An anion-sensitive H+-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esclekntum) fruit. The H'-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by N03-, N,N'-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H'-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K. for the H-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H'-ATPase are very similar to those described for a number of plant cell tonoplast H'-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.
ABSTRACICitrate transport across the membrane of tomato fruit tonoplast vesicles was investipted. In the tonoplast vesicles, ["Cqmethylamine uptake was stimulated 10-fold by MgATP and strongly inhibited by NO3-.Under identical experimental conditions, ["Cqcitrate uptake was inhibited by 5 millimolar free Mg2", and this inhibition was reversed in the presence of ATP, presumably by ATP chelation of free Mg2. No evidence was obtained in support of energy-linked ATP stimulation of citrate uptake. Citrate uptake showed saturation kinetics, and was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid and by other orgnic acids. The pH-dependence of uptake suggested that citrate3 was the transported species. Our results indicate that citrate transport across the tomato fruit tonoplast occurs by facilitated diffusion of citrate>. The carrier shares some features in common with anion channels in that it is relatively nonspecific for organic acids and is inhibitable by 4,4'-diisothyocyano-2,2'-stilbenedisulfonic acid.One of the major organic acids present in tomato fruit tissue is citric acid (6,14). This acid accumulates to concentrations of 6 to 30 mmol per kg fresh weight in the whole fruit, and of 10 to 34 mmol per kg fresh weight in the pericarp tissue (6). It is likely that this high level of citric acid is largely confined to and indeed concentrated in the vacuole, and therefore must be transported across the tonoplast. The goal of the present study was to elucidate the mechanism(s) by which citric acid is transported into the vacuole of developing tomato fruit.Although information on accumulation of organic acids in plant cell vacuoles is scarce, a few studies have contributed valuable insight into mechanisms that are operative in some plants. Luttge and Ball (7) Based on these studies, it appeared that any of several mechanisms could be operative in citrate transport across the tomato tonoplast, including simple diffusion, facilitated diffusion, or carrier-mediated active transport, possibly by an antiport mechanism. It also appeared that the functional mechanism could be either dependent upon, or independent of the electrochemical gradient for H+ established by the tonoplast H+-ATPase. MATERIALS AND METHODSPlant Material. Tomato fruit (Lycopersicon esculentum cv UC82 were harvested from greenhouse or field grown plants (depending on availability) at the mature green stage (approximately 40 d after pollination).Membrane Preparation. Tomato fruit tonoplast membranes were prepared as previously described (11) except that a 16%/ 30% discontinuous sucrose gradient interface was collected to increase yield of tonoplast vesicles. Protein Determination. Protein was determined by the method of Schaffner and Weismann (13).Citrate Uptake Assays. The typical reaction mix for these assays consisted of 50 mM Tris/Mes (pH 7.0), 50
Incorporation of FN into IGF-I-loaded collagen+Vicryl membranes reduced IGF release from collagen and increased the migration of human gingival fibroblasts. The new membrane may promote healing and reformation of the dentogingival junction.
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