There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H+-translocating inorganic pyrophosphatase (tonoplast H+-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (Mr) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinitylabeling, sequencing, and immunological procedures, that the major polypeptide associated with the H+-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H+-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under

The Hf-translocating inorganic pyrophosphatase (H+-PPasc) of Beta vacuolar membrane (tonoplast) vesicles has been purified by 90-fold from detergent-solubilized membranes and the MgPPi-binding subunit identified by affinity labeling. The purified enzyme has a specific activity of 1100 pmol/mg.h and contains one prevalent M, = 64000 polypeptide which strictly copurifies with activity.

The HW-translocating inorganic pyrophosphatase (H -PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca2" and a number of other divalent cations (Co2, Mn2", Zn2+ ATPase2 (EC 3.6.1.3) and a H+-PPase (EC 3.6.1.1) (28). The two pumps have been purified to near-homogeneity by independent techniques (6,18,25,29).It is now clear that both the vacuolar H+-ATPase and H+-PPase mediate electrogenic H+ translocation into the same intracellular compartment. Individual mechanically isolated vacuoles display both ATP-and PPi-dependent inward currents (12), and the steady-state transmembrane pH difference and electrical potential difference generated by the ATPase and PPase in isolated vacuolar vesicles interact nonadditively and are subject to kinetic control by a common H+-electrochemical potential difference (14,28). The continuous and simultaneous operation of both pumps in vivo might, however, appear redundant. Therefore, it is important to know whether the two enzymes are under independent cytoplasmic control. This question is relevant not only for tonoplast energization, but also for gaining a deeper understanding of the general metabolic role of PPi.PPi is becoming increasingly recognized as an important metabolite in plant cells, where it is present in the cytosol at concentrations of between 200 and 300 Mm (32, 34). Consideration of two of the best-characterized (soluble) PPi-dependent enzymes in plants-UDP-glucose pyrophosphatase and PPi fructose-6-P-1-phosphotransferase-indicates that modulation of cellular PPi levels could have profound metabolic consequences. Thus, the cytosolic reactions catalyzed by UDP-glucose pyrophosphatase and PPi fructose-6-P-1-phosphotransferase are close to equilibrium in physiological conditions (34). Diminished cytosolic PPi would therefore be expected to favor the formation of fructose-6-P from fructoseThe vacuolar membrane (tonoplast) of higher plant cells contains two primary H+ pumps: a vacuolar-type H+-

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The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1-4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono-fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.

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