Immunohistochemical Detection of Plasma Membrane H ATPase in Hypocotyls Immunohistochemical detection was performed following previous methods with modifications. Transverse sections of hypocotyl sections on microscope slides were heated in phosphatebuffered saline for 1 min at 105 C for antigen retrieval. The sections were blocked in a blocking solution of 3% bovine serum albumin fraction V in phosphatebuffered saline for 1 h at room temperature and then incubated overnight at room temperature with anti H ATPase and preserum diluted 1:1,000 in blocking solution. After washing of the sections, they were incubated at room temperature for 3 h with goat anti rabbit IgG conjugated to Alexa Fluor 488 diluted 1:1,000 in blocking solution. After washing, the transverse sections were observed with a fluorescence microscope and images were captured with a CCD camera system . The plasma membrane H ATPase, a member of the superfamily of P type ATPases, which are characterized by the formation of phosphorylated intermediates during catalysis, has 10 transmembrane segments and N and C termini in the cytosol .
ATP-competitive STAT inhibitor selleckchem The H ATPase is a ubiquitous enzyme from fungi to vascular plants and is a functional monomer with a molecular mass of about 100 kD that can form a dimer or hexamer . The H ATPase actively transports H out of the cell, coupled with ATP hydrolysis, and creates an electrochemical gradient of H across the plasma membrane for energizing substance transport, coupled with many secondary transporters, the maintenance of membrane potential, and pH homeostasis . Indeed, the H ATPase has been shown to be an essential enzyme in yeast and Arabidopsis plants . The structure of the H ATPase is highly conserved from fungi to the vascular plants, apart from the C terminal region. In vascular plants, including one of the most basal of vascular plant lycophytes, Selaginella moellendorffii, the C terminal region of the H ATPase, consisting of around 100 amino acids, is known as an autoinhibitory domain and contains a penultimate Thr .
On the other hand, the plasma membrane H ATPases in yeast, red algae , and green algae lack such a C terminus, and the length of the C terminus varies among species . Here, we define the H ATPase having the C terminal region containing the penultimate Thr as a pT H ATPase and others as the non pT H ATPase. Taken together, the pT H ATPases probably did exist in the last Sinomenine common ancestor of liverworts and other land plants. However, when pT H ATPase appeared in the evolution of plants remains unknown. The H ATPase is known to be regulated by physiological signals at both transcriptional and posttranscriptional levels . Posttranslational regulation of the pT H ATPase has been studied extensively.