Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

The present study describes the biochemical characteristics of an acid -fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag⁺ and Hg²⁺ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one 1 2-linked xylose residue, probably one fucose residue 1 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid -fructosidase of the sweet-pepper fruit.Abbreviations F -fructosidase - ConA concanavalin A - DEAE diethylaminoethyl - DTNB dithionitrobenzoic acid - endo F endo--N-acetylglucosamidase F - endo H endo--N-acetylglucosamidase H - NEM N-ethylmaleimide - PCMB parachloromercurobenzoate - PNGase glycopeptide-N-glycosidase - TFMS trifluoromethane sulfonic acid This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.     

Deuterium-labelled indole-3-acetic acid neo-synthesis in plantlets and excised roots of maize

Synthesis of indole-3-acetic acid (IAA), using stable-isotope incorporation, was investigated in Zea mays L. Incorporation of ²H from ²H₂O into IAA molecules was shown to occur in intact plantlets and excised primary roots cultured in vitro. This demonstrates the de-novo formation of IAA, a process which is quantitatively well defined and is initiated early in germination.Abbreviations IAA indole-3-acetic acid     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive -carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of -carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive -carotene accumulation, greatly enhances -carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive -carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive -carotene accumulation in D. bardawil.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

The SKS of the KMSKS signature of class I aminoacyl-tRNA synthetases corresponds to the GKT/S sequence characteristic of the ATP-binding site of many proteins

On the basis of protein modification studies and primary structure comparison, we propose that the SKS sequence within the KMSKS signature of the class 1 aminoacyl-tRNA synthetases corresponds to the GKT(or S) sequence considered as a signature of the nucleotide triphosphate-binding site of many proteins.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.     

The influence of lecithin on plasma choline concentrations in triathletes and adolescent runners during exercise

An investigation was carried out on the effect of lecithin (phosphatidylcholine, 90%) on the plasma choline concentrations during continuous strain in 10 top level triathletes (4 women and 6 men), trial I, and 13 excellent adolescent runners (3 girls and 10 boys), trial II. Venous blood, collected before and immediately after the race, was separated and plasma was assayed by an improved high performance liquid chromatography method for choline. Each study comprised three experiments. In trial I the triathletes performed two periods of bicycle exercise each lasting 2 h at an average speed of 35 km · h^–1, and in the second study (trial II) the subjects were subjected to severe physical stress on two occasions during cross-country races of durations between 30–60 min according to their ages. The participants received either a placebo or 0.2 g lecithin · kg body mass^–1, 1 h before each exercise. As a control the same dose of lecithin was administered without any exercise (both trials I and II). Bicycle exercise without lecithin supply decreased plasma choline concentrations in all the triathletes, on average by 16.9% (P0.01). When lecithin was given before exercise, average plasma choline concentrations remained at the same level as the initial values. The supply of lecithin without exercise led to a significant increase of the plasma choline concentrations, on average by 26.9% (P0.01). In trial II, when running without a supply of lecithin, the mean plasma choline concentrations in the adolescent runners remained stable which may have been due to the duration of the physical stress. When lecithin was given before exercise, plasma choline concentrations increased, on average by 18.9% (P0.01). The administration of lecithin without exercise led in these participants to an increase in plasma choline concentrations, on average by 54% (P0.001).It was found from the present study that a combination of both lecithin intake and hard physical stress prevented in most subjects a decrease in plasma choline and this could affect performance.     

Distribution of NADPH-diaphorase activity in rat paravertebral,prevertebral and pelvic sympathetic ganglia

Summary Paravertebral (superior cervical and stellate), prevertebral (coeliac-superior mesenteric, inferior mesenteric) and pelvic (hypogastric) sympathetic ganglia of the rat were investigated by enzyme histochemistry to ascertain the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity. In the paravertebral ganglia the majority of the sympathetic neuronal perikarya contained lightly and homogeneously distributed formazan reaction product but there was a range of staining intensities amongst the neuron population. In contrast, in the prevertebral ganglia, intense NADPH-diaphorase staining was present in certain neurons. Firstly, a population of neurons of the coeliac-superior mesenteric ganglion complex were surrounded by densely NADPH-diaphorase-positive baskets of fibres and other stained fibres were seen in interstitial nerve bundles and in nerve trunks connected to the ganglion complex. Secondly, in both the inferior mesenteric ganglion and hypogastric ganglion there were many very intensely NADPH-diaphorase positive neurons. Stained dendritic and axonal processes emerged from these cell bodies. In both ganglia this population of neurons was smaller in size than the lightly stained ganglionic neurons and commonly had only one long (presumably axonal) process. The similarity of these highly NADPH-diaphorase-positive neurons with previously described postganglionic parasympathetic neurons in the hypogastric ganglion is discussed.