CONICET | Buscador de Institutos y Recursos Humanos

PMCA is alarge membrane protein that its function is to couple the hydrolysis of an ATPmolecule to the extrusion of a calcium ion from the cytoplasm to the extracellularmedium against its concentration gradient. PMCA has not crystallized yet, givenits complex structure and large number of trans-membrane segments. It is anessential ATPase for the homeostasis of the cytoplasmic concentration of Ca2+.By homology with other P-ATPases and for its sequence we know that this proteinhas 10 transmembrane segments, an Actuator domain that by its movement causesthe conformational change, a N domain that has the nucleotides binding site anda P domain that is where the aspartic amino acid is phosphorylated after thehydrolysis of ATP. In theabsence of calcium, this pump is in a equilibrium between E1 and E2. In thepresence of calcium, this ion can bind to E1 in its high affinity site, whichis oriented to the cytoplasmic side. Then the enzyme can bind ATP and hydrolyzeit to form the phosphorylated intermediate E1P. By a conformational change theenzyme passes to the E2P intermediate. Now, the calcium site is oriented to theextracellular medium and has a low affinity. So, the calcium is release andthen the phosphate, and the enzyme can start a new reaction cycle. One way tomeasure enzyme activity is by spectrophotometric methods revealing thephosphate released by time and by protein concentration, and another is measuringthe amount of phosphorylated intermediates formed from ATP labeled withradioactive phosphorus.We can studythe PMCA using different inhibitory molecules that fix the pump in differentintermediates of the P-ATPase reaction cycle. We use aluminium which is thethird most abundant element in the earth's crust, it is very used since it hasbeneficial characteristics. Aluminum is an element that has a small ionic radiuswith 3 positive charges, so it has a high charge / mass ratio. It is describedthat can bind strongly to amino acids and cause protein crosslinking. It isinvolved in deterioration of neurodegenerative disorders. And also in thesedisorders there are reported failures in calcium homeostasis.First westudy the phosphate release as a function of time in the presence of increasingconcentrations of aluminium. We fitted a decreasing hyperbola to theexperimental data and we calculated the inhibition constant. 8,3To clarifywhether the aluminum is joining the calcium or magnesium site we made theactivity curves as a function of calcium (at saturating concentrations of Mg)in the absence and in the presence of increasing concentrations of aluminium. Thenwe fit growing hyperbola to the expetimental data and we obtained the constantsof calcium in function of aluminum and the maximum speed values. Calciumconstant values do not vary significantly as a function of aluminum whilemaximum velocity decreases.This effectmay be due to an non-competitive inhibition or to an irreversible binding ofaluminum to the enzyme. To checkwhich hypothesis was correct we decided to do a dilution experiment. For thiswe incubate the enzyme in the presence of increasing concentrations ofaluminum. And in one case we dilute 10 times to measure the activity. What we gotwas the same behavior in the diluted as in the undiluted, so we conclude thataluminum binds irreversibly in our conditions.To continuewith the study of the partial reactions of the cycle, we measure the amount ofphosphorylated enzyme as a function of Al concentration. We observed that theamount of phosphorylated intermediate increased hyperbolically as a function ofaluminum. We fitted a growing hyperbola to the experimental data and calculatethe constant that is similar to the inhibition constant we showed above. thismeans that this is the step involved in the inhibition. The aluminum is may beinhibiting the enzyme in some of these intermediates and thus preventing thereaction cycle. Todistinguish between these two intermediaries we did studies of proteolysis withproteinase K. Depending on the conformation of the enzyme, I will havedifferences in accessibility to sites of protease cleavage. In all cases that Ishow you here, we incubate the protein 4 minutes with the proteinase. First,what we did was to see if we saw differences between the protein in E1 withcalcium and E2 in the absence of calcium. We can see in both cases a band of 94kDa that comes from a cut in the glutamic 348 that is very exposed as in othersP ATPase. In the E1 conformation a cut is quickly produced in the N terminalthat produces P127, and another cut in the C terminal that produces P 87. Indicatingthat E1 is in a more open conformation that exposes these two sites, while inE2 these fragments are not produced in the same time of proteolysis, indicatingthat these sites are less exposed. We did the same but for the enzyme in thepresence of Lanthanum that we know that stabilizes the conformation E1P, andthe pattern of proteolysis is similar to E1 as we expected. When doing it inthe presence of Aluminum we saw that we only observed PMCA and P94, being aconformation with less exposure to proteolysis like E2. With these results wesuggest that the fixed protein in an E2P compatible intermediary. To followwhat we wanted to see is if it was possible to happen at the cellular level. Forthat we measured variations of cytoplasmic calcium with a fluorescent probecalled Fluo4AM. What I show you here is the areas under the curve thatrepresents intracellular calcium in a time interval after extracellularstimulation with calcium. The larger the area, the more intracellular calcium,the less the capacity to extrude calcium. As we did the experiments inconditions where the cytoplasmic calcium exit mechanisms of both the reticulumand the membrane are inhibited, we can say that what we see could be for thePMCA inhibition by akuminium. And finally by confocal fluorescencemicroscopy with a probe that binds aluminum, Lumogallion, I show that in ourconditions, aluminum is interacting inside the cell.

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