Sugar nucleotides dissipate ATP-generated transmembrane pH gradient in Golgi vesicles from suspension-cell protoplasts ofChenopodium rubrum L. |
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Authors: | Maria Gogarten-Boekels Johann Peter Gogarten Friedrich-Wilhelm Bentrup |
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Institution: | (1) Botanisches Institut I der Justus-Liebig-Universität, Senckenbergstraße 17-21, D-6300 Giessen, Federal Republic of Germany;(2) Present address: Thimann Laboratoires, University of California, 95064 Santa Cruz, CA, USA |
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Abstract: | A microsomal vesicle fraction (GV) markedly enriched by the Golgi marker enzyme latent inosine diphosphatase (IDPase) has been isolated from photoautotrophic suspension-cell protoplasts ofChenopodium rubrum L. Addition of ATP creates a substantial pH gradient across the GV membrane as measured by accumulation of acridine orange. The GV showed a density of 1.14 g·cm-3 by equilibrium density centrifugation on sucrose gradients. Coincidence of acridine-orange accumulation and IDPase activity was confirmed on Percoll gradients. Formation of the pH gradient half-saturates at 0.3 mM MgATP, peaks at pH 7, and is competitively inhibited by ADP (k
i 0.1 mM), but not by Pi; it is hardly inhibited by orthovanadate, quickly dissipated by monensink
2=18 nM), nigericin (k
1/2=25 nM), and sluggishly by N-ethylmaleimide (k
1/2 35 M). Inhibition by KNO3 (k
1/2 6.7 mM) is incomplete (60%). Uridine 5 -diphosphate (UDP)-glucose, UDP-galactose, but not UDP-mannose and the pertinent sugars, dissipate the ATP-generated pH gradient (k
1/2 10–20 mM UDP-glucose; optimum pH at 7.8). This UDP-glucose activity is accompanied by release of Pi, but not of glucose or sucrose. UDP-glucoseinduced Pi release from the GV saturates (k
1/2=1 mM UDP-glucose; optimum pH at 7) and is completely inhibited by the anion-channel blocker 4,4 -diisothiocyano-2,2 -stilbene disulfonic acid (DIDS;k
1/2=140 M). The GV incorporates UDP-U-14C]glucose into an acid-labile, alkaline-stable macromolecular compound; this process is like-wise inhibited by DIDS. We propose a model including, inter alia, a UDP-glucose/uridine-5 -monophosphate translocator and a phosphate-permeable anion channel to operate in Golgi vesicles ofChenopodium rubrum.Abbreviations AO
acridine orange
- DIDS
4,4 -diisothiocyano-2,2 -stilbene disulfonic acid
- FCCP
carbonyl cyanidep-trifluoromethyoxyphenyl hydrazone
- GV
Golgi-vesicle-enriched microsomal fraction
- IDPase
mosine diphosphatase |
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Keywords: | Chenopodium Golgi vesicle Proton ATPase Protoplast Sugar nucleotide Suspension cell |
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