Differential Sensitivities towards Inhibitors and SH-Compounds of H +-ATPase and H +-Pyrophosphatase Associated with Intact Vacuoles from Sweet Sorghum ( Sorghum bicolor [L.] Moench) Stem Parenchyma

Abstract

Intact vacuoles isolated from sweet sorghum ( Sorghum bicolor [L.] Moench) stem parenchyma protoplasts were found to be stable for more than 4 days. Purity and vitality of the organelle fraction allowed its use for a comparative study of H + -ATPase and -pyrophosphatase, the tonoplast energizing systems. In mature storage tissue, PP i ase exhibited far less stability than ATPase, and PP i -dependent H + -transport accounted for only 12-–slightly, and was unaffected by PCMBS. Furthermore, the two tonoplast-associated enzyme complexes revealed different sensitivities towards other inhibitors (DCCD, DIDS, DES). ATPase-driven H + -pumping was inhibited highly specifically by the macrolide antibiotic bafilomycin A 1 with 50% inhibition at 10 −10 M. The inhibition occurred directly after application, and a protection by substrate, M 2+ or Cl − could not be observed. ATP hydrolysis was less sensitive to bafilomycin, with 50% inhibition at 10 −9 M. Tonoplast PP i ase activity, in contrast, remained unaltered up to 10 −6 M bafilomycin. A similar insensitivity to the antibiotic applied for the postulated Ca 2+ / H+ -anti porter at the tonoplast: When maximum ΔpH formation was completed and active H + -pumping was suppressed by 10 −9 M bafilomycin A 1 , a gradual decrease in the ΔpH by increasing amounts of Ca 2+ , but not Mg 2+ , up to concentrations of about 6 μM free calcium could be observed. Higher concentrations of Ca 2+ , however, destroyed the integrity of the sorghum stem parenchyma vacuoles.