Parvalbumin-immunoreactive proteins in the nervous system of planarians

Summary 1. Using immunological methods, we have identified parvalbumin-like material in the triclade flatworms,Polycelis nigra, Polycelis auriculata, Crenobia alpina, Dugesia tahitiensis, andDugesia polychroa.2. Western immunoblot analysis of these five species revealed heat stable parvalbumin-immunoreactive proteins between 32 kD and 44 kD.3. Proteins at 19 and 32 kD revealed intense labeling with⁴⁵Ca²⁺.4. Double immunodiffusion of planarian supernatants showed complete fusion of precipitates, indicating immunological relatedness of the parvalbuminlike material among the species investigated.5. Immunocytochemical studies exhibit parvalbumin-immunoreactive material exclusively in neurons supporting the notion of an early evolutionary appearance of these proteins in the nervous system.     

Streptozotocin-Induced Diabetes Mellitus and Further Nimodipine Treatment Do Not Change Calcium Currents in Rat Sensory Neurons within Early Stages of the Disease

Earlier, considerable prolongation of the depolarization-induced Ca²⁺ transients was demonstrated in primary sensory neurons of rats with streptozotocin (STZ)-induced diabetes mellitus. To analyze the nature of this effect, we examine possible changes in the characteristics of voltage-operated calcium channels. Neither the amplitude of Ca²⁺ currents provided by both high- and low-voltage activated calcium channels nor the respective current densities significantly changed within the early stages of diabetes mellitus. In rats treated with nimodipine, also no significant changes in the calcium channel activity were observed. Only in the case of a decrease in the external calcium concentration was some drop in the Ca²⁺ current amplitude observed. We conclude that within the early stages of diabetes mellitus there are no significant modifications in the structure of the membrane of primary sensory neurons manifested in the expression of Ca²⁺ channels, which might be responsible for the observed rapidly occurring changes in calcium signalling, cytosolic Ca²⁺ accumulation, and synaptic plasticity.     

An Ibotenate-Selective Metabotropic Glutamate Receptor: Mediates Protein Phosphorylation in Cultured Hippocampal Pyramidal Neurons

Abstract: Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca²⁺ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120-and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and ionotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca²⁺ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca²⁺ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca²⁺ but not in 50 nM extracellular Ca²⁺. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphates in both normal and low extracellular Ca²⁺. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca²⁺. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50–60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml),-suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation. These results suggest that the stimulation of phosphorylation in pyramidal neurons by glutamate occurs predominantly through the activation of an ibotenate-selective metabotropic glutamate receptor.     

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca²⁺ ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg²⁺, Ba²⁺ or K⁺ could not replace this requirement for Ca²⁺. The presence of K⁺ did not enhance the Ca²⁺-dependent swelling response. When the Ca²⁺-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca²⁺-channelblocker Verapamil and La³⁺ inhibited R-induced swelling. It is proposed that R causes the opening of Ca²⁺-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.Abbreviations EDTA ethylenediaminetetraacetic acid - FR far-red light - nov non-osmotic-volume - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light     

Effect of calcium on synthesis of dipicolinic acid inPenicillium citreoviride and its feedback resistant mutant

Dipicolinic acid synthesis inPenicillium citreoviride strain 3114 was inhibited by Ca²⁺ ions, but not by Ba²⁺, Cu²⁺or Fe²⁺. Among the metals tested, only Zn²⁺ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca²⁺ or Zn²⁺. A mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca²⁺ complex showed cross-resistance to inhibition by dipicolinic acid: Zn²⁺. Both 3114 and271 33-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca²⁺, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca²⁺ did not inhibit thede novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca²⁺ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca²⁺ complex, mutant differed from the parent in three other aspectsviz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca²⁺ ions in the medium and (iii) Mg²⁺ requirement for the mutant increased three fold. Higher requirement of the Mg²⁺ could be partially relieved by Ca²⁺ during secondary metabolism. The results support the inference thatde novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca²⁺complex.     

Partial Characterization of a Novel Cardioinhibitory Peptide from the Brain of the Snail Helix aspersa

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin.2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure : H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH₂. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-ammo acid sequence of HCIP is identical to that of FMRFamide family peptides.3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified .     

Outgrowth morphology and intracellular calcium of crustacean neurons displaying distinct morphologies in primary culture

Peptidesecreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca²⁺ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium ([Ca²⁺]_i) have been implicated in regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal [Ca²⁺]_i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological tapes can extend out processes over a [Ca²⁺]_i range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high k⁺ saline led to increases in [Ca²⁺]_i in soma, neurite, and lamellipodium of veiling neurons. Increase were great for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb [Ca²⁺]_i were used to assess the role of [Ca²⁺]_i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested addition of 100 μMCD²⁺, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd²⁺. Branching neurons were unaffected by Cd²⁺. Cd²⁺ at lower levels (10 μM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 μM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal [Ca²⁺]_i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing [Ca²⁺]_i with respect to regenerative outgrowth, but not its form. 1994 John Wiley & Sons, Inc.     

