The binding site of the strongly bound Eu3+ in Eu(3+)-regenerated bacteriorhodopsin.

A Scatchard plot for the strongly bound Eu3+ to deionized bacteriorhodopsin (bR) was made using a method based on measuring the concentration of unbound Eu3+ from its fluorescence intensity. The results suggest that the first mole of Eu3+ added to a mole of bR is strongly bound by displacing 2-3 protons. In order to reconcile this result with the previous time-resolved fluorescence studies on Eu(3+)-regenerated bR, which showed the presence of 3 sites of comparable binding constants, one is forced to conclude that the emission from the strongly bound Eu3+ is completely quenched, e.g. by energy transfer to the retinal. For this to take place, the Eu3+ must be within a few A from the retinal, i.e. within the retinal pocket (the active site). The possible importance of this conclusion to the deprotonation mechanism of the protonated Schiff base, the switch of the proton pump in bR, is discussed.

A Fourier transform infrared spectrometry study of the reactions of phosphatidylcholines with gaseous N2O5 and NO2.

The liquid lining of the alveolar region of the lung contains a surfactant which lowers the surface tension. The major active surface-tension-lowering compounds are phosphatidylcholines, some of which contain unsaturated fatty acid components. In order to determine whether these unsaturated moieties react with the gaseous air pollutant N2O5, which may be present in urban atmospheres at concentrations up to 15 ppb, phosphatidylcholines adsorbed on glass at 25 degrees C were exposed to mixtures of approximately 2 Toor (approximately 2600 ppm) N2O5 in 1 atm of air or argon in the gas phase. Nitronitrates were identified as products of the reactions of N2O5 with beta-oleoyl-gamma-palmitoyl L-alpha-phosphatidylcholine (OPPC) and dioleoyl L-alpha-phosphatidylcholine (DOPC) using Fourier transform infrared (FTIR) spectrometry and in the case of DOPC, fast atom bombardment mass spectrometry. FTIR studies also show that 2 Torr (approximately 2600 ppm) NO2 in 1 atm of air reacts with OPPC and DOPC to give new bands tentatively identified as nitronitrates. Finally, HNO3 was shown to react with OPPC, DOPC, and the saturated dipalmitoyl L-alpha-phosphatidylcholine to give products tentatively identified as nitrate salts and glycerol. These studies suggest that inhaled N2O5, if it reaches the alveolar region, is likely to react with unsaturated C = C groups in surfactant to form nitronitrates.

Nature of the individual Ca binding sites in Ca-regenerated bacteriorhodopsin.

The binding constants, K(1) and K(2), and the number of Ca(2+) ions in each of the two high affinity sites of Ca(2+)-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K(1) and K(2), it was found that two hydrogen ions are released for each Ca(2+) bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K(s), for the binding of Ca(2+) to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K(s) and its pH dependence with those of K(1) and K(2) showed that the site corresponding to K(s) is to be identified with that of K(2). This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca(2+) that causes the blue to purple color change.Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.

Effect of linoleic acid hydroperoxide on endothelial cell calcium homeostasis and phospholipid hydrolysis.

The relationship between intracellular free calcium ion concentrations ([Ca2+]i) and release of arachidonic acid from membrane phospholipids following peroxidation was examined in rabbit aortic endothelial cells treated with linoleic acid hydroperoxide (LOOH). LOOH (0.1-0.4) mumol/10(6) cells) caused a rapid and dose-dependent transient increase in [Ca2+]i in the presence of extracellular Ca2+ that remained elevated over baseline for 15 to 30 s. In the absence of extracellular Ca2+, LOOH also evoked a transient increase in [Ca2+]i of lesser magnitude which immediately returned to basal (or below basal) levels. In this regard, the rise in intracellular Ca2+ after LOOH or vasopressin (AVP) treatments involved, at least in part, related intracellular pools that in each case was followed by influx of extracellular Ca2+. The intracellular membrane sources known to be affected by vasopressin were not directly involved. Most notably, the LOOH evoked rise in [Ca2+]i was not associated with release of IP3, suggesting that the source of intracellular Ca2+ is not IP3-sensitive pools. However, pretreatment with LOOH strongly inhibited the rise in [Ca2+]i upon subsequent addition of AVP or LOOH and the extent of such inhibition was dependent on the availability of free intracellular Ca2+ and presence of extracellular Ca2+. These findings suggest that reuptake of Ca2+ into intracellular membrane pools is reduced in the presence of LOOH and/or the availability of Ca2+ from agonist-sensitive sites is inhibited by LOOH. An increase in free 20:4 levels was found after LOOH treatment that was only partly prevented using intracellular Ca2+ chelators which maintained [Ca2+]i at basal levels after LOOH treatment. These findings suggest that LOOH induction of phospholipid hydrolysis proceeds following small transients in [Ca2+]i that are considerably less than that evoked by agents such as AVP, approximating basal Ca2+ concentrations. Inhibition of LOOH-induced lipid peroxidation by vitamin E also prevented the rise in [Ca2+]i and 20:4 release indicating that phospholipid hydrolysis is dependent, at least in part, on membrane lipid peroxidation. Inhibition of protein kinase C (PKC) completely blocked LOOH-induced release of 20:4 but had little effect on the LOOH-induced rise in [Ca2+]i, suggesting an indirect relationship between LOOH-induced membrane Ca2+ signalling events, with intervention via PKC-mediated induction of phospholipid hydrolysis. A rapid and progressive translocation of PKC to the membrane fraction was evident after LOOH addition over the time course corresponding to the maximal release of 20:4 which was also inhibited by vitamin E.(ABSTRACT TRUNCATED AT 400 WORDS)