Auxin Receptors of Maize Coleoptile Membranes Do Not Have ATPase Activity

Cross, John W.; Briggs, Winslow R.; Dohrmann, Ulrike; Ray, Peter M.

doi:10.1104/pp.61.4.581

Cited by 77 publications

(26 citation statements)

References 17 publications

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“…While the plasma membrane ATPase consists of a single catalytic peptide (22), it may well require other peptides for efficient proton transport, in a manner analogous to the F1F0-ATPase (14). The protein that binds auxin is not the catalytic subunit (6), but might be a proton channel peptide (3) or a regulating peptide.…”

Section: Discussionmentioning

confidence: 99%

Auxin Regulation of a Proton Translocating ATPase in Pea Root Plasma Membrane Vesicles

Gabathuler¹,

Cleland²

1985

Plant Physiol.

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ABSTRACrPea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K, of about 0.23 millimolar for MgATP, is only slightly affected by K' or Cl-and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO3-> Cl-) and has a K. of about 0.7 millimolar. Auxins decrease the K., to about 0.35 millimolar, with no significant effect on the V,,,., while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.The plasma membrane of plant cells contains an ATPase which is responsible for the electrogenic export of protons to the apoplastic solution (26). Its activity can be studied with isolated, inside-out plasma membrane vesicles, since they can retain the ability to transport protons in response to MgATP (25,27,29 on wet filter paper. After 3 d, the apical (2 cm) tips of the roots were harvested.Isolation of Sealed Microsomal Vesicles. All procedures were carried out at 4°C. The roots were first chopped with a razor blade and then homogenized using a mortar and pestle in 1 ml/ g fresh weight of buffer 1 (6% [w/w] sorbitol, 3 mm EDTA, 0.1 % BSA, 25 mM Tris, 20 mM mercaptoethanol, pH 7.0, with Mes). The homogenate was strained through four layers ofcheesecloth, the residue was rehomogenized in 1 ml/g fresh weight of buffer I and refiltered. The total filtrate was centrifuged at 6,000g for 20 min. The supematant was then centrifuged at 1 l0,OOOg for 30 min (Rotor type 30, Beckman L5-50 centrifuge) to obtain the microsomal pellet. The pellet was resuspended in 5 ml of buffer I and layered over a discontinuous Dextran T70 gradient consisting of 7 ml of 10%, 7 ml of 6%, and 7 ml of 4% Dextran T70 (w/w) in buffer I. This was centrifuged at 70,000g for 2 h (Rotor SW 27). The membrane bands at the interfaces were removed with a Pasteur pipette (fraction A, 0/4% interface; B, 4/6%; C, 6/10%) and diluted with buffer 11 (4.5% sorbitol, 25 mm Tris, pH 7.0, with Mes). After centrifugation at 1 l0,OOOg for 30 min, each pellet was resuspended in buffer II (except where indicated) and was used immediately for enzyme and proton translocation experiments.Enzyme Assays. ATPase activity was assayed in 0.5 ml containing 25 mM Tris-Mes pH 7.0, 10 mm MgSO4, 0.1 mM ammonium molybdate, 1 mM NaN3, 0.067 to 2.5 mm NaATP, and salts as indicated. The presence of sodium ions in NaATP did not affect the results, as Tris-ATP gave identical results. Reactions were initiated by addition of membrane protein (10-50 jig). After 10 to 30 min at 30°C, the reaction was stopped with 1 ml ice-cold 0.5% ammonium molybdate + 0.1% SDS in 0.72 N H2SO...

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Section: Discussionmentioning

confidence: 99%

Auxin Regulation of a Proton Translocating ATPase in Pea Root Plasma Membrane Vesicles

Gabathuler¹,

Cleland²

1985

Plant Physiol.

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“…The reactions were started by adding 0.5 mM ADP or PPi and proceeded for 15 min at 37°C. Phosphate released was determined as described by Cross et al [22].…”

Section: Adpase and Ppiase Activitymentioning

confidence: 99%

ADP‐ or pyrophosphate‐dependent proton pumping of pea stem tonoplast‐enriched vesicles

Macrı̀

Vianello

1987

FEBS Letters

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Pea stem tonoplast-enriched vesicles have an ADPase and PPiase activity capable of generating a transmembrane proton gradient (ApH) and an electric potential (A~,). Both proton translocating activities have a pH optimum around 6.5, are Mg2+-dependent, require the presence of a monovalent cation (K +, Rb ÷ or Cs +) and of a permeant anion, such as NO3, CI or Br-. They are almost completely inhibited by 50 #M DIDS, DES and DCCD, 50% inhibited by 100/zM molybdate and unaffected by Na3VO4 or KNO3. Hexokinase and ATP do not prevent H÷-ADPase and H+-PPiase activity, thus indicating that these functions are not caused by an ATP-dependent proton pumping and that they have catalytic sites different from those of H÷-ATPase, respectively. On the basis of these characteristics, ADP-and PPi-dependent proton translocating activities seem carried out by a similar enzyme complex which appears different from the NOa-inhibited, VOW--insensitive H+-ATPase.

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“…Our interpretation of this is indicated in Figure 1. Neither IAA (Cross et al, 1978) nor fusicoccin (TognoH et al, 1979;Stout and Cleland, 1980) appear to bind directly with the membrane ATPase, although in the case of fusicoccin the receptor may be that part of a multi-unit enzyme complex which regulates catalytic activity .…”

Section: Molecular Site Of Action Of Fusicoccinmentioning

confidence: 99%

“…Tbere is no evidence bearing on these questions, but tbere is the precedent of the gibberellins, which were first known as fungal toxins. Auxin and fusicoccin must influence H+ secretion by different mecbanisms (Cross et al, 1978, and references tberein) but it is not excluded tbat tbe ultimate site of activating the H"*" pump is the same; nor is it excluded that tbey are different.…”

Section: Activation Of the A Tpase By Fusicoccin In Vivomentioning

confidence: 99%

Regulation of Plant Cell Growth: The Changing Perspective

Hanson

Trewavas

1982

New Phytologist

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Summary This article outlines the main features of growth regulation by auxin and places them side‐by‐side with the physiological properties of fusicoccin. The fundamental role of the plasma membrane located H+/K+ ATPase in growth responses is examined and the information about this enzyme briefly reviewed. The effects of wounding on the activity of this enzyme are detailed and it is concluded that growth in excised tissue segments ceases not from lack of hormone but from impairment of ATPase function. An additional conclusion of major importance for hormone studies is reached; that is, that the experimentally‐realized reactivation of growth by auxin in excised tissues represents largely an accelerated recovery from injury. Furthermore the calmodulin/Ca2+ system may be an essential intermediary in this recovery step. Finally, the major area of ignorance, predisposition to growth, is considered.

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Auxin Receptors of Maize Coleoptile Membranes Do Not Have ATPase Activity

Cited by 77 publications

References 17 publications

Auxin Regulation of a Proton Translocating ATPase in Pea Root Plasma Membrane Vesicles

Auxin Regulation of a Proton Translocating ATPase in Pea Root Plasma Membrane Vesicles

ADP‐ or pyrophosphate‐dependent proton pumping of pea stem tonoplast‐enriched vesicles

Regulation of Plant Cell Growth: The Changing Perspective

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