Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis.

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.

Cucumber hypocotyls respond to cutin monomers via both an inducible and a constitutive H(2)O(2)-generating system

Hypocotyls from etiolated cucumber (Cucumis sativa L.) seedlings were gently abraded at their surface to allow permeation of elicitors. Segments from freshly abraded hypocotyls were only barely competent for H(2)O(2) elicitation with fungal elicitor or hydroxy fatty acids (classical cutin monomers). However, elicitation competence developed subsequent to abrasion, reaching an optimum after about 4 h. This process was potentiated in seedlings displaying acquired resistance to Colletotrichum lagenarium due to root pretreatment with 2,6-dichloroisonicotinic acid or a benzothiadiazole. Induction of competence depended on protein synthesis and could be effected not only by surface abrasion, but also by fungal spore germination on the epidermal surface or by rotating the seedlings in buffer. Inhibitor studies indicated that the inducible mechanism for H(2)O(2) production involves protein phosphorylation, Ca(2+) influx, and NAD(P)H oxidase. In contrast, a novel cucumber cutin monomer, dodecan-1-ol, also elicited H(2)O(2) in freshly abraded hypocotyls without previous competence induction. This finding suggests the presence of an additional H(2)O(2)-generating system that is constitutive. It is insensitive to inhibitors and has, in addition, a different specificity for alkanols. Thus, dodecan-1-ol might initiate defense before the inducible H(2)O(2)-generating system becomes effective.

Cutin monomers and surface wax constituents elicit H2O2 in conditioned cucumber hypocotyl segments and enhance the activity of other H2O2 elicitors

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface and cut segments were conditioned to develop competence for H2O2 elicitation. Alkaline hydrolysates of cutin from cucumber, tomato, and apple elicited H2O2 in such conditioned segments. The most active constituent of cucumber cutin was identified as dodecan-1-ol, a novel cutin monomer capable of forming hydrophobic terminal chains. Additionally, the cutin hydrolysates enhanced the activity of a fungal H2O2 elicitor, similar to cucumber surface wax, which contained newly identified alkan-1,3-diols. The specificity of elicitor and enhancement activity was further elaborated using some pure model compounds. Certain saturated hydroxy fatty acids were potent H2O2 elicitors as well as enhancers. Some unsaturated epoxy and hydroxy fatty acids were also excellent H2O2 elicitors but inhibited the fungal elicitor activity. Short-chain alkanols exhibited good elicitor and enhancer activity, whereas longer-chain alkan-1-ols were barely active. The enhancement effect was also observed for H2O2 elicitation by ergosterol and chitosan. The physiological significance of these observations might be that once the cuticle is degraded by fungal cutinase, the cutin monomers may act as H2O2 elicitors. Corrosion of cutin may also bring surface wax constituents in contact with protoplasts and enhance elicitation.

Competence for Elicitation of H2O2 in Hypocotyls of Cucumber Is Induced by Breaching the Cuticle and Is Enhanced by Salicylic Acid.

To study H2O2 production, the epidermal surfaces of hypocotyl segments from etiolated seedlings of cucumber (Cucumis sativus L.) were gently abraded. Freshly abraded segments were not constitutively competent for rapid H2O2 elicitation. This capacity developed subsequent to abrasion in a time-dependent process that was greatly enhanced in segments exhibiting an acquired resistance to penetration of their epidermal cell walls by Colletotrichum lagenarium, because of root pretreatment of the respective seedlings with 2,6-dichloroisonicotinic acid. When this compound or salicylic acid was applied to abraded segments, it also greatly enhanced the induction of competence for H2O2 elicitation. This process was fully inhibited by 5 [mu]M cycloheximide or 200 [mu]M puromycin, suggesting a requirement for translational protein synthesis. Both a crude elicitor preparation and a partially purified oligoglucan mixture from Phytophthora sojae also induced, in addition to H2O2 production, a refractory state, which explains the transient nature of H2O2 elicitation. Taken together, these results suggest that the cucumber hypocotyl epidermis becomes conditioned for competence to produce H2O2 in response to elicitors by a stimulus resulting from breaching the cuticle and/or cutting segments. This conditioning process is associated with protein synthesis and is greatly enhanced when substances able to induce systemic acquired resistance are present in the tissue.

Insulin stimulatory action on amino acid uptake in bovine adrenal cortex or glomerulosa zone.

1. Insulin stimulated the [1-14C] methylaminoisobutyric acid and [1-14C] aminoisobutyric acid uptake in the bovine adrenal cortex or in the glomerulosa zone through the A system. 2. Verapamil nullified the insulin stimulatory action indicating that this hormonal action is probably related to the voltage-dependent Ca2+ channels.

Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients.

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.

Fast and sensitive protein staining with colloidal acid violet 17 following isoelectric focusing in carrier ampholyte generated and immobilized pH gradients.

A new method is described for fast and sensitive staining of proteins following isoelectric focusing in carrier ampholyte and immobilized pH gradient polyacrylamide gels. After fixation with trichloroacetic acid the gels are stained for 5-10 min with 0.1-0.2% colloidal Serva Violet 17 (generic name: Acid Violet 17; Color Index No. 42,650) in 10% w/v phosphoric acid. After staining for only 0.5-3 min, major zones, corresponding to 100-500 ng protein, are visible without destaining on a weak background. Detection of minor components requires destaining with 3% w/v phosphoric acid for 5-80 min depending on gel thickness (120-500 microns) and type of support (fabric reinforced versus gels backed to a polyester film). For selected pH marker proteins (bovine serum albumin, carbonic anhydrase, horse myoglobin) a staining sensitivity of 1-2 ng/mm2 protein is found. Dye elution from stained fabric reinforced gels with 50% v/v dioxane-water, followed by absorbance measurements, results in a linear relationship over a range of 1-100 micrograms marker proteins. Staining with collodial Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges. Staining with the collodial dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazardous.