A comparison of the low-density-lipoprotein receptor from bovine adrenal cortex, rabbit and rat liver and adrenal glands by lipoprotein blotting

Kroon, Paulus A.; Thompson, G M; Chao, Yu‐Sheng

doi:10.1042/bj2230329

Cited by 40 publications

(15 citation statements)

References 29 publications

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“…The result was similar to that o f previous ly reported LDL receptors in terms of human fibroblasts or bovine adrenal LDL receptors [17,24].…”

Section: Discussionsupporting

confidence: 81%

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Immunohistochemical and Biochemical Detection of Low Density Lipoprotein Receptors in Cultured Rat Mesangial Cells

Ito¹,

Chimata²,

Oka³

et al. 1995

Nephron

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The expression of low density lipoprotein (LDL) receptors by cultured rat mesangial cells was demonstrated using immunohistochemical and biochemical methods. Using a mouse monoclonal anti-human LDL receptor antibody, the immunofluorescence technique was applied to cultured mesangial cells and showed granular staining. Immunoblotting analysis of the membrane fraction of cultured mesangial cells showed a single band with a protein of approximately 130,000 molecular weight. In addition, [¹²⁵I]LDL binding and the uptake of [¹²⁵I]LDL by cultured mesangial cells were studied. Binding and uptake both reached an equilibrium after about 30 min of incubation. A 50% reduction in [¹²⁵I]LDL (20 μg protein/ml) binding and uptake occurred when unlabelled LDL was added to cultures at 20-40 μg protein/ml. Addition of high density lipoprotein without apoE to the culture medium did not induce competitive inhibition of [¹²⁵I]LDL binding and uptake by mesangial cells. These findings suggest that cultured mesangial cells have the capacity to express an LDL receptor that regulates the cellular uptake of LDL.

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“…The result was similar to that o f previous ly reported LDL receptors in terms of human fibroblasts or bovine adrenal LDL receptors [17,24].…”

Section: Discussionsupporting

confidence: 81%

“…3). which coincided with that previously reported for LDL receptors in human liver and bovine adrenal LDL re ceptors [17,24],…”

Section: Inwnmoblotting Analysis O F Mesangia! Cellssupporting

confidence: 76%

Immunohistochemical and Biochemical Detection of Low Density Lipoprotein Receptors in Cultured Rat Mesangial Cells

Ito¹,

Chimata²,

Oka³

et al. 1995

Nephron

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“…Apart from a possible increase in the rate of secretion of apoB by livers from cholesterol-fed rabbits, two other factors may contribute to this modest increase in accumulation of VLDL total protein: (i) more VLDL is expected to accumulate in perfusates from cholesterol-fed rabbits, even if the rates of secretion of VLDL were the same, because very few of the secreted lipoproteins would be taken up by the suppressed hepatic LDL receptors in cholesterol-fed rabbits (20,36) …”

Section: Discussionmentioning

confidence: 99%

Molecular cloning of partial cDNAs for rabbit liver apolipoprotein B and the regulation of its mRNA levels by dietary cholesterol.

Kroon¹,

DeMartino²,

Thompson³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Apolipoprotein B (apoB) is the major protein of plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Here we report the molecular cloning of cDNAs for rabbit liver apoB, by use of the expression vector Xgtll, and the use of these cDNAs to study the regulation of apoB mRNA levels by dietary cholesterol. The j-galactosidaseapoB fusion proteins expressed by recombinant clones were identified with guinea pig anti-rabbit LDL antibodies. The cloned cDNAs hybridized to an 18-kilobase mRNA that was present in liver and intestine. Slot blot analysis showed that this mRNA was not present in other tissues studied, with the possible exception of kidney. When rabbits are fed a highcholesterol diet, they develop severe hypercholesterolemia.Most of the excess cholesterol is contained in .3-VLDL, a cholesteryl ester-rich lipoprotein that contains apoB and apoE. We addressed the question of whether increased apoB mRNA levels, and by inference increased apoB synthetic rates, are responsible for the accumulation of ,l-VLDL. A comparison of apoB mRNA levels showed that cholesterol-fed rabbits had lower liver apoB mRNA levels than control rabbits. We suggest that the accumulation of plasma 83-VLDL in cholesterol-fed rabbits is not due to an increased production of fi-VLDL but solely due to a suppression of hepatic LDL receptors.Apolipoprotein B (apoB) plays a central role in cholesterol metabolism. It is an obligatory constituent of chylomicrons, very low density lipoproteins (VLDL), and low density lipoproteins (LDL) (1). Circulating apoB exists in two forms which are immunologically related. ApoB-100 has a molecular weight of about 400,000 and is made in the liver, whereas apoB-48 has a molecular weight of about 200,000 and is made in the intestine (2, 3). The liver secretes apoB-100 into circulation as a structural protein of triacylglycerol-rich VLDL, which is converted to cholesterol-rich LDL by the action of lipoprotein lipase (4). Despite the abundance of apoB in plasma, its structure and biosynthesis have not been well-characterized. This in large part is due to its size, its insolubility when delipidated, its tendency to aggregate, and its susceptibility to oxidation. Some progress has recently been made, however, with the identification and sequencing of partial cDNA clones for human and rat apoB (5-10). The concentration of plasma LDL is strongly correlated with the risk of coronary heart disease. Two factors regulate the plasma concentration of LDL: (i) the rate of VLDL synthesis and (ii) the receptor-dependent removal of LDL from circulation. The importance of the LDL receptor in the regulation of plasma cholesterol levels has been demonstrated (11). However, the question of whether the production rate of apoB contributes to elevated LDL cholesterol levels remains unclear, although evidence from a number of studies suggests that alterations in the regulation of apoB synthesis may contribute to hypercholesterolemia and atherosclerosis (12)(13)(14).To address the question of the regulation...

