| Research Abstract |
Plant vacuolar membrane contains two distinct proton pumps, namely inorganic pyrophosphatase and ATPase. The vacuolar pyrophosphatase catalyses the PPi hydrolysis and the proton translocation into vacuole. Two proton pumps create a low internal pH and provide an energy source of the secondary transport systems of the vacuolar membrane. In this study, the molecular properties of the enzymes were investigated.
The vacuolar pyrophosphatase was purified from mung bean hypocotyls and shown to consist of a single protein of 73 kDa. An antibody to the 73-kDa protein inhibited the PPi hydrolysis of enzyme. DCCD, which is thought to inhibit ATPases by blocking proton conductance, binds to the 73-kDa protein and inhibits the pyrophosphatase. Thus the 73-kDa protein is an actual H^+-translocating pyrophosphatase. The pyrophosphatase functions as a homodimer comprised of 73-kDa subunit. The enzyme requires Mg^<2+> for its stability and activity. Ca^<2+>, however, strongly inhibits the pyrophosphatase. The rate of inhibition by Ca^<2+> depended on the concentration of Ca-PPi complex, and the apparent K_i for CaPPi was about 17 uM.
The vacuolar ATPase was also purified from mung bean and shown to consist of nine different subunits (68, 57, 44, 38, 37, 32, 16, 13 and 12 kDa). Except for the 16-kDa subunit, the other subunits were released from the membrane by treatment with KSCN. These eight subunits may compose the hydrophilic part of the ATPase, and the DCCD-binding subunit of 16 kDa composes the proton channel. Enzymatic properties of the ATPase and pyrophosphatase were also analyzed.
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