Effect of pH and MgCl₂ on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria

Abstract

Binding of purine nucleotides to the uncoupling protein (UCP) was investigated in membrane particles prepared from brown fat mitochondria of cold-acclimated rats. Mitochondrial membranes were separated from soluble protein with Lubrol WX and treated with 3 M urea at basic pH. The resulting membrane vesicles were permeable to GDP and contained up to 3 nmol UCP/mg protein with unchanged nucleotide binding, as compared to the mitochondria (GDP/UCP ratio = 1.0; pKd GDP = 6.0 at pH 7.0). UCP bound nucleotides to one type of specific binding sites, located exclusively on the cytosolic side of the mitochondrial membrane. The binding affinity of guanine nucleotides was 3-18-times higher than that of the corresponding adenine nucleotides, when measured in membrane particles from cold-acclimated rats, hamsters, and guinea pigs. The pH-dependent binding affinities of GDP and ADP attained a maximum at pH 5.0-6.0 (pKd GDP = 6.8, pKd ADP = 5.8) and were decreased by a factor of 10(2) at pH 4.0 and pH 8.0, respectively, whereas the binding affinity of ATP was maximal at pH 4.0 (pKd = 7.0) and was decreased by a factor of 10(3) at pH 7.5. Participation of the protein binding center in nucleotide interaction with UCP in the membrane was highly pH-dependent. Mg2+ modified the number of binding sites engaged at a given nucleotide concentration by complex binding of nucleotides; the Kd for Mg.GTP2- and Mg.GDP- was 20-50-times lower than that of the free nucleotides.

Native UCP1 Displays Simple Competitive Kinetics between the Regulators Purine Nucleotides and Fatty Acids

“…Although this value is low compared with the values normally considered when the inhibition is expressed in terms of total GDP (21), the value is unexpectedly high compared with the binding affinity for GDP originally reported (ϳ0.005 mM) (23) and confirmed in the detailed analyses of Rafael et al (24) (ϳ0.001 mM at pH 7.0) and Huang and Klingenberg (40) (0.003 mM at pH 7.0). Apparently, the interaction between GDP and UCP1 is different when it is studied in non-respiring mitochondria (as in binding studies) compared with when the brown-fat mitochondria are respiring (as studied here).…”

“…The nominal levels of the oleate added were 10, 20, Because only the free purine nucleotide (ATP, ADP, GDP, or GTP) (i.e. not the Mg 2ϩ -nucleotide complex) is relevant for nucleotide binding to UCP1 (23)(24)(25), the values for free GDP were used in graphs and for calculation of kinetic parameters. Free GDP and Mg 2ϩ concentrations in the incubation medium at 37°C and pH 7.2 were calculated by use of a computer program for making buffers of a defined ion concentration, BAD 4 (26).…”

“…These levels transferred to the free levels of 0. 24 Concentration-response curve data were analyzed with the general fit option of the KaleidaGraph application for MacIntosh for adherence to simple Michaelis-Menten kinetics,…”

“…1). As it has been established that it is the free purine nucleotides (and not their Mg 2ϩ complexes) that interact with UCP1 (23)(24)(25), the corresponding free GDP concentrations were calculated. As Mg 2ϩ may interact, for example, with oleate (34), the solutions were adjusted so that free Mg 2ϩ concentrations remained similar (0.2-0.3 mM).…”

Temperature-dependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm,Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation

“…UCPs are the members of anion carrier family, which are located in the inner mitochondrial membrane and can facilitate proton transport into the matrix (Goglia and Skulachev, 2003). Their activity appears to be tightly regulated with free fatty acids and superoxide acting as stimulators, and purine nucleotides, especially GDP -as inhibitors in all organisms studied so far, including plants, mammals and protists [ (Rafael et al, 1994;Navet et al, 2005;Talbot and Brand, 2005;Dlaskova et al, 2006;Vercesi et al, 2006) and references therein]. Antioxidant protection has been proposed as a key function for UCP2 and 3; these proteins were found to be upregulated in response to pro-oxidant conditions, and elevated oxidative damage was observed in genetic or functional knockouts for these proteins (Nedergaard et al, 2001;Cadenas et al, 2002;McLeod et al, 2005).…”