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“…In addition, as a general rule, the rate of transfer is dependent upon the apparent molecular weight of the protein, and higher molecular weight polypeptides leave the gel slower than lower molecular weight proteins and necessitate a longer blotting time [13]. As demonstrated in several studies using SDS-PAGE [3, 4, 7-11, 14, 151, the LDL receptor is present as a single protein band with an apparent Mr ranging from approximately 130000 to 160000 depending on the animal species [14] or tissue [8], and on the electrophoresis conditions [ 151. Due to the high molecular weight, the LDL receptor transfer to nitrocellulose requires long blotting times (about 16 h), since accelerating voltages are not recommended.…”

Section: Introductionmentioning

confidence: 97%

“…The solubilized LDL receptor is able to retain its binding capacity [6] in spite of the denaturing conditions of electrophoresis and the electrophoretic transfer to nitrocellulose [7]. In this way, LDL bound to blotted receptors can be detected directly with high sensitivity by autoradiography [8] or the streptavidin-biotinylated peroxidase complex [9] after incubation with '251-labeled LDL or biotin-modified LDL. LDL receptors also can be detected by incubation of the blotted protein with radiolabeled antibodies like Ig G-C7 [lo], IgG-MAC 188 [ll], IgG-15C8, IgG-HL1, or IgG-4A4 [12], which bind with high affinity and specificity to the LDL-receptor protein.…”

Section: Introductionmentioning

confidence: 99%

Horizontal semi‐dry electroblotting for the detection of the low density lipoprotein receptor in solubilized liver membranes

Himber

1993

Electrophoresis

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A high efficiency transfer of the low density lipoprotein (LDL) receptor proteins from polyacrylamide slab gel onto immobilizing nitrocellulose membranes using the horizontal semi-dry electrophoretic system is described. The transfer of the LDL receptors from solubilized rat liver microsomes was performed between two graphite plate electrodes in a continuous buffer system containing methanol and sodium dodecyl sulfate. The protein transfer was achieved in only 150 min at a constant current of 0.8 mA/cm2 at room temperature with very low Joule heat development. The homogeneous electric field yield between the two electrode plates produced a satisfactory transfer of the LDL-receptor protein band in spite of its high molecular weight, and only few protein traces remained in the polyacrylamide gel after blotting. This improved method allows a rapid and quantitative transfer of the LDL receptors without protein denaturation, since the specific binding activity of the blotted receptor is retained as demonstrated by ligand-blotting and immunoblotting.

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A comparison of the low-density-lipoprotein receptor from bovine adrenal cortex, rabbit and rat liver and adrenal glands by lipoprotein blotting

Cited by 40 publications

References 29 publications

Immunohistochemical and Biochemical Detection of Low Density Lipoprotein Receptors in Cultured Rat Mesangial Cells

Immunohistochemical and Biochemical Detection of Low Density Lipoprotein Receptors in Cultured Rat Mesangial Cells

Molecular cloning of partial cDNAs for rabbit liver apolipoprotein B and the regulation of its mRNA levels by dietary cholesterol.

Horizontal semi‐dry electroblotting for the detection of the low density lipoprotein receptor in solubilized liver membranes

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