Accumulation of sucrose in vacuoles isolated from red beet tissue

Vacuoles were isolated from red beets (Beta vulgaris L.) by slicing the tissue and separated using a discontinuous dextran gradient centrifugation. The uptake of sucrose against a concentration gradient into the dextran-impermeable [³H]-H₂O space of these organelles was studied using silicone layer filtering centrifugation on both fluorometric and ¹⁴C-measurement of sucrose. The rate is 24 nmol sucrose (unit betacyanin)^-1 h^-1 and appears to be stimulated by ATP to an uptake rate of 34 nmol. Control experiments with slices cut from red beet tissue and incubated with [¹⁴C]sucrose gave comparable results. An ATPase activity dependent on both Mg²⁺ and K⁺ seems to be localized at the inner surface of the tonoplast. This activity is strongly inhibited by EDAC and tartrate and there is no effect of oligomycin, whereas a slight stimulation was caused by DCCD.Abbreviation CCCP carbonylcyanide-m-chlorophenylhydrazone - EDAC ethyl-3(3-dimethylaminopropylcarbodiimide) - EDTA ethylenediamine tetraacetic acid - fr.wt. fresh weight - HEPES n-2-hydroxyethylepiperazine-n-2-ethanesulfonic acid - MES 2(n-morpholino)ethane sulfonic acid - P_i inorganic phosphate - Tris tris-(hydroxymethyl)-aminomethanDedicated to A.L. Kursanov, Moscow, on his 75th birhday