The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x M017) roots were investigated. Azide (0.1-10 miflimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 milUimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by KV. K -stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K+ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.fungal (3, 4) cells. At higher concentrations (usually >50 .tM), vanadate has been shown to inhibit ATPase activities (8,13,26,35) and ion transport (8, 9, 16) in various plant tissues. However, at these concentrations, vanadate also inhibits plant and animal phosphatases (22,32,36). Mitochondrial ATPase activity is not affected by up to 500 ,UM vanadate, but higher concentrations (1 mM) have been reported to inhibit respiration and oxidative phosphorylation in rat liver mitochondria (11). Vanadate is clearly not a specific inhibitor of plasma membrane-ATPase, but it may be useful if used in combination with other inhibitors.Mitochondrial (and/or chloroplast) ATPase, acid phosphatase from the vacuole, and other phosphatases which readily utilize ATP as a substrate have caused considerable problems in studies on plasma membrane ATPase (14). It seems that molybdate is an inhibitor of phosphatase (34,36), although the specificity of molybdate for phosphatase over ATPase has not been reported. Azide is among several potent inhibitors of mitochondrial type, F1-ATPases (4).In this paper, we report the results of an investigation designed to compare the effects of vanadate, molybdate, and azide on ATPase and phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn roots. It seems that plasma membrane ATPase activity can be distinguished by its sensitivity to vanadate and insensitivity to molybdate or azide.Progress in elucidating the role of plasma membrane-associated ATP phosphohydrolase (ATPase) in ion transport has been limited by the lack of a specific inhibitor of the enzyme. The availability of such an inhibitor is important for studies seeking to correlate effects on ATPase activity with changes in ion tra...

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full‐size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris‐tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS‐PAGE is a simplified method in which the same buffer is used for both the gel and the electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels. Curr. Protoc. Cell Biol. 37:6.1.1‐6.1.38. © 2007 by John Wiley & Sons, Inc.

Electrophoresis is used to separate complex mixtures of proteins, (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates) to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit contains protocols for standard Laemmli gels, electrophoresis of peptides and small proteins, continuous SDS‐PAGE ultrathin gels, multiple single‐concentration gels, gradient gels, and multiple gradient gels.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.