Deficient mitochondrial Ca(2+) buffering in the Cln8(mnd) mouse model of neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses (NCLs) are a group of genetic childhood-onset progressive brain diseases characterized by a decline in mental and motor capacities, epilepsy, visual loss and premature death. Using patch clamp, fluorescence imaging and caged Ca²⁺ photolysis, we evaluated the mechanisms of neuronal Ca²⁺ clearance in Cln8^mnd mice, a model of the human NCL caused by mutations in the CLN8 gene. In Cln8^mnd hippocampal slices, Ca²⁺ clearance efficiency in interneurons and, to some extent, principal neurons declined with age. In cultured Cln8^mnd hippocampal neurons, clearance of large Ca²⁺ loads was inefficient due to impaired mitochondrial Ca²⁺ uptake. In contrast, neither Ca²⁺ uptake by sarco/endoplasmic reticulum Ca²⁺ ATPase, nor Ca²⁺ extrusion through plasma membrane was affected by the Cln8 mutation. Excitotoxic glutamate challenge caused Ca²⁺ deregulation more readily in Cln8^mnd than in wt neurons. We propose that neurodegeneration in human CLN8 disorders is primarily caused by reduced mitochondrial Ca²⁺ buffering capacity.     

Multi-target novel neuroprotective compound ITH33/IQM9.21 inhibits calcium entry, calcium signals and exocytosis

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca²⁺ entry and neuronal Ca²⁺ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K⁺-evoked [Ca²⁺]_c transients with an IC₅₀ of 5.31 μM. At 10 μM, the compound decreased the amplitude and area of the Ca²⁺ transient elicited by challenging single fura-2-loaded cells with high K⁺, by 40% and 80%, respectively. This concentration also caused a blockade of K⁺-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca²⁺ currents (IC₅₀ = 6.52 μM). At 10 μM, ITH/IQM also inhibited the Ca²⁺-dependent outward K⁺ current, leaving untouched the voltage-dependent component of I_K. The inward Na⁺ current was unaffected. Inhibition of depolarisation-elicited Ca²⁺ entry, [Ca²⁺]_c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

Simultaneous recording of cytosolic Ca2+ levels inDidinium andParamecium during aDidinium attack onParamecium

Summary The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca²⁺ level in each cell. However, these putative increases in cytosolic Ca²⁺ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca²⁺ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca²⁺ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca²⁺ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca²⁺ rise inDidinium, theParamecium also showed an increase in cytosolic Ca²⁺. This is the first simultaneous recording of changes in the Ca²⁺ level during a predator-prey interaction in ciliates. The possible roles of these Ca²⁺ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.Abbreviations AED aminoethyldextran - P_i inorganic phosphate - FITC fluorescein isothiocyanate     

Ca2+ reversibly inhibits active rotation of chloroplasts in isolated cytoplasmic droplets ofChara

Summary The effects of Ca²⁺ and other cations on chloroplast rotation in isolated cytoplasmic droplets ofChara were investigated by iontophoretically injecting them. Chloroplast rotation stopped immediately after Ca²⁺ injection and recovered with time, suggesting the existence of a Ca²⁺-sequestering system in the cytoplasm. The Ca²⁺ concentration necessary for the stoppage was estimated to be >10^–4M. Sr²⁺ had the same effect as Ca²⁺. Mn²⁺ and Cd²⁺ induced a gradual decrease in the rotation rate with low reversibility. K⁺ and Mg²⁺ had no effects. Ba²⁺ had effects sometimes similar to Ca²⁺ or Sr²⁺ and sometimes similar to Mn²⁺ or Cd²⁺.Reversible inhibition by Ca²⁺, together with its specificity, strongly supports the hypothesis that a transient increase in the Ca²⁺ concentration in the cytoplasm upon membrane excitation directly stops the cytoplasmic streaming inCharaceae internodes (Hayama et al. 1979).     

