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()

Intact vacuoles isolated from sweet sorghum (Sorghum bicolor L.! Moen ch) 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 tonopla st energizing systems. In mature storage tissue, PP(i)ase exhibited fa r less stability than ATPase, and PP(i)-dependent H+-transport account ed for only 12-15% of the ATP-induced pumping rate. Whereas ATPase sho wed high sensitivity towards SH-modifying compounds, tonoplast PP(i)as e activity was affected by DTT or NEM only slightly, and was unaffecte d 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 A1 with 50 % inhibition at 10(-10 ) M. The inhibition occurred directly after application, and a protect ion by substrate, Mg2+ or Cl-could not be observed. ATP hydrolysis was less sensitive to bafilomycin, with 50 % inhibition at 10(-9) M. Tono plast PP(i)ase activity, in contrast, remained unaltered up to 10(-6) M bafilomycin. A similar insensitivity to the antibiotic applied for t he postulated Ca2+/H+-antiporter at the tonoplast: When maximum DELTAp H formation was completed and active H+-pumping was suppressed by 10(- 9) M bafilomycin A1, a gradual decrease in the DELTApH by increasing a mounts of Ca2+, but not Mg2+, up to concentrations of about 6 muM free calcium could be observed. Higher concentrations of Ca2+, however, de stroyed the integrity of the sorghum stem parenchyma vacuoles.