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Isolated rabbit hepatocytes incorporated [35S]methionine into cellular and secreted apolipoprotein B (apo-B), and [3H]glycerol into cellular and secreted triacylglycerol and phospholipids. Newly synthesized apo-B was incorporated into rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), cis-Golgi and trans-Golgi membranes and was preferentially transferred into the lumen of the RER with specific radioactivities ten times those in the membrane. Radiolabelled apo-B did not equilibrate with pre-existing unlabelled apo-B, and pools of different specific radioactivities were established in different subcellular fractions. Only a small fraction of the newly synthesized apo-B was transferred to the Golgi lumen. In pulse-chase experiments, most of the newly synthesized apo-B in the RER membrane and the RER lumen was degraded. [3H]Glycerol was incorporated into triacylglycerol and phospholipids in the lumen of the RER, SER, cis-Golgi and trans-Golgi. However, in contrast with apo-B, all of the radiolabelled lipids in the lumen of the RER, SER and cis-Golgi were transferred to the trans-Golgi lumen or secreted. Analysis of the lipid composition of the lumenal content fractions suggests that, although very-low-density-lipoprotein (VLDL) lipids are present in the endoplasmic reticulum lumen, a large fraction of these is not associated with apo-B. Collectively these observations suggest that assembly of apo-B into complete VLDL is not cotranslational, that most lipids become associated with apo-B late in the endoplasmic reticulum compartment and that the lipids are further modified in the Golgi lumen.

Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteins proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o-phenanthroline was reversed by the addition of zinc ions. Oleate, o-phenanthroline and leupeptin also stimulated secretion of radiolabelled apo-B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion.

The aim of this study was to investigate the types and characteristics of chylomicron precursors in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low fat diet, newly synthesized and immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/ very low density lipoproteins (light particles). After feeding fat, newly synthesized, and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminalradiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.Dietary lipids are digested in the small intestine, and the products are transferred across the brush border of the enterocytes. Triacylglycerols (TAG) 1 are resynthesized and assembled into chylomicrons, which are released into the lamina propria and move via the lymph into the blood. The ability of enterocytes to assemble and secrete chylomicrons is modulated by a variety of factors including the amount and composition of the dietary fats (reviewed in Refs. 1-3).Chylomicrons consist of droplets of TAG with some cholesterol ester, stabilized by a shell of phospholipids, cholesterol, and protein (1-3). The major protein of chylomicrons is apolipoprotein-B48 (apoB48), a truncated form of apoB100 produced through post-transcriptional editing of the mRNA of apoB100, the characteristic protein of very low density lipoproteins (VLDL), which transport endogenous lipid from the liver (4 -6). The intracellular events in the assembly of VLDL and chylomicrons revealed by electron microscopic studies are basically similar (7-9). ApoB is synthesized by bound ribosomes in the rough endoplasmic reticulum (RER), the lipid components are synthesized in the smooth endoplasmic reticulum (SER), and assembly of the lipoprotein particle occurs within the lumen of the ER/Golgi compartment. There is evidence for a two-step assembly of VLDL in which small, dense apoB-containing particles are formed initially in the lumen of the RER and...

Very low density lipoproteins (VLDL), the vehicle of transport of triacylglycerol from the liver, consist of a triacylglycerol/ cholesterol ester core stabilised by an outer shell of phospholipid, cholesterol and proteins. Apolipoprotein B (apo-B) is the major non-exchangeable protein of VLDL and is essential for their secretion. Recently, we have investigated the intracellular distribution of apo B in rat liver using cell fractionation and quantitative immunoblotting (1).Although apo B is a secreted protein with a signal sequence its co-translational and post-translational behaviour is not typical. More that 90% of the apo B of rat liver r.e.r and s.e.r. and 15% of that of the trans-Golgi is associated with the membrane rather than in the cisternal space (1). We have recently shown that in rabbit liver the membrane bound form of apo B is at the cytosolic side of the e.r. and Golgi (2-3). These observations are consistent with those of other investigators (4,5) and have led to the idea that newly synthesised apo B may have two possible fates. Under metabolic conditions promoting tracylglycerol synthesis and secretion, apo B is channelled into the lumen of the e.r./Golgi compartment for packaging with VLDL and secretion. When lipid synthesis is reduced, then apo B is channelled into the membrane bound pool and degraded. We have now tested this hypothesis by following the movement of newlysynthesised apo B between intracellular sites in hepatocytes freshly isolated by collagenase perfusion and incubated in suspension under conditions in which VLDL secretion is modulated. Three conditions were used. Freshly isolated hepatocytes in HEM containing 1% bovine serum albumin were, (i) incubated in the presence of added oleate; (2-bound to 1% fatty acid free bovine serum albumin); (ii) incubated in the absence of oleate (with 1% albumin) or (iii) hepatocytes were isolated from livers of rats fed 1% orotic acid in their diet for seven days and incubated as in (i). Orotic acid blocks secretion of apo B containing lipoproteins (6).There was no detectable secretion of apo B-48 or B-100 by cells from orotic acid treated liver incubated in the presence of oleate.On addition of oleate to normal cells secretion of apo B-48 was stimulated and was linear for approximately one hour at 4 pg/h/mg cell protein for apo B-48 and 1.2 pg/h/mg cell protein for apo B-100. The apo B mass was 3.75 pg apo B-100 per mg cell protein and 10.4 pg of apo B-48.The apo B mass (pg/mg of fraction protein) of subcellular fractions (r.e.r., s.e.r., cis-Golgi and trans-Golgi) was similar in preparations from normal liver and from livers of orotic acid fed rats. Values were as reported previously (1). Apo

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

Summary. The phospholipid content of different activated partial thromboplastin time (APTT) reagents was determined and compared to heparin sensitivity. The seven reagents included were those most widely used amongst participants of the U.K. National External Quality Assessment Scheme (NEQAS) at the time of study. Heparin sensitivity was assessed using the APTT ratios obtained by more than 300 NEQAS participants on ®ve plasmas prepared from patients receiving unfractionated heparin. The concentrations of three neutral lipids and six phospholipids present in the seven APTT reagents were determined by high-performance thin-layer chromatography (HPTLC) and densitometry. Both the concentrations and the relative percentages of individual phospholipid components varied markedly between reagents. The total phospholipid concentration included a 12-fold range from 16 to 205 mg/ml. Phosphatidylserine (PS) was completely lacking from one reagent prepared from vegetable material and ranged from 3 to 22 mg/ml in the other six reagents containing extracts from animal tissue. The concentration of phosphatidylcholine ranged from 3 to 109 mg/ml. There was no demonstrable relationship between the concentration of any individual lipid components and heparin sensitivity. However, the relative percentage phospholipid composition was important since a lower % of PS or phosphatidylinositol (PI) correlated with increasing heparin sensitivity.