Makoto Ushimaru

Stimulation of p‐nitrophenylphosphatase activity of Na+/K+‐ATPase by NaCl with oligomycin or ATP

It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain‐sensitive and K+‐dependent p‐nitrophenylphosphatase (pNPPase) activity of Na+/K+‐ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+‐binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+‐ATPase by p‐nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+‐occluded E1 state of Na+/K+‐ATPase, it seemed that the Na+‐occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high‐energy phosphoenzyme formed from ATP with Na+, is also the Na+‐occluded E1 state, it is suggested that the Na+‐occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between α subunits. Therefore, membrane‐bound Na+/K+‐ATPase would function as at least an (αβ)2‐diprotomer with interacting α subunits at the phosphorylation step.

The dimeric form of Ca2+-ATPase is involved in Ca2+ transport in the sarcoplasmic reticulum

To identify the functional unit of Ca(2+)-ATPase in the sarcoplasmic reticulum, we assessed Ca(2+)-transport activities occurring on sarcoplasmic reticulum membranes with different combinations of active and inactive Ca(2+)-ATPase molecules. We prepared heterodimers, consisting of a native Ca(2+)-ATPase molecule and a Ca(2+)-ATPase molecule inactivated by FITC labelling, by fusing vesicles loaded with each type of Ca(2+)-ATPase. The heterodimers exhibited neither Ca(2+) transport nor ATP hydrolysis, suggesting that Ca(2+) transport by the Ca(2+)-ATPase requires an interaction between functional Ca(2+)-ATPase monomers. This finding implies that the functional unit of the Ca(2+)-ATPase is a dimer.

The Involvement of L-Type Amino Acid Transporters in Theanine Transport

L-Theanine has favorable physiological effects in terms of human health, but the mechanisms that transport it to its target organs or cells are not completely defined. To identify the major transport mechanisms of L-theanine, we screened for candidate transporters of L-3H-theanine in several mammal cell lines that intrinsically express multiple transporters with various specificities. All of the cells tested, T24, HepG2, COS1, 293A, Neuro2a, and HuH7, absorbed L-3H-theanine. Uptake was significantly inhibited by the addition of L-leucine and by a specific inhibitor of the system L transport system, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). L-3H-Theanine uptake occurred mostly independently of Na+. These results indicate that L-theanine was taken up via a system L like transport system in all of the cells tested. Additionally, in experiments using cells stably expressing two system L isoforms, LAT1 and LAT2, we found that the two isoforms mediated L-theanine transport to similar extents. Taken together, our results indicate that L-theanine is transported mostly via the system L transport pathway and its isoforms.

Ca(2+) transport of Ca(2+)-ATPase of the sarcoplasmic reticulum in an ADP-sensitive phosphoenzyme state.

To understand the energetics of Ca(2+)-transporting adenosine triphosphatase (Ca(2+)-ATPase), it is important to determine the energy consumption step. To do this, we measured the dissociation of Ca(2+) from Ca(2+)-ATPase into Ca(2+)-loaded vesicles. We observed that 45Ca(2+) added to the outside of the vesicles accumulated in the 40Ca(2+)-loaded vesicles after the addition of ADP and ATP. The acceleration of 45Ca(2+) accumulation increased by 1.4-fold after the addition of 1 microM ADP. Under the same conditions, Ca(2+)-dependent phosphate liberation was not observed, and all of the active phosphoenzymes were in the ADP-sensitive phosphoenzyme (E(1)P) state. These results indicated that the ADP stimulated 45Ca(2+) accumulation by the ADP-ATP exchange reaction and that this ADP-ATP exchange reaction did not pass through the ADP-insensitive phosphoenzyme state. Therefore, we demonstrate that one Ca(2+) ion dissociates at the E(1)P state, which does not correspond with the phosphoenzyme conversion, that is the energy consumption step in the E(1)-E(2) model.

On the Error of the Dixon Plot for Estimating the Inhibition Constant between Enzyme and Inhibitor.

In textbook treatments of enzyme inhibition kinetics, adjustment of the initial inhibitor concentration for inhibitor bound to enzyme is often neglected. For example, in graphical plots such as the Dixon plot for estimation of an inhibition constant, the initial concentration of inhibitor is usually plotted instead of the true inhibitor concentration. Justification for this approximation should be presented to students, because in certain conditions it can be inaccurate.

Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca(2+)-ATPase.

BACKGROUND SERCA maintains intracellular Ca2+ homeostasis by sequestering cytosolic Ca2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:19-cis) and linoleamide (18:29,12-cis), induce an increase in intracellular Ca2+ levels, which might be caused by their inhibition of SERCA. METHODS Three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca2+-ATPase activity of SERCA was examined. RESULTS The Ca2+-ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC50 15-53μM) and partially by oleamide (IC50 8.3-34μM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:19-trans), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca2+ and the apparent maximum velocity of Ca2+-ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45μM in linoleamide and from 1.6 to 55μM in oleamide depending on the isoform. CONCLUSIONS Linoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca2+ affinity of the enzyme. GENERAL SIGNIFICANCE Our findings imply a novel role of these PFAAs as modulators of intracellular Ca2+ homeostasis via regulation of SERCA activity.