seedlings, two cytosolic and two plasma membrane-associated. The two cytosolic and two plasma membrane variants can be separated on the basis of their affinity for binding to heperin. Both the cytosolic and the plasma membrane heparin-binding forms have apparent molecular weights of about 50-70 kDa by size exclusion chromatography. The two heparin-binding forms have been partially purified. The partially purified enzymes are activated by micromolar calcium and are specific for phosphorylated phosphoinositides; in their substrate specificities they resemble mammalian phospholipases C s.

Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

“…We report here a kinetic study of the partially purified, membrane-associated enzyme using different approaches. Few reports regarding the biochemical characterization of plant PLC are available (Tate et al, 1989;Melin et al, 1992;Pical et al, 1992;Yotsushima et al, 1992Yotsushima et al, , 1993Hirayama et al, 1995;Huang et al, 1995;Shi et al, 1995;Kopka et al, 1998). To our knowledge, this is the first report in which different assays were used to characterize this enzyme in plants.…”

“…PLC is a family of isoenzymes that has been classified into three groups: ␤, ␥, and ␦ (Rhee and Bae, 1997). In plants several studies have reported the biochemical presence of this enzyme (McMurray and Irvine, 1988;Tate et al, 1989;Melin et al, 1992;Pical et al, 1992;Yotsushima et al, 1992Yotsushima et al, , 1993Huang et al, 1995;De Los Santos-Briones et al, 1997). Different genes for PLC have also been cloned (Hirayama et al, 1995(Hirayama et al, , 1997Shi et al, 1995;Yamamoto et al, 1995;Kopka et al, 1998), all of them resembling the ␦ type.…”

Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba

“…TCA was removed, the pH was adjusted to 8.0, and the sample was concentrated 2-fold by evaporation under nitrogen and reconstitution in H,O. Receptor-binding activity was assayed by the usual method, and hydrolysis of added [3H]Ins(1,4,5)P3 was monitored by scintillation counting following chromatographic separation of [3H]Ins(1,4,5)P3 from [3H]Ins(1, 4)P2 on Amiprep trimethylaminopropyl SAX minicolumns (Amersham) as described by Huang et al (1995).…”

Involvement of phosphoinositide turnover in tracheary element differentiation in Zinnia elegans L. cells

“…Phospholipase C assay PLC activity was assayed by a modification of the method of Huang et al (1995). From 1.0·10 6 to 1.5·10 6 cells were collected by centrifuging at 20 g for 10 min.…”