Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?
The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.
Roberts
G
G
División de Biofisica, Instituto de Investigación Médica M. y M. Ferreyra, Córdoba, Argentina.
Berberián
G
G
Beaugé
L
L
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D002523:Edible Grain / Q000201:enzymology*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D008274:Magnesium; D011188:Potassium
D007527:Isoenzymes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium