Identification of high density lipoprotein binding proteins in mature adipocyte plasma membranes.
High density lipoprotein (HDL) binding proteins were identified in nonreduced detergent extracts of plasma membranes or crude membrane fractions of rat adipocytes by ligand blotting. Using 125I-labelled human apolipoprotein-E-free HDL ([125I]HDL3), two binding proteins in adipocyte membranes were detected with apparent molecular masses of 122 and 88 kilodaltons (kDa), respectively. The binding of HDL3 to both binding proteins was abolished by pronase treatment and was inhibited by excess unlabelled HDL3. Excessive unlabelled low density lipoprotein reduced the binding of [125I]HDL3 to the 122-kDa binding protein relatively less than that to the 88-kDa binding protein. Polyclonal antisera against purified rat apolipoprotein A-I (apoA-I) effectively inhibited the binding of HDL3 to adipocyte membranes. Affinity-purified antibodies against rat apoA-I also revealed two HDL-binding proteins in rat adipocyte and liver plasma membranes preincubated with rat HDL. The sizes of the HDL-binding proteins in adipocyte plasma membranes detected by anti-apoA-I were similar to those detected by radiolabelled ligand blotting and their counterparts in rat liver plasma membranes. The study demonstrates two HDL-binding proteins, distinguishable by apparent molecular masses and ligand binding affinity, in plasma membrane proteins of mature rat adipocytes using radiolabelled ligand and immunoligand blotting techniques. The results suggest that apoA-I is involved in the interactions between HDL and both variants of HDL-binding proteins.
Shen
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Department of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Canada.
Angel
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A
D017667:Adipocytes / Q000737:chemistry*; D000818:Animals; D016632:Apolipoprotein A-I / Q000276:immunology / Q000502:physiology; D002352:Carrier Proteins; D002462:Cell Membrane / Q000737:chemistry*; D007106:Immune Sera / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL / Q000378:metabolism; D053440:Lipoproteins, HDL3; D008099:Liver / Q000737:chemistry; D008297:Male; D008970:Molecular Weight; D016601:RNA-Binding Proteins; D051381:Rats; D017208:Rats, Wistar; D018110:Receptors, Lipoprotein / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D016632:Apolipoprotein A-I; D002352:Carrier Proteins; D007106:Immune Sera; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D016601:RNA-Binding Proteins; D018110:Receptors, Lipoprotein; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein