Comparison of different methods for LDL isolation and radioiodination on liver LDL receptor binding in vitro.
Lipoproteins were isolated either by immunoaffinity chromatography (LDL and VLDL) or ultracentrifugation (LDL). Purified lipoproteins were labeled with 123I using either Iodogen or iodine-monochloride (IC1) each followed by purification with gel-chromatography or dialysis (total of 4 combinations). Lipoprotein-concentrations of 0.1-6 micrograms protein/mL were used for direct binding assays investigating the specific binding of labeled lipoproteins (in the presence of a 50-fold excess of unlabeled lipoproteins) to human liver apo-B, E-receptors. In separate experiments displacement of bound 123I-lipoproteins (labeled by the methods mentioned) by unlabeled ones was studied. The binding capacities estimated by Scatchard analysis were similar to each other (141-163 ng protein bound/mg liver plasma membrane protein) independent from the method used for isolation and labeling. Also the affinity constants were very similar and ranged from 0.9 to 1.7 micrograms protein/L. It is concluded that immunoaffinity chromatography or ultracentrifugation for isolation of lipoproteins and the Iodogen or IC1-method for radiolabeling can be recommended to be equally good for in vitro receptor investigation.
Virgolini
I
I
Atherosclerosis Research Group (ATK), Austrian Academy of Sciences.
Angelberger
P
P
Lupattelli
G
G
Li
S R
SR
Pidlich
J
J
Molinari
E
E
Sinzinger
H
H
D000328:Adult; D001055:Apolipoproteins B / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008566:Membranes / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*; D014461:Ultracentrifugation
D001055:Apolipoproteins B; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL; D011973:Receptors, LDL