Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1.

Abstract

To study the assembly of newly synthesized lipids with apoprotein A1, we administered [2-3H]glycerol to young chickens and determined the hepatic intracellular sites of lipid synthesis and association of nascent lipids with apoprotein A1. [2-3H]glycerol was rapidly incorporated into hepatic lipids, reaching maximal levels at 5 min, and this preceded the appearance of lipid radioactivity in the plasma. The liver was fractionated into rough and smooth endoplasmic reticulum and Golgi cell fractions. The isolated cell fractions were further subfractionated into membrane and soluble (content) fractions by treatment with 0.1 M Na2CO3, pH 11.3. At various times, the lipid radioactivity was measured in each of the intracellular organelles, in immunoprecipitable apoprotein A1, and in materials that floated at buoyant densities similar to those of plasma lipoproteins. Maximal incorporation occurred at 1 min in the rough endoplasmic reticulum, at 3-5 min in the smooth endoplasmic reticulum, and at 5 min in the Golgi cell fractions. The majority (66-93%) of radioactive glycerol was incorporated into triglycerides with smaller (4-27%) amounts into phospholipids. About 80% of the lipid radioactivity in the endoplasmic reticulum and 70% of that in the Golgi cell fractions was in the membranes. The radioactive lipids in the content subfraction were distributed in various density classes with most nascent lipids floating at a density -<1.063 g/ ml. Apoprotein A1 from the Golgi apparatus, obtained by immunoprecipitation, contained sixfold more nascent lipids than did that from the endoplasmic reticulum. These data indicate that [2-3H]glycerol is quickly incorporated into lipids of the endoplasmic reticulum and the Golgi cell fractions, that most of the nascent lipids are conjugated with apoproteins A1 in the Golgi apparatus, and that very little association of nascent lipid to apoprotein A1 occurs in the endoplasmic reticulum.High density lipoprotein (HDL) t represents one of the major classes of plasma lipoproteins. Plasma HDLs are small, spherical particles, -8-10 nm diam, which float at a density of between 1.063 and 1.21 g/ml and consist of a layer of phospholipids, free cholesterol, and apoproteins surrounding a membrane lipid core composed mainly of cholesterol esters and triglycerides (8,16,20,28,46).In mammals the study of HDL synthesis and secretion is complicated by the fact that there is more than one site of apoprotein and lipid synthesis. In addition, there are several Abbreviations used in this paper. HDL, high density lipoprotein; RER and SER, rough and smooth endoplasmic reticulum, respectively; VLDL, very low density lipoprotein. apoproteins that are shared by both very low density lipoprorein (VLDL) and HDL (8,16,19,20,46). In recent years, the avian species has attracted considerable attention as an animal model for the study of the biosynthesis and secretion of HDL. It has been reported that in young chickens, plasma HDL is 92% of the total serum lipoproteins (33), that 90% of the protein content of H...

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

“…For our experiments, the method of Ehrenreich et al [1973] based on discontinuous sucrose gradients was followed, and we isolated a so-called heavy, an intermediate and a light Golgi fraction at interphases corresponding to densities of 1.148-1.116, 1.116-1.077, and 1.077-1.031 g/ml. Our Golgi fractions were 8-27 times enriched in GT, similar to other chicken hepatic preparations [Banerjee and Redman, 1984;Gavazova et al, 1986]. Approximately 12% of GT was recovered in the three Golgi fractions.…”

Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-1.

“…HDL was floated from the adjusted serum between densities 1.063-1.21 g/ml, dialyzed against 0.15 mM NaCl, 0.01 M sodium phosphate (pH 7.4) at 4"C and delipidated as previously described (3). Rabbit antiserum to HDL apoproteins was prepared and tested for its specificity as previously described (4). This antiserum was used to isolate nascent Apo A-I from the liver and serum.…”

Intracellular Lipidation of Newly Synthesized Apolipoprotein A-I in Primary Murine Hepatocytes

“…However, it still remains unclear whether apoA-I synthesized in the hepatocyte is lipidated intracellularly and/or at the cell surface. Banerjee and Redman (10,11) showed that in the avian hepatocyte the initial lipidation of the protein occurs in Golgi fractions but the newly formed lipoprotein particles are not immediately mature.…”