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- Berg, Otto, et al.
(author)
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Cooperative binding of monodisperse anionic amphiphiles to the i-Face : Phospholipase A2-paradigm for interfacial binding
- 2004
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:25, s. 7999-8013
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Journal article (peer-reviewed)abstract
- Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A2 (PLA2) to form the premicellar complexes (Ei#) are characterized to discern the short-range specific interactions. Typically, Ei# complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (Ei#, i = 1, 2, 3). In the presence of calcium, the dissociation constant K1 for the E1# complex of PLA2 with decyl sulfate (CMC = 4500 μM) is 70 μM with a Hill coefficient n1 = 2.1 ± 0.2; K2 for E2# is 750 μM with n2 = 8 ± 1, and K3 for E3# is 4000 μM with an n3 value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of Ei#; (c) Ki and ni do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the Ei# complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in Ei#. The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of Ei#. We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E* and Ei# aggregates is discussed.
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