ISSN:
1432-1424
Keywords:
H+-ATPase
;
plant plasma membrane
;
reconstitution
;
vanadate
;
red beet
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Summary Characteristics of the native and reconstituted H+-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H+-ATPase retained characteristics similar to those of the native plasma membrane H+-ATPase following reconstitution into proteoliposomes. ATPase activity and H+ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO 3 − was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H+ transport showed a pH optimum of 6.5, required a divalent cation (Co2+〉Mg2+〉Mn2+〉Zn2+〉Ca2+), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H+-ATPases were stimulated by monovalent ions. Native plasma membrane H+-ATPase showed an order of cation preference of K+〉NH 4 + 〉Rb+〉Na+〉Cs+〉Li+〉choline+. This basic order was unchanged following reconstitution, with K+, NH 4 + , Rb+ and Cs+ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H+ transport, both enzymes required a cation such as K+ for maximal transport activity, but were stimulated preferentially by Cl− even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H+-ATPase.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01871063
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