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Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies.

ABSTRACT: The low density lipoprotein (LDL) receptor has been purified to homogeneity from rabbit liver by a combination of DEAE-Sephacel chromatography, LDL-Sepharose 4B chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The receptor protein had a pI of 4.45 and an Mr of 120 x 10(3)-125 x 10(3) in SDS gels under non-reducing conditions. Incubation of the LDL receptor with neuraminidase decreased its Mr to 105 x 10(3)-110 x 10(3) and increased its pI from 4.45 to 5.25. The purified receptor exhibited all the properties of the membrane-bound receptor including Ca2+-dependent binding of rabbit and human LDL but not of methylated LDL or high density lipoprotein. The amount of LDL receptor present in rabbit liver was measured by a quantitative blotting procedure employing a newly developed rat anti-receptor monoclonal antibody. The affinity and specificity of this monoclonal antibody allowed the quantification of the LDL receptor in detergent extracts of liver homogenate, thus eliminating the loss of receptor associated with the preparation of membrane fractions prior to receptor assay. Livers from adult female New Zealand White rabbits contained 149 +/- 13 ng of LDL receptor/mg of liver protein. Administration of pharmacological doses of 17 alpha-ethinyloestradiol raised the concentration of LDL receptor in liver to 312 +/- 25 ng/mg of liver protein.

SUBMITTER: Gherardi E

PROVIDER: S-EPMC1149314 | biostudies-other | 1988 Jul

REPOSITORIES: biostudies-other

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Rat monoclonal antibodies to rabbit and human serum low-density lipoprotein.

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| S-EPMC1149129 | biostudies-other

Immunoassay of bovine and human low-density-lipoprotein receptors using monoclonal antibodies.

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Probing of the expression of the low-density lipoprotein receptor in vivo using an anti-receptor monoclonal antibody.

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High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice.

Project description:The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein ((125)I) and in the cholesteryl ester (CE) moiety ([(3)H]). The metabolism of (125)I-/[(3)H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([(3)H]). In contrast, in LDLR(-/-) mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR(-/-) mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR(-/-) mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.

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Cysteamine Decreases Low-Density Lipoprotein Oxidation, Causes Regression of Atherosclerosis, and Improves Liver and Muscle Function in Low-Density Lipoprotein Receptor-Deficient Mice.

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Model IgG monoclonal autoantibody-anti-idiotype pair for dissecting the humoral immune response to oxidized low density lipoprotein.

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| S-EPMC3326269 | biostudies-literature

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