A rapid quenching a apparatus permitting to mix for different microvolume solutions

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A preparation of pig kidney Na^+ K^+-ATPase showed changes in fluorescence energy transfer between probes bound to the alpha-subunit. Excitation of BIPM probe, which was covalently bound to Cys-964, and excitation of FITC probe at Lys-501 gave different FITC fluorescence intensity changes at 520 nm in BIPM-FITC doubly labeled enzyme accompanying formation of reaction intermediates. The data suggest that Fluorescence energy transfer from the BIPM to the FITC probe accompanies the processes of migration of Na^+ and K^+ in the pump molecules. The dynamic fluorescence changes after phosphorylation and dephosphorylation seem to reflect changes in the binding state or the process of migration of these ions.The Lys-480 in the alpha-subunits of Na^+, K^+-ATPase from pig kidneys was specifically modified with PLP or AP2PL probes in the presence of NaCl. The data show that these probes can sense molecular events related to the formation of phosphoenzymes induced by AcP and presumably to the form … More ation of Mg-Na-ATP-enzyme complex. The data also suggest that PLP or AP_2PL probes at Lys-480 in the presence of Na^+ and Mg^<2+> do not affect the transphosphorylation from AcP to Asp-369 to form phosphoenzymes byt that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg^<2+>.When pig stomach membrane H^+, K^+-ATPase preparations were incubated with [gamma-^<32>P]ATP and Mg^<2+> with vanadate, ^<32>P was incorporated into the alpha-chain of H^+, K^+-ATPase to a steady-state level of approximately 0.7 mol of phosphotyrosine (Tyr (P)) /mol of phosphoenzyme intermediates. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatment solubilized ^<32>P-containing peptides from the alpha-chain almost completely. A reverse-phase column chromatography of the supernatant gave two peaks of ^<32>P-peptide with similar total radioactivities.The comparison of the recovery of amino acid from each Edman cycle showed that the phosphorylation of Tyr^<10> occurred preceding the phosphorylation of Tyr^7. These data and others suggested the presence of a novel membrane bound enzyme system to participate in reversible phosphorylation of boty Tyr residues in the alpha-chain of H^+, K^+-ATPase. Less