Spatial and cellular localization of calcium-dependent protease (CDP II)inAllomyces arbuscula

Abstract

Immunogold labeling of calcium-dependent neutral protease II (CDPII) with specific antibodies in near median longitudinal ultrathin sections of Allomyces arbuscula showed that the enzyme is predominantly localized in the growing hyphal and rhizoidal apices. The tips in both cell type had more enzyme than the distal regions and showed a gradient distribution. Labeling of the ultrathin sections and western blot analysis of purified subcellular fractions showed that CDPII is mainly cytosolic. Catalytic activity of the enzyme measured with synthetic substrate (Bz-Arg-pNA) showed that 90% of its activity is present in the soluble fraction, although a small amount is associated with the nuclei (0.2%), plasma membranes (0.7%) and microsomes (3.9%). This association is discussed in the context of the functional role of the enzyme and its possible localized activation. Western blot analysis of the crude extract and indirect immunofluorescence of the fixed permeabilized hypahe after treatment with CDPII showed that theα-tubulin is a specific target of the enzyme.

Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene

“…Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e but a small amount is associated with plasma membrane and also present in the nuclei (Ojha and Barja, 2003). In this report we present the results on characterization of the gene coding for CDP II, its expression during development and show that this enzyme belongs to clan CD of C 14 family cysteine proteases, recently designated as metacaspase, present in fungi, protists and plants (Uren et al, 2000).…”

“…The presence of high level of transcript in the spores did not correlate with the total protein. The spores had relatively low level of proteins and no enzyme activity or antigen reacting to CDP II-specific antibodies in Western blots (Huber and Ojha, 1994;Ojha, 1996a) of spore cell-free extracts (Huber and Ojha, 1994;Ojha and Barja, 2003).…”

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: Cross-reaction with proteins other than spectrin

“…Proteins from unstained mono-and bi-dimensional gels were electrophoretically transferred to nitrocellulose membranes (BA85, pore size 0.45 lm) using the transfer buffer described by Burnette (1981) and immunoblotted as described earlier (Ojha and Barja, 2003). The membrane was blocked overnight at 4°C with 5% BSA in TBS-Tween, incubated for 2 h at room temperature with polyclonal anti-chicken ab-spectrin (Sigma S1390) or anti-N. crassa a-actinin antibody at 1:800 and 1:500 dilution in blocking buffer.…”

“…Primary anti-chicken spectrin-antibody and secondary goat anti-rabbit fluorescein-isothiocyanate (FITC, green, Sigma F9887) antibodies were used at dilutions of 1:50 and 1:100 in phosphate buffer (Ojha and Barja, 2003). The fluorescent signal was analysed with a Leica TCS SP2 AOBS confocal microscope.…”

Differentiation inside multicelled macroconidia of Fusarium culmorum during early germination

“…Swelling is certainly not merely water uptake alone, but is characterised by active incorporation of compounds into the cell wall (Bartnicki-Garcia and Lippman, 1977), and by the synthesis of protein and RNA (Ojha and Barja, 2003;Van Etten et al, 1983). During polarised growth, extension of the fungal cell occurs at a restricted area at the tip of the hypha (Parton et al, 1997), and results in elongation of the germ tube.…”