Joan A. Higgins

A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol

We describe a new method for the rapid fractionation of plasma lipoproteins, which makes use of a new non-ionic, iodinated, density gradient medium, iodixanol, commercially available as Optiprep(TM). The method is simple: plasma or serum is mixed with iodixanol followed by centrifugation in a vertical or near vertical rotor. Separation of VLDL, LDL and HDL can be achieved in 3 h and the lipoprotein fractions are comparable in density and composition with those prepared using conventional salt based gradients. Each class of lipoprotein can be removed in a single fraction, or a profile of lipoprotein distribution can be obtained using a gradient fractionator. Because the medium is inert, fractions from the gradient can be analysed by agarose gel electrophoresis or assayed for lipid content or apolipoprotein composition by SDS-PAGE without removing the iodixanol. Small differences in electrophoretic mobility of HDL and LDL across several gradient fractions suggest that subfractionation of these classes may occur. The new method is simple, rapid and versatile with potential application for preparation of lipoproteins and for analysis of lipoprotein profiles in the research or clinical laboratory.

Biogenesis of endoplasmic reticulum phosphatidylcholine: Translocation of intermediates across the membrane bilayer during methylation of phosphatidylethanolamine

Phosphatidylethanolamine of rat liver microsomes is rapidly methylated by S-adenosyl[methyl-14C]methionine to produce phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. Using phospholipase C as a probe, on both opened (0.4% taurocholate or French pressure cell treatment) and unopened microsomes, it is demonstrated that phosphatidylcholine is labelled in the inner leaflet of the bilayer and, to a greater extent, in the outer leaflet. Phosphatidyl-N,N-dimethylethanolamine is labelled in the outer leaflet and in a pool sequestered from phospholipase C in open and closed vesicles. Phosphatidyl-N-monomethylethanolamine is labelled in a similarly sequestered pool. When microsomes containing labelled phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine were incubated with unlabeled S-adenosylmethionine, these phospholipids were methylated to produce phosphatidylcholine in the outer leaflet. This metabolism was inhibited by S-adenosylhomocysteine. Trypsin treatment of unopened microsomes inhibited 95% of the incorporation of 14CH3 into the outer leaflet of the bilayer with no effect on incorporation into sequestered phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. Therefore, sequestered phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine are apparently synthesized by enzymes located at the inner surface of the microsomal membranes. These observations suggest that initial methylation of phosphatidylethanolamine takes place at the inner surface of the microsomes and that phosphatidyl-N-monomethylethanolamine is transferred to the outer leaflet to produce phosphatidylcholine. However, phosphatidyl-N-monomethylethanolamine is also methylated at the inner leaflet to produce phosphatidylcholine which does not equilibrate with that of the outer leaflet. Phosphatidylcholine of both the inner and outer bilayer leaflets is uniformly labelled by injection of [14C]methionine, in vivo.

Evidence that during very low density lipoprotein assembly in rat hepatocytes most of the triacylglycerol and phospholipid are packaged with apolipoprotein B in the Golgi complex

Rat liver lipids were labelled by an intraportal injection of [3H]palmitic acid followed by isolation of rough and smooth endoplasmic reticulum or ‘cis’ or ‘trans’‐enriched Golgi fractions. The preparations were separated into membrane and contents and the apolipoprotein B of the content fractions was immunoprecipitated. More than 90% of the labelled triacylglycerol and phospholipid secreted into the blood immunoprecipitated with apolipoprotein B. Under the same experimental conditions 8, 12, 27 and 59% of the lipids of the rough, smooth, ‘cis‐Golgi’ and ‘trans‐Golgi’ contents, respectively, were immunoprecipitated. Thus, the ‘trans‐Golgi’ region appears to be the major intracellular site of assembly of apolipoprotein B with triacylglycerol and phospholipid.

Direct Evidence for a Two-step Assembly of ApoB48-containing Lipoproteins in the Lumen of the Smooth Endoplasmic Reticulum of Rabbit Enterocytes

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 luminal-radiolabeled 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.

Asymmetric distribution of phosphatidylethanolamine in the endoplasmic reticulum demonstrated using trinitrobenzenesulphonic acid as a probe

At pH 7.4 approximately one third of the phosphatidylethanolamine (PE) of rat liver microsomes is labelled by trinitrobenzenesulphonic acid (TNBS). The same fraction of the PE was labelled, when a fixed concentration of microsomes were incubated with concentrations of TNBS from 1.5 mM to 12 mM, or when the TNBS concentration was fixed at 3.0 mM and the microsomal protein varied between 1.2 and 12.0 mg. Microsomes incubated with TNBS remain closed indicated by retention of mannose-6-phosphatase latency, retention of labelled vesicular contents and by the appearance of the vesicles in the electron microscope. When the microsomal vesicles were opened by alkaline pH or after passage through the French pressure cell the % of PE labelled increased up to 90% of the total. The small % remaining unlabelled may be due to some vesicles remaining closed or to steric hindrance by the relatively bulky label on both phospholipid and protein. Phospholipase C hydrolyses approximately one third of the PE in closed microsomal vesicles. After treatment of microsomes with phospholipase C the % PE available for labelling by TNBS decreased and was inversely proportional to the % PE hydrolysed. These results suggest that the same pool of PE is available for either hydrolysis by phospholipase C or for labelling by TNBS, and that this pool is that of the outer leaflet of the microsomal membrane bilayer.

Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.

Capacitation and the acrosome reaction in guinea pig spermatazoa increase the availability of surface aminophospholipids for labelling by trinitrobenzene sulphonate

Guinea pig spermatozoa isolated from the epididymis were capacitated by incubation in phosphate buffered saline at 37 degrees C for 5h. and the acrosome reaction induced by addition of calcium chloride (10mM). There was no significant difference between the phospholipid compositions of freshly prepared, capacitated or acrosome reacted spermatozoa. However, the % of the aminophospholipids available to trinitrobenzene sulphonate in the external medium was increased significantly in capacitated and acrosome reacted spermatozoa compared with freshly isolated spermatozoa. These observations are consistent with the morphological observations of others and suggest that on capacitation there is a change in the arrangement of phospholipids in the plasma membrane of spermatozoa to increase the concentration of fusogenic phospholipids in the outer leaflet.

A role for smooth endoplasmic reticulum membrane cholesterol ester in determining the intracellular location and regulation of sterol-regulatory-element-binding protein-2

Cellular cholesterol homoeostasis is regulated through proteolysis of the membrane-bound precursor sterol-regulatory-element-binding protein (SREBP) that releases the mature transcription factor form, which regulates gene expression. Our aim was to identify the nature and intracellular site of the putative sterol-regulatory pool which regulates SREBP proteolysis in hamster liver. Cholesterol metabolism was modulated by feeding hamsters control chow, or a cholesterol-enriched diet, or by treatment with simvastatin or with the oral acyl-CoA:cholesterol acyltransferase inhibitor C1-1011 plus cholesterol. The effects of the different treatments on SREBP activation were confirmed by determination of the mRNAs for the low-density lipoprotein receptor and hydroxymethylglutaryl-CoA (HMG-CoA) reductase and by measurement of HMG-CoA reductase activity. The endoplasmic reticulum was isolated from livers and separated into subfractions by centrifugation in self-generating iodixanol gradients. Immunodetectable SREBP-2 accumulated in the smooth endoplasmic reticulum of cholesterol-fed animals. Cholesterol ester levels of the smooth endoplasmic reticulum membrane (but not the cholesterol levels) increased after cholesterol feeding and fell after treatment with simvastatin or C1-1011. The results suggest that an increased cellular cholesterol load causes accumulation of SREBP-2 in the smooth endoplasmic reticulum and, therefore, that membrane cholesterol ester may be one signal allowing exit of the SREBP-2/SREBP-cleavage-regulating protein complex to the Golgi.

Asymmetric synthesis and transmembrane movement of phosphatidylethanolamine synthesised by base-exchange in rat liver endoplasmic reticulum.

Using trinitrobenzenesulphonic acid (TNBS) as a probe we have observed that phosphatidylethanolamine (PE) formed by base-exchange is initially concentrated in the cytosolic leaflet of the membrane bilayer. At 2 min, the specific activity of the PE in this leaflet was 3-times that of the PE in the cisternal leaflet. After 30 min, the specific activities of the two pools of PE, determined with either phospholipase C or TNBS, were similar. Transbilayer movement of PE was slow at low temperature, prevented by EDTA and restored by the addition of calcium ions after EDTA treatment. Trypsin treatment of microsomes, under conditions in which the vesicles remained closed, inhibited the incorporation of ethanolamine into PE by 87%. The cytosolic location of the ethanolamine base-exchange enzyme is consistent with the initial concentration of newly synthesised PE at this site prior to its transmembrane movement to the cisternal leaflet.

Membrane-bound apolipoprotein B is exposed at the cytosolic surface of liver microsomes.

We have used a competitive enzyme‐linked immunoassay with a panel of monoclonal antibodies to probe the topography of the membrane‐bound form of apolipoprotein B (apo B) in rabbit microsomes. All epitopes investigated were found to be expressed at the cytosolic side of the microsomal membrane under conditions in which the vesicles remained sealed. These results indicate that the membrane‐associated form of apolipoprotein B is either at the cytosolic side of the endoplasmic reticulum membrane or integrated into the membrane. From this site apo B may be translocated to the lumen for assembly into VLDL or may be degraded.