Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L.

Abstract

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with N03-sensitive and vanadate-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg2", a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.A great deal of information has been accumulated on the proton ATPase located on the tonoplast from several plants (1,7,19,27). The majority of the publications involve measurements carried out on intact vacuoles isolated by the osmotic lysis of enzymically isolated protoplasts under conditions that preserve the integrity of the tonoplasts (1 1, 26).To get more insight into the biochemical and molecular properties of the tonoplast ATPase and the membrane per se, we need membranes both of high purity and in a large quantity sufficient for analytical uses. For this purpose, tonoplast-enriched fractions are separated from crude microsomal membranes by centrifugation on a linear or discontinuous sucrose density gradient or a dextran density gradient (2,7,8,10,16,18,20).ATPases are known to be associated with wide variety of membrane systems in plant cells. Recently, ATPases have reported to be associated with ER (4, 5), Golgi membranes (6), and plastid envelopes (15), and they have been partially characterized. The tonoplast-enriched fractions separated after a density gradient are usually contaminated with those endomembranes associated with ATPases. Tonoplasts could also be separated in a high purity from the intact vacuoles isolated from enzymically prepared protoplasts following an osmotic lysis. However, there ' Partly supported by Science.is difficulty in getting a large quantity of the tonoplasts.This paper reports the isolation of tonoplast from etiolated seedlings of Vigna radiata by a method in which sorbitol was used as an osmotic stabilizer throughout crude membrane preparation prior to a sucrose density gradient centrifugation. This method has permitted us to isolate tonoplast in a high purity and in a large quantity on subsequent sucrose density gradient.MATERIALS AND METHODS Plant Materials. Seeds of Vigna radiata...

Impairment of Tonoplast H+-ATPase as an Initial Physiological Response of Cells to Chilling in Mung Bean (Vigna radiata [L.] Wilczek)

“…The chill-induced decline of the H+-ATPase activity and the impairment of the proton-translocating function, whether reversible or irreversible, may therefore result in a perturbation of the cellular compartmentations of solutes and ions, especially protons and Ca2 . In contrast to the ATPase, the tonoplast PPase and the proton-pump activities were rather stable for chilling periods up to 3 d. As reported earlier (29), mung bean tonoplast H+-ATPase activity was not stimulated by the addition of 0.016% Triton X-100 into the reaction mixture, whereas the plasma membrane ATPase was stimulated severalfold by the addition of the detergent (28), suggesting that these isolated membrane vesicles were mostly in a normal sidedness. In purified tonoplast vesicles, on the other hand, the PPase activity was observed to be latent and stimulated dramatically by the addition of 0.015% detergent into the reaction mixture (data not shown), suggesting an insideout orientation to the vesicles.…”

Comparison of Temperature Dependency of Tonoplast Proton Translocation between Plants Sensitive and Insensitive to Chilling

“…Released Pi was quantified by the Fiske-SubbaRaw (5) method. Assays of other enzymes were carried out according to the methods reported earlier (14). Protein was quantified according to the dye method of Bradford (2) using BSA as a standard.…”

Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals

“…The pelleted microsomes were resuspended (1.5-2 mg protein/mL) in 50 mM Hepes (pH 7.0) containing 0.2 M sucrose. Ten milliliters of this suspension were layered over 30 mL of a linear 15% to 45% (w/w) sucrose gradient and centrifuged at 85,000g for 3 or 16 h. Two-milliliters fractions were collected from the top of the gradient by upward displacement using 60% (w/w) sucrose and assayed for marker enzymes as described previously (22,28,29). NADH:Cyt c reductase was assayed by measuring LA550 at 25°C in a reaction mixture containing 25 sodium orthovanadate or 50 mM KNO3 in a total volume of 1 ml.…”