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Fluorescent Properties of Model Chromophores of Tyrosine-66 Substituted Mutants of Aequorea Green Fluorescent Protein (GFP). --(KOJIMA, S.; OHKAWA, H.; HIRANO, T.; MAKI, S.; NIWA, H.; OHASHI, M.; INOUYE, S.; TSUJI, F. I.; Tetrahedron Lett. 39 (1998) 29, 5239-5242; Dep. Appl. Phys. Chem., Univ. Electro-Commun., Chofu, Tokyo 182, Japan; EN)

To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Sepharose 4B in a Ca(2+)-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylglycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca(2+)-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca(2+)-dependent LB-binding protein identified in the bronchoalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body.

The annexins are a family of highly homologous Ca(2+) and phospholipid binding proteins. The expressive amounts of several annexins have been shown to alter in certain pathological states such as brain ischemia and Alzheimer's disease. It has been demonstrated that ethanol induces cytotoxicity, which results in brain damage. In this study, we examined the relationship between ethanol-induced cytotoxicity and the intrinsic amount of annexins using cell lines (rat glioma C6 cells and human adenocarcinoma A549 cells). A decrease in the mitochondrial enzyme (dehydrogenase) activity, which is widely used to measure cytotoxicity, was observed with a high concentration of ethanol (200 mM or more) after a 24-h exposure in both C6 and A549 cells. Western blot analysis revealed that the amount of annexin IV was augmented in both cells by ethanol, whereas levels of annexins I and V were unchanged. The amount of annexin IV was augmented with increasing concentration of ethanol. The overexpression of annexin IV in C6 cells by transfection with annexin IV-DNA enhanced ethanol-induced cell lesion and was accompanied by NFkappaB activation. Thus, it might be indicated that the amount of annexin IV is selectively augmented and this augmentation facilitates the development of cell lesion by ethanol.