Cellulose in algal cell wall: an "in situ" localization.

In order to ascertain the presence and the in muro localization of cellulose, the enzyme-gold affinity test was applied to algal cell walls. The high specificity of affinity cytochemistry allowed us, by using the enzyme cellulase, to confirm the available biochemical data and to give a map of the cellulose localization in different algal groups. Taking into account the complex skeletal polysaccharide structure and composition of the algal cell walls, this method proved to be a reliable tool in this field.

Mobilization of Ca2+ by cyclic ADP-ribose from the endoplasmic reticulum of cauliflower florets.

The NAD+ metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca2+ release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca2+ release was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+: These vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2+ release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling.

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate.

Higher plants share with animals a responsiveness to the Ca(2+) mobilizing agents inositol 1,4,5-trisphosphate (InsP(3)) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular (45)Ca(2+) flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca(2+)-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca(2+) with a K(1/2) = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca(2+) pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca(2+) pathway distinguishes it from the InsP(3)- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca(2+) release is insensitive to heparin and 8-NH(2)-cADPR, specific inhibitors of the InsP(3)- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca(2+) release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca(2+) release pathway is independent of cytosolic free Ca(2+) and therefore incapable of operating Ca(2+)-induced Ca(2+) release. In contrast to the sea urchin system, the NAADP-gated Ca(2+) release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca(2+) mobilization pathways and Ca(2+) release sites might contribute to the generation of stimulus-specific Ca(2+) signals in plant cells.

Functional conservation of calreticulin in Euglena gracilis.

Calreticulin is the major high capacity, low affinity Ca2+ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca2+ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca2+ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca2+ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phylogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca2+ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore ii) that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca2+ binding function of calreticulin from Euglena traces the functional history of Ca2+ second messenger signal transduction pathways deep into eukaryotic evolution.

Primary structure of the N-linked carbohydrate chains of Calreticulin from spinach leaves.

Calreticulin is a multifunctional Ca(2+)-binding protein of the endoplasmic reticulum of most eukaryotic cells. The 56 kDa Calreticulin glycoprotein isolated from spinach (Spinacia oleracea L.) leaves was N-deglycosylated by PNGase-F digestion. The carbohydrate moiety was isolated by gel permeation chromatography and purified by high-pH anion-exchange chromatography. The fractions were investigated by 500 MHz 1H-NMR spectroscopy, in combination with monosaccharide analysis and fast-atom bombardment-mass spectrometry. The following carbohydrate structure could be established as the major component (Man8GlcNAc2): (sequence see text) Heterogeneity was demonstrated by the presence of two minor components being Man7GlcNAc2 lacking a terminal residue (D1 or D3), compared to the major component. A cross-reactivity with an antibody against the endoplasmic reticulum retention signal HDEL was also found.

Plant calreticulin is specifically and efficiently phosphorylated by protein kinase CK2.

Calreticulin isolated from spinach leaves has been specifically phosphorylated in vitro by protein kinase CK2 while animal calreticulin from rabbit liver is not a substrate of this kinase under the same conditions. Phosphoserine is the only phosphoamino acid detected. High affinity binding (Km = 4.4 microM) and a nearly stoichiometric incorporation of phosphate was determined. Partially purified spinach calreticulin is phosphorylated at the same site(s) by a copurifying protein kinase sharing biochemical properties very similar if not identical to those of mammalian CK2. Other plant calreticulins isolated from Liriodendron tulipifera appear to be also phosphorylated by CK2.

Metabolic activation of benzo[a]pyrene in two fetal mouse hepatocyte lines: induction of DNA adducts and micronuclei.

We have studied the metabolic competence of two non-transformed epithelial-like cell lines derived from fetal mouse liver, C 6 and C 2.8, to activate the promutagen benzo[a]pyrene by measuring both the induction of DNA adducts through the nuclease P1-enhanced 32P-postlabeling assay and the formation of micronuclei. The pattern and level of DNA adducts detected in C 6 and C 2.8 cells treated with benzo[a]pyrene were compared with those obtained in human peripheral blood lymphocytes treated with the same compound and with [3H]anti-benzo[a]pyrene diolepoxide. In both the cell lines and in human lymphocytes we observed a consistent induction of distinct DNA adducts. In C 6 and C 2.8 cells, the most evident adduct showed a position similar to that of the main adduct induced by [3H]-anti-benzo[a]pyrene diolepoxide in human lymphocytes. In addition, benzo[a]pyrene caused a significant increase of micronucleated C 6 and C 2.8 cells, whereas the frequency of micronuclei did not increase in CHO cells treated, for comparison, in the same way.

Evidence that spinach leaves express calreticulin but not calsequestrin.

The presence of either calreticulin (CR) or calsequestrin (CS-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca(2+)-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn(2+) (i.e., under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca(2+). Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca(2+)-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.