Purification of an elicitor-induced glucan synthase (callose synthase) from suspension cultures of French bean (Phaseolus vulgaris L.): purification and immunolocation of a probable M(r)-65,000 subunit of the enzyme.
Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of relative molecular masses (M(r)) 55,000 and 65,000, the latter being in excess. The M(r)-65,000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the M(r)-55,000 subunit is likely to represent the catalytic subunit while the M(r)-65,000 polypeptide is a possible regulatory subunit. The M(r)-65,000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates, at the plasma membrane-wall interface of wounded cells and in papillae in infected cells.
McCormack
B A
BA
Division of Biochemistry, School of Biological Sciences, Royal Holloway and Bedford New College, University of London, Surrey, UK.
Gregory
A C
AC
Kerry
M E
ME
Smith
C
C
Bolwell
G P
GP
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004790:Enzyme Induction; D007887:Fabaceae / Q000201:enzymology*; D005656:Fungal Proteins / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins; D016253:Microscopy, Immunoelectron; D008861:Microsomes / Q000201:enzymology*; D003904:Mitosporic Fungi; D008970:Molecular Weight; D010946:Plants, Medicinal; D029702:Schizosaccharomyces pombe Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase