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Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca2+ -binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca2+ . Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca2+ -dependent binding of recoverin to Phenyl-Se- pharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca2+ ]free (10 uM) than at low [Ca2+ ]freee(10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca2+ ]free in recoverin and at low [Ca2+ ]freee in GCAP2. Such different changes of hydrophobicity evoked by Ca2+ ap­pear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.
Retinal lipids of crayfish, kept at 4°C under continuous darkness for 3 weeks, consisted mainly of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); sphingomyelin (SM), phosphatidylinositol (PI) and phosphatidylserine (PS) were minor contributors. PI, involved in the phototransduction cascade, never reached greater concentrations than 7% of the total. High concentrations of polyunsaturated fatty acids (PUFA) such as 20:4n-6, 20:5n-3 and 22:6n-3 (DHA, docosahexaenoic acid) were present in PC, PE and PS, but scarce in SM and PL In retinae of crayfish kept at 4°C in darkness for 3 weeks and then exposed to white light (6 h; ca. 4,500 lx), SM and PS remained seemingly unaffected. PC, however, significantly decreased within 10 min to 65% of the initial value and 50% at 180 min. To study the reduction of PC, lipids of retinae suspended in physiological solution with/without phospholipase C (PLC) and phospholipase A2 (PLA2) inhibitors such as DMDA (=DEDA), manoalide, ET-I8-OCH3, and U-73122 were measured. Only free fatty acids (FFA) of retinae with inhibitors of PLA2 like DMDA and manoalide decreased. Retinae irradiated by white light for 3 h displayed a significant reduction of PC, compared with those that had remained in continuous darkness. However, the PC of retinae with PLA2-inhibitors was not decreased by light. Our results provide evidence that not only photoreceptor cell PLC, but also PLA2 is activated by light.
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