Effect of Vanadate, Molybdate, and Azide on Membrane-Associated ATPase and Soluble Phosphatase Activities of Corn Roots

Gallagher, Sean R.; Leonard, Robert T.

doi:10.1104/pp.70.5.1335

Cited by 409 publications

(232 citation statements)

References 33 publications

(55 reference statements)

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“…Azide-sensitive ATPase and Cyt c oxidase, both inner mitochondrial membrane markers, showed 4.5% and 5.6% contamination, respectively. Vanadate-inhibited ATPase has been used as a plasma membrane marker when molybdate is present as an inhibitor of phosphatase (2,14). In these experiments, 17.4% of the plasma membranes were present in the vacuolar fraction.…”

Section: Resultsmentioning

confidence: 94%

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Proton Transport in Isolated Vacuoles from Corn Coleoptiles

Mandala

Taiz²

1985

Plant Physiol.

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Vacuoles were isolated from corn coleoptile protoplasts and ATPdependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of l'4Clmethylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATPstimulated methylamine uptake and a-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, the activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl1, and inhibited by NO3-, carbonyl cyanide-m-chlorophenylhydrazone, N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. We conclude that the microsomal proton pump previously charcterized in com coleoptiles Plant Physiol 70: 1738-1742) is derived from the tonoplast.The importance of electrogenic proton pumping ATPases in the transport of solutes across the plasma membrane and tonoplast of plant cells is being increasingly recognized. A cationstimulated Mg2+-ATPase on the plasma membrane is thought to be an electrogenic proton pump (25), and recent attempts to purify and reconstitute plasma membrane proteins have supported this idea (28). The presence of a proton-pumping ATPase on the tonoplast has been more controversial, although recent studies with isolated, intact vacuoles have led to the identification of a tonoplast ATPase in several systems (2-4, 7, 17, 19, 29-31). Investigations of the transport properties of sealed microsomal membranes have indicated that vesicles capable of in vitro ATPdependent proton transport can be isolated from a number of different plant tissues (26 and references therein). The bulk of this activity appears to be due to a novel ATPase which is distinguishable from other known ATPases (plasma membrane, mitochondrial, and chloroplast), but is similar to the recently characterized tonoplast ATPase.We have previously shown that microsomal membranes from corn coleoptiles have ATP-dependent proton pumping activity which is stimulated by Cl-, has a slightly alkaline pH optimum,

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Section: Resultsmentioning

confidence: 94%

“…After a 30-min incubation at 37°C, released Pi was determined (13). Vanadate-inhibited ATPase was assayed at pH 6.5 in the presence of 1 mm Na-molybdate to inhibit phosphatase activity (2,14). Azide-inhibited ATPase was determined at pH 8.5.…”

Section: Methodsmentioning

confidence: 99%

Proton Transport in Isolated Vacuoles from Corn Coleoptiles

Mandala

Taiz²

1985

Plant Physiol.

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“…The plasma membrane and the other membrane fraction were analysed for enzyme activities that serve as markers for various plant membranes, including the plasma membrane, tonoplast, mitochondria, Golgi and endoplasmic reticulum (ER). Speci®c markers include: the VO 4± sensitive Ptype ATPase activity (marker for plasma membrane and ER), NO 3± sensitive V-type ATPase activity (marker for tonoplast) (Gallagher and Leonard, 1982), Mg +2 -depedent inosine diphosphatase (marker for Golgi) (Ray et al, 1969), antimycine A insensitive NADHdependent cytochrome c reductase (marker for ER) (Lord et al, 1973) and cytochrome c oxidase (marker for mitochondria) (Hodges and Leonard, 1972).…”

