Rat Low Density Lipoprotein Research Articles

Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.

Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.

Radioiodination of human low density lipoprotein: A comparison of four methods

A comparison has been made of four labelling techniques used to radioiodinate human low density lipoprotein (LDL). 1. (1) Chloramine T iodination at pH 7.4 was 20–25% efficient and gave a product immunologically indistinguishable from native LDL. Approximately 30% of the incorporated radioactivity, however, was found in LDL lipids, and the metabolic decay of the labelled complex in rats did not obey first order kinetics. Radiolabelling at pH 10 reduced the uptake of 125I into lipids to 10% but also cut the overall incorporation of radioiodine by a factor of 7. 2. (2) Lactoperoxidase labelling and (3) conjugation with iodinated N-succinimidyl-3-(4-hydroxyphenyl)propionate were highly efficient (100%), but incorporation of radioactivity into the lipid moiety was unacceptably high (approximately 30%). 3. (4) The efficiency of iodine monochloride labelling was highly reproducible and the product was immunologically indistinguishable from native LDL. Incorporation of radioactivity into the lipid moiety was less than 4% when the I/protein ratio of the product was kept at or below 1 : 1. Decay of the radiolabelled LDL in rats was monoexponential.

A New Serum Lipoprotein Associated Erythrocyte Antigen which Reacts with a Monoclonal IgM

A monoclonal IgM (IgM) was found to react with stored human red blood cells (SHRBC) and human low density lipoprotein (LDL). IgM Re Fab fragment was reactive. SHRBC antigen was present, in a hidden state, in all fresh human ABO erythrocytes studies; it was not present in fresh or stored animal RBC. Cross reactivity of SHRBC and LDL antigens against IgM Re and IgM Re Fab was demonstrated. In contrast with SHRBC antigen, LDL antigen has no species specificity and was also found in rabbit and rat LDL. Furthermore, cross-reacting soluble inhibitors were also found in human saliva, and in a small molecule fraction issued from human and rabbit serum. The IgM Re also reacted with human and animal glomerular membrane. These findings are suggestive of two closely related but not identical antigens: a human specific SHRBC antigen and a LDL-associated, SHRBC-like antigen, which is a secreted, non-human specific substance. The former is part of the human RBC structure, the latter is a soluble molecule which may be found in saliva and in serum where it may be free or bound to LDL; it also binds to glomerular membranes.

An Appraisal of Methods for Calculating the Fractional Catabolic Rate of Low Density Lipoprotein in Turnover Studies in Humans

An Appraisal of Methods for Calculating the Fractional Catabolic Rate of Low Density Lipoprotein in Turnover Studies in Humans

Surface binding and interiorization of homologous and heterologous serum lipoproteins by rat aortic smooth muscle cells in culture

Rat aortic smooth muscle cells in culture were incubated with rat or human iodinated low and high density lipoprotein at 5–50 μg/ml for 3 h. With the homologous lipoproteins, 25–49% of total cellular protein radioactivity was trypsin releasable and was considered as surface-bound radioactivity, while the balance represented cellular uptake. The ratio of surface-bound to cellular label was higher when the cells were incubated with human lipoproteins and was about 9:1 with human high density lipoprotein. Cellular uptake of rat low density lipoprotein was about twice that of rat high density lipoprotein, while degradation of labeled protein, which had presumably followed protein uptake, was similar and ranged from 20 to 25% of protein uptake in 3 h. Experiments designed to test the effect of cell density on lipoprotein uptake have shown that the uptake was related inversely to cell density. Thus, the lower lipoprotein uptake encountered in the rat smooth muscle cells, compared to that described for human fibroblasts (Goldstein, J.L. and Brown, M.S. (1974) J. Biol. Chem. 249, 5153–5162), could be due in part to the much lower cell density used in the latter studies, as well as to cell type and species difference.

Subcellular Localization of “B” Apoprotein of Plasma Lipoproteins in Rat Liver

The B apoprotein is thought to be essential for the synthesis and secretion of triglyceriderich lipoproteins from mammalian liver. Antibodies were prepared against rat low density lipoproteins (LDL, density 1.025-1.045) and very low density lipoproteins (VLDL). By Ouchterlony immunodiffusion and Immunoelectrophoresis, the major determinant shared by these antisera had the characteristics of the B apoprotein. Fab fragments were prepared by the method of Porter and conjugated to horseradish peroxidase (HRP) by the two step method of Avrameas and Ternynck. Male Long-Evans rats (250-300 gm) were fasted overnight, anesthetized with Brevital (Lilly), and perfused through the portal vein with fresh 4% formaldehyde in 0.135 M sodium phosphate buffer.

Disk electrophoresis of isolated rat-serum lipoproteins

1. 1. Disk electrophoresis was performed in 3.75% polyacrylamide gel with ultra-centrifugally isolated rat-serum lipoprotein fractions. These included the very low density, low density and two high density lipoprotein fractions. 2. 2. Four different stains, namely, sudan black B, amido black 10B, oil red O and iodine were employed and the results indicated the presence of at least seven lipo-protein components among the various fractions. Densitometry has been conducted on the gel patterns and has proved useful in the identification of the several disk-electrophoretic components. 3. 3. By comparison of the rat-serum lipoprotein patterns with those obtained with human-serum lipoproteins, it was concluded that the rat low density lipoproteins contained components which were probably smaller in size than those of the human-serum low density lipoproteins. The possibility that one of these components might represent the rat high density lipoprotein component (HDL 1, d = 1.050) has been indicated. 4. 4. The principal component of rat high density lipoprotein (1.125 < d < 1.21) migrated slower than the principal component of the rat high density lipoprotein (1.063 < d < 1.125).