Highly sensitive detection of net hydrogen charged into austenitic stainless steel with secondary ion mass spectrometry.

Secondary ion mass spectrometry (SIMS) is used to detect local distributions of hydrogen in various materials. However, it has been well-known that it is extremely difficult to analyze net hydrogen (H(N)) in metals with SIMS. This was because hydrogen, which is originated from moisture (H(2)O), hydrocarbon (C(x)H(y)) or other organic materials (C(x)H(y)O(z)) existing on a sample surface or in the SIMS chamber, is simultaneously detected in the SIMS measurement of the H(N), and the H(N) and the background-originated hydrogen (H(BG)) cannot be distinguished in a SIMS profile. The effective method for reductions and determinations of the H(BG) in hydrogen measurements of metallic materials with the SIMS method has not been established. The present paper shows an effective method for reduction and estimation of H(BG) in SIMS analyses of hydrogen charged into type 316 L austenitic stainless steel, and an accurate estimation method of the net charged hydrogen. In this research, a silicon wafer is sputtered by a primary ion beam of a SIMS near an analyzed area (silicon sputtering method) to reduce H(BG). An uncharged type 316 L sample was prepared for estimation of H(BG) in SIMS measurements of the hydrogen-charged sample. The gross intensities of hydrogen between the hydrogen-charged sample and the uncharged sample were compared. The gross intensities of hydrogen of the uncharged sample (26.8-74.5 cps) were much lower than the minimal gross intensities of hydrogen of the hydrogen-charged sample (462-1140 cps). Thus, we could reduce the H(BG) enough to estimate the hydrogen charged into the type 316 L sample. Moreover, we developed a method to determine intensities of H(BG) in the measurement of the hydrogen-charged sample by estimating the time-variation of hydrogen intensities in the measurements of the uncharged sample. The intensities of the charged hydrogen can be obtained by subtracting the estimated intensities of the H(BG) from the gross intensities of hydrogen of the hydrogen-charged sample. The silicon sputtering method used to reduce H(BG) and the determination method for H(BG) in this research can be applied to the accurate hydrogen analysis for other various metallic materials.

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.

Synthesis of ATP by the simple addition of ADP to the p-nitrophenyl phosphate-prepared phosphoenzyme of the sarcoplasmic reticulum Ca2+-ATPase.

The sarcoplasmic reticulum Ca2+-ATPase was phosphorylated with either p-nitrophenyl phosphate (pNPP) or with ATP in the presence of Ca2+, under the condition that the free energy change of pNPP hydrolysis is less than that of ATP, DeltaG'(pNPP)

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.