Characteristics of the isolated purine nucleotide binding protein from brown fat mitochondria.
The isolation of the purine nucleotide binding protein (NbP), the putative uncoupling protein, from hamster brown adipose tissue mitochondria and some of its functional characteristics are described. (1) Among various detergents tested, Triton is the most suitable; the total GDP binding capacity can be recovered after solubilization by Triton and is rather stable in this extract. (2) For separation of NbP from the ADP/ATP carrier, differences in the solubilizing conditions and the stability at room temperature between both proteins are exploited. The preparation is substantially free of ADP/ATP carrier. (3) The purified NbP has a binding capacity for 16 mumol of GDP/g of protein, corresponding to a 16-fold purification from mitochondria. (4) In sodium dodecyl sulfate-polyacrylamide gel electrophoresis in single band of Mr 32 000 is found. A dimer structure is suggested from chemical cross-linking, from the binding capacity for GDP, and from the previously reported centrifugation equilibrium. (5) The isolated NbP preparation consists of Triton-protein-phospholipid mixed micelles with a Stokes radius of 60.5 A as determined by gel filtration. The Triton binding is 1.9 g/g of protein, and the phospholipid binding is 0.2 g/g of protein. (6). The amino acid composition has a polarity index of 43.5%. The N-terminal peptide has the sequence Val-Asp-Pro-Thr-Thr-Ser-Glu-Val. (7) The affinity of NbP for different purine nucleotides decreases in the order GTP greater than GDP greater than ATP greater than ITP greater than ADP greater than IDP. The affinity for the monophosphates is 100 time lower. (8) Photooxidation and the lysine reagent 2,4,6-trinitrobenzenesulfonic acid decrease the binding capacity without influencing the affinity of the unaffected sites. GDP protects against photooxidation.
D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D006224:Cricetinae; D003902:Detergents; D066298:In Vitro Techniques; D008647:Mesocricetus; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D011487:Protein Conformation; D011685:Purine Nucleotides / Q000302:isolation & purification* / Q000378:metabolism
D003902:Detergents; D011685:Purine Nucleotides