Section: Membrane Isolation and Fractionationmentioning

confidence: 99%

The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface

Chiou¹,

Liu²,

Harrison³

2001

The Plant Journal

185

145

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SummaryThe movement of phosphate from the soil into plant root cells is the ®rst of many crucial transport events required to supply phosphorous (P) to cells throughout the plant. In addition to the ability to acquire phosphate from the soil, the majority of the vascular plants are able to form arbuscular mycorrhizal associations in which phosphate may be delivered to the cortex via a fungus. Previously, we cloned two phosphate transporter genes, MtPT1 and MtPT2 from Medicago truncatula roots. Complementation of a yeast phosphate transport mutant revealed that MtPT1 is a functional phosphate transporter and Northern analyses revealed that MtPT1 is expressed exclusively in roots (Liu et al., 1998, Mol. Plant-Microbe Interact. 11, 14±22). Utilising an antibody speci®c for MtPT1, we have analysed the accumulation and spatial expression patterns of the MtPT1 transporter. MtPT1 transcript and protein levels show close correlation and increase dramatically in the roots in response to phosphate starvation. MtPT1 protein levels decrease in roots during development of a symbiosis with arbuscular mycorrhizal (AM) fungi, indicating that this transporter is not involved in symbiotic phosphate transport. Membrane fractionation and analysis of a MtPT1/GFP fusion protein revealed that MtPT1 is located in the plasma membrane, while in situ hybridisation and immunolocalisation demonstrate the presence of MtPT1 transcripts and protein in the epidermal cells and root hairs of M. truncatula roots. MtPT1 shows expression patterns consistent with a role speci®cally in the acquisition of phosphate from the soil and is distinct from the other phosphate transporter of this class described to date.

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“…The ATP was present as the Tris salt (pH 6.5) converted from the sodium salt by treatment with Dowex 50-W ion-exchange resin (H+ form) according to the method of Hodges and Leonard (1974). Vanadate-inhibited K+,Mg2+-ATPase was determined as the difference of the activity in the presence and absence of 0.1 mM Na3V04 (Gallagher and Leonard, 1982).…”

Section: Subcellular Marker Enzyme Assaysmentioning

confidence: 99%

Cellular Localization of Protoporphyrinogen-Oxidizing Activities of Etiolated Barley (Hordeum vulgare L.) Leaves (Relationship to Mechanism of Action of Protoporphyrinogen Oxidase-Inhibiting Herbicides)

1993

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Seven-day-old, etiolated barley (Hordeum vulgare 1. var Post) leaves were fractionated into crude and purified etioplast, microsomal, and plasma membrane (PM) fractions. Protoporphyrinogen oxidase (Protox) specific activities of crude etioplast, purified etio- reduced the Protox activity of all fractions, except that microsomal Protox activity was slightly stimulated by NADPH. Ascorbate, CSH, or a combination of the two reductants enhanced Protox inhibition by AFM, and AFM inhibition of Protox was greatest in all fractions with DTT. NADPH enhanced AFM inhibition significantly only in etioplast fractions. Uroporphyrinogen I (Urogen I) and coproporphyrinogen I (Coprogen I) oxidase activities were found in all fractions; however, etioplast fractions had significantly more substrate specificity for protoporphyrinogen IX (Protogen IX) than the other fractions. Urogen I and Coprogen I oxidase activities were unaffected by AFM in all fractions, and 2 mM DTT almost completely inhibited these activities from all fractions. Diethyldithiocarbamate inhibited PM Protox activity by 62% but had less effect on microsome and little or no effect on etioplast Protox. juglone and duroquinone stimulated microsomal and PM Protox activity, whereas the lesser effect of these quinones on etioplast Protox activity was judged to be due to PM and/or microsomal contaminants. These data indicate that there are microsomal and PM Protogen IX-oxidizing activities that are not the same as those associated with the etioplast and that these activities are not inhibited i n vivo by AFM. In summary, these data support the view that the primary source of high protoporphyrin IX concentrations in AFM-treated plant tissues is from Protogen IX exported by plastids and oxidized by AFM-resistant extraorganellar oxidases.

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Effect of Vanadate, Molybdate, and Azide on Membrane-Associated ATPase and Soluble Phosphatase Activities of Corn Roots

Cited by 409 publications

References 33 publications

Proton Transport in Isolated Vacuoles from Corn Coleoptiles

Proton Transport in Isolated Vacuoles from Corn Coleoptiles

The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface

Cellular Localization of Protoporphyrinogen-Oxidizing Activities of Etiolated Barley (Hordeum vulgare L.) Leaves (Relationship to Mechanism of Action of Protoporphyrinogen Oxidase-Inhibiting Herbicides)

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