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Cooperative binding of monodisperse anionic amphiphiles to the i-Face: Phospholipase A2-paradigm for interfacial binding
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
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2004 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 25, 7999-8013 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2004. Vol. 43, no 25, 7999-8013 p.
National Category
Biological Sciences Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-70290DOI: 10.1021/bi0497650OAI: oai:DiVA.org:uu-70290DiVA: diva2:98201
Available from: 2005-04-20 Created: 2005-04-20 Last updated: 2011-02-18Bibliographically approved

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