No abstract

No abstract

The optical probes 9-aminoacridine, quinacrine, and bis (3-propyl-5-oxoisoxazol-4-yl) pentamethine oxonol (OX-VI) were used to measure the formation of pH and electric potential gradients in vesicles believed to be derived from corn root tonoplast membranes. Under certain conditions the quenching of fluorescence of 9-aminoacridine was shown to be quantitatively related to A pH, and the shift in the absorbance spectrum (measured as absorbance difference at 610 and 580nm) of OX-VI shown to be quantitatively related to Ar In the absence of chloride ions, a positive interior membrane potential of approximately +100mV was formed upon the addition of 5 mM Mg/ATP to a suspension of membrane vesicles. The addition of C1-salts reduced AO and stimulated the formation of ApH. In the presence of 50mMCI-, a ApH of approximately 1.1 units was established following the addition of 5 mM Mg/ATP. The kinetics of C1-activation of H + transport could be resolved into a linear and a saturable component, with a K~ for the saturable component between 4 and 5 mM. CI-inhibition of A~, showed similar kinetics, indicating that C1-activates electrogenic H § transport as a permeant anion. The biphasic kinetics suggest two pathways for CI-permeation, with the linear component attributable to permeation through the lipid bilayer and the saturable component attributable to permeation through an anion channel. This model is supported by the effects of the anion channel blockers, 4-acetamido-4'-isothiocyano-2,2'-stilbenedisulfonic acid (SITS) and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), which abolished the saturable component but not the linear component of CI-stimulated H + transport.CI-also stimulated ATPase activity associated with the membrane vesicles both in the absence and presence of gramicidin. In the presence of gramicidin the kinetics of C1 stimulation of ATPase activity were adequately described by a Michaelis-Menten function with a K m for CIbetween 4 and 5 mM. This result indicated a direct role for CI-in activating the H+-ATPase in addition to its apparent role as a permeant anion. The similarity in K m values for CI-stimulation of the ATPase and for the saturable component of CI-stimulation of H + transport suggests that the H+-ATPase may be closely associated with a CI-channel and that together they catalyze the nearly electroneutral transport of H § and CI-in vivo.

No abstract

Microsomal membes isolated from red beet (Beta ulgatns L.) storage tissue were found to contain high levels of ioophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicdin and coincded with a peak of NO3-sensitive ATPase activity. At In the last few years several groups have reported the existence of an anion-sensitive, proton-translocating ATPase in a microsomal membrane fraction from plant cells (3,8,11,13,16,21). There have also been several reports describing an ATPase associated with isolated plant vacuoles (1,15,(26)(27)(28) been suggested that this anion-sensitive H+-ATPase is of tonoplast origin (11,16). In order to confirm a tonoplast origin for the anion-sensitive H+-ATPase, this study used membrane isolation procedures and H+-transport assays developed for studying the corn root, anion-sensitive H+-ATPase (3, 1 1) to identify a similar ATPase in red beet microsomal membranes. This tissue was chosen for several reasons: (a) Briskin and Poole (7) have recently shown that microsomal membranes can be readily isolated from this tissue in high yields and substantially free of nonspecific phosphatase activity, (b) intact vacuoles can be easily isolated from this tissue (14) and, (c) a tonoplast ATPase has already been characterized in this tissue (1, 28). These advantages allowed the identification ofan anion-sensitive H+-translocating ATPase in red beet microsomal membranes which could be compared directly to the ATPase activity associated with isolated vacuoles. The results support a tonoplast origin for the anionsensitive H+-ATPase and further identify a second, vanadatesensitive H+-translocating ATPase in the red beet microsomal membranes, which is presumably of plasma membrane origin. MATERIALS AND METHODSPlant Material. Red beets (Beta vulgaris L.) were obtained commercially. Care was taken to purchase beets with fresh leafy tops to ensure that they were freshly harvested. The tops were removed and the storage tissue (swollen hypocotyl) stored at 4°C until use, but not longer than 1 month.Membrane Preparation. Microsomal membranes were prepared essentially as described by Briskin and Poole (7)