title: Differential regulation of hexameric and dodecameric hemocyanin from A. leptodactylus.
abstract: The oxygen binding properties of hemocyanins are regulated on a short time scale by effectors such as l-lactate, urate and protons, and on longer time scales by expression of the different types of subunits. For Astacus leptodactylus it was shown previously that acclimation to higher temperatures leads to increased levels of a 6-meric hemocyanin species, whereas at lower temperatures the 12-meric form prevails. Here we show that the temperature dependence of the two forms supports the idea, that the maintenance of high affinity towards oxygen is the driving force for the differential expression of these hemocyanins. Furthermore, the two different types of hemocyanin differ not only in the affinity to oxygen, but also with respect to their interaction with l-lactate: while the 12-meric form displays a normal shift in oxygen affinity upon the addition of l-lactate this allosteric regulation is absent in the 6-meric form. Exclusive binding of l-lactate to the 12-meric form was supported by isothermal titration calorimetry. These results indicate that l-lactate binds either at the interface between the two hexamers or at subunit α' which is responsible for the formation of the 12-mers and is not present in the 6-meric form. Urate has a comparable effect on the oxygen affinity of 6-meric and 12-meric forms and also binds to a similar extent to the oxygenated state as determined by isothermal titration calorimetry. Thus, urate and l-lactate do not seem to share the same binding sites. Interestingly, urate binding sites with no allosteric effect seem to exist, which is unusual. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
authors.affiliation: Institute for Molecular Biophysics, University of Mainz, Jakob-Welder-Weg 26, Mainz, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Kölsch
authors.forename: Julia
authors.initials: J
authors.lastname: Hörnemann
authors.forename: Clemens
authors.initials: C
authors.lastname: Wengenroth
authors.forename: Nadja
authors.initials: N
authors.lastname: Hellmann
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001001:Anura / Q000378:metabolism*; D001665:Binding Sites; D006433:Hemocyanins / Q000737:chemistry* / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D055503:Protein Multimerization; D011522:Protons; D013696:Temperature; D014527:Uric Acid / Q000378:metabolism*
chemical_list: D007773:Lactates; D011522:Protons; D014527:Uric Acid; D006433:Hemocyanins; D010100:Oxygen
keywords: Allosteric interaction; Effectors; Hemocyanin; ITC; Oxygen affinity; PO; Temperature adaptation; isothermal titration calorimetry; phenoloxidase