Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

Benson, Michael J.; Tipton, Charles M.

doi:10.1104/pp.62.2.165

Cited by 22 publications

(15 citation statements)

References 15 publications

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“…The literature contains a number of reports of the purification of an ATPase activity from the plant plasma membrane (4,7,32). Benson and Tipton (4) claim to have purified the K+-stimulated ATPase from a membrane fraction from corn roots, but the enzyme described in their report had a low mol wt, poor substrate specificity and was insensitive to inhibitors of the plasma membrane ATPase.…”

Section: Discussionmentioning

confidence: 99%

“…However, the specific activity for ATP hydrolysis reported by Tognoli et aL (32) was so low (less than 1 ,umol/mg * h) that it is likely that the majority of the ATPase activity was inactivated by their method of preparing the membrane fraction. In each report (4,7,32), enzyme activity is retained after treatments that might be expected to remove lipids from a lipid-requiring protein and thus inactivate such a protein. The enzymes studied in these reports are probably nonspecific phosphatase contaminants of the membrane fraction used.…”

Section: Discussionmentioning

confidence: 99%

“…Despite rapid advances in purification and characterization of membrane transport proteins from biological membranes (15,18,27,35) there is no example of a purified, well characterized transport protein from the plasma membrane of higher plants. However, there are several reports of the purification of a putative transport ATPase (4,7,32). Benson and Tipton (4) report purification of a low mol wt ATPase activity from a membrane fraction from corn roots, but they provide no evidence that this enzyme was associated with the plasma membrane and did not represent the activity of a nonspecific phosphatase.…”

mentioning

confidence: 99%

“…However, there are several reports of the purification of a putative transport ATPase (4,7,32). Benson and Tipton (4) report purification of a low mol wt ATPase activity from a membrane fraction from corn roots, but they provide no evidence that this enzyme was associated with the plasma membrane and did not represent the activity of a nonspecific phosphatase. Similarly, Cross et al (7) report that a low mol wt ATP hydrolyzing activity with poor substrate specificity was released from a microsomal membrane fraction from corn coleoptiles by Triton X-100.…”

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confidence: 99%

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Solubilization and Partial Purification of the Adenosine Triphosphatase from a Corn Root Plasma Membrane Fraction

DuPont¹,

Leonard²

1980

Plant Physiol.

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The K+-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-fl-D-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent- octyl-fl-D-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Solubilization and Partial Purification of the Adenosine Triphosphatase from a Corn Root Plasma Membrane Fraction

DuPont¹,

Leonard²

1980

Plant Physiol.

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“…However, the enzyme has poor substrate specificity and its relationship to ion uptake is obscure (10). K+ stimulation of the purified enzyme is thus a continuous and positively cooperative function of external KCI concentration (5). A comparison of the kinetic properties of the solubilized enzyme with that of the reconstituted, membrane-bound enzyme should be awaited, however.…”

Section: Resultsmentioning

confidence: 99%

Stimulation of Plasmalemma Adenosine Triphosphatase from Oat Roots by Inorganic and Organic Cations: Concentration-Dependence, Selectivity, and Sites

Håvarstein¹,

Nissen²

1981

Plant Physiol.

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K-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 m;limolar KCI. The phases obeyed MlchaeLs-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 nillilar. Stimulation by alkali cations was in the order K+ > Rb+ > Na+ > Cs' > Li' or in a closely related sequence. The specificity reflected differences in ,,.,, not in affinity (K.'). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 mllmolar appeared monophasic rather than biphasic. Substitution on the quatemary nitrogen of the amino alcohols decreased their effectiveness, as did extension and brhing of the chain. Stimulation was maximal at about pH 7 both for K' and choline.The kinetics of K' stimulation are multiphasic, not cooperative, as was also found for uptake. The ATPase is also stimulated by organic cations, but the difference in kinetics indicates the existence of separate sites for stimulation and transition.Membrane-bound ATPases have been implicated in the transport of ions in plants (17,18,30). Particularly close correlations exist between a K+-stimulated ATPase and uptake of K+ across the plasmalemma (13,27,28). The role of this enzyme in ion transport remains unclear, however (25, 43).The present paper examines some unresolved questions concerning this relationship. The complex kinetics of ion uptake and ATPase stimulation have been described variously as cooperative (17,27,28) MATERIALS AND METHODS Seedlings of oat (Avena sativa L. cv. Titus) were grown in 0.5 mM CaC12 for 6 to 8 days at 16 to 22 C in the dark (40).A plasmalemma-enriched membrane fraction was isolated as described by Hodges and Leonard (19) with some minor modifications (32). The excised roots were ground with a mortar and pestle in a solution of 250 mm sucrose, 3 mm EDTA, 2.5 mm DTT, and 25 mm Tris-Mes buffer (pH 7.2). The filtered homogenate was centrifuged successively at 13,000g for 15 min and 40,000g for 30 min. The 40,000g pellet was suspended in 34% sucrose (w/w) and layered onto a discontinuous gradient consisting of 20 ml of 45% sucrose (w/w), 8 ml of 40%o sucrose, and 8 ml of 34% sucrose. All sucrose solutions contained 1 mm DTT and 1 mM Tris-Mes (pH 7.2). The gradient was centrifuged for 2 h at 80,000g in a Spinco SW 27 rotor, and the membrane fraction was collected at the 34/ 40% sucrose interface. For most experiments the membrane fraction was frozen at -18 C and used the following day (cf. Figs. 1 and 2).ATPase activity was measured in a l-ml volume containing 3 mM ATP (Tris salt, pH 6.5), 30 mm Tris-Mes (pH 6.5), 3 mM MgSO4, varying concentrations of monovalent inorganic or organic cations (as chlorides), and 10 to 25 ,tg of membrane protein.Reactions, initiated by the addition of ATP, were carried out at 38 C in a shaking water bath for 30 to 45 min, depending on the amount of protein. (ATPase activity was linear for at least 1 h, and the ATP concentrations remained essentially constant.) Pi released was de...

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Purification and Characterization of a divalent cation-activated ATP-ADPase from pea stem microsomes

Tognoli¹,

Marrè²

1981

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

Cited by 22 publications

References 15 publications

Solubilization and Partial Purification of the Adenosine Triphosphatase from a Corn Root Plasma Membrane Fraction

Solubilization and Partial Purification of the Adenosine Triphosphatase from a Corn Root Plasma Membrane Fraction

Stimulation of Plasmalemma Adenosine Triphosphatase from Oat Roots by Inorganic and Organic Cations: Concentration-Dependence, Selectivity, and Sites

Purification and Characterization of a divalent cation-activated ATP-ADPase from pea stem microsomes

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