Effect of divalent cations on succinate transport in Rhizobium tropici,R. leguminosarum bv phaseoli and R. loti

Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C₄-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca²⁺ or Mg²⁺. However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca²⁺ or Mg²⁺ had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca²⁺ to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca²⁺ and Mg²⁺. A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.The authors are with the Division of Biochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Avda, Italia 3318, 11.600 Montevideo, Uruguay     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Determination of the growth and solubilization capabilities of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> T1

The growth capability of Trichoderma harzianum Rifaii Tl was tested on Malt Extract and Czapeks Dox agar containing different concentrations of Cu²⁺, Zn²⁺, Mn²⁺, Fe²⁺ and Ca²⁺. The T. harzianum Tl isolate was observed to produce mycelia and spores in various mineral-containing media. It showed the lowest tolerance to Ca²⁺ and the highest tolerance to Fe²⁺. Solubilization capability of T. harzianum Tl for some insoluble minerals via acidification of medium has been tested on MnO₂, CuO, Fe₂O₃ and metallic Zn. T. harzianum Tl was able to solubilize MnO₂ and metallic Zn in a liquid medium.     

Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum

Concentrations of adenine and pyridine nucleotides and the associated charge values were examined in extracts of mycelium of Penicillium notatum during vegetative growth and reproductive development promoted by the addition of Ca²⁺ (10 mmol dm^-3). The significant increase in adenylate energy charge promoted by Ca²⁺ was due to a fall in intracellular AMP and a concomitant rise in ATP concentration. Intracellular concentrations of NADH and NAD fell within 1 h of the addition of Ca²⁺. The catabolic reduction charge was unchanged by Ca²⁺ whilst the anabolic reduction charge increased in Ca²⁺-induced mycelium due to lowered intracellular NADP concentration. Reduced concentration of NADPH in Ca²⁺-induced mycelium, relative to the vegetative controls, lowered the phosphorylated nucleotide fraction. The results are discussed in relation to metabolic economy during morphogenesis in P. notatum.     

Calcium Signalling in Rat Brain Neurons and Differentiation of PC-12 Cells Induced by Application of a Probiotic Product

The effects of a biotechnological probiotic product, PP, produced by food fermentation with Lactobacilli (US patent approved), on the growth of neurites in rat pheochromocytoma cells (PC-12) and on calcium responses of rat brain neurons were studied in culture. The PP increased the length of neurites in PC-12 cells, resulting in an irreversible differentiation of cancerous cells into neuron-like structures. Moreover, a change in the neurotransmitter phenotype of differentiated cells was found; some cells, such as excitatory neurons, began to respond to glutamate application by increasing [Ca²⁺] _i . The PP directly activated PC-12 cells and neurons by the release of Ca²⁺ from the intracellular stores in a steady manner. The PP also stimulated the entry of Ca²⁺ into the cells in a Ca²⁺ add-back protocol, which was considerable upon washing out of PP. Thus, the products of Lactobacillus metabolism, such as those in PP, can act as a neuronal growth factor and manifest clear pharmacological reactions at the cellular level. By comparison, commercial lyophilized probiotic bacteria also induced a Ca²⁺ rise in neurons, but not in PC-12 cells. Some neurons did not respond to probiotic bacteria, and some neurons responded with some delay. Upon wash out of probiotic bacteria, a huge entry of Ca²⁺ into the cells was observed. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 284–293, May–June, 2005.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.     

Identification and characterisation of Ca-pectate binding peroxidases inArabidopsis thaliana

The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca²⁺ and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca²⁺-mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca²⁺-pectate but not to Ca²⁺-alginate, a polyuronate gel similar to Ca²⁺-pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca²⁺-pectate and Ca²⁺-alginate was also assessed. It appeared that 3 of them exhibited a Ca²⁺-pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca²⁺-alginate was much weaker than to Ca²⁺-pectate, confirming the specificity of the interaction with the pectic structure.     

Characterization of cell surface material removed by water and NaCl washing ofParacoccus denitrificans grown under conditions of divalent cation deficiency and sufficiency

Paracoccus denitrificans grown on complex medium deficient in Mg²⁺ and Ca²⁺ are rendered lysozyme susceptible by washing with NaCl, whereas cells grown in a succinate-salts medium (Mg²⁺ and Ca²⁺ sufficient) or complex medium supplemented with Mg²⁺+Ca²⁺ are not. The material released by water washing of cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ was characterized by a high protein content. There was a high lipid: protein ratio and an appreciable amount of 3-deoxyoctulosonic acid in the material released by NaCl washing of cells grown under all conditions, indicating release of outer membrane material. The lipid ornithine: lipid phosphorous ratios of NaCl wash from cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ were 0.54 and 0.34, respectively. Although NaCl washing removed outer membrane material from cells grown under all conditions, only divalent cation deficient cells were rendered lysozyme susceptible. This might be explained by the increased outer membrane ornithine-containing lipid to phospholipid ratio in these cells yielding a more permeable outer membrane.