The effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors.     

Effects of estrogen administration on the lipoproteins and apoproteins of the chicken.

The plasma lipoproteins of estrogen-treated and untreated sexually immature hens have been compared with respect to their concentration in plasma, protein and lipid composition, particle size, and and apoprotein composition. Administration of diethylstilbestrol resulted in a 400-fold rise in the concentration of very low density lipoprotein (VLDL), a 70-fold rise in low density lipoprotein (LDL), and a marked reduction in high density lipoprotein (HDL) protein. It also resulted in the production of LDL and HDL which were enriched in triacylglycerol, while the proportion of cholesterol in all three lipoprotein fractions decreased. In contrast to the lipoproteins from untreated birds, lipoproteins of density less than 1.06 g/ml from estrogen-treated birds were not clearly separable into discrete VLDL and LDL fractions, but appeared to be a single ultracentrifugal class. The apoprotein composition of VLDL and LDL from untreated birds differed from each other; however, the apoprotein patterns of VLDL and LDL from estrogen-treated birds were indistinguishable: both contained a large amount of low molecular weight protein in addition to the high molecular weight component that predominates in the untreated state. The apoprotein composition of HDL was also markedly altered by estrogen administration: the 28,000 mol. wt. protein (apo A-I) decreased in amount from 65% to less than 5% of the total, while a low molecular weight (Mr = 14,000) protein and as yet poorly defined high molecular weight components became predominant. These observations indicate that the hyperlipidemia induced by estrogen administration is accompanied by marked alterations, both qualitative and quantitative, in the plasma lipoproteins.     

Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions

Two procedures were used to isolate hepatocytic Golgi fractions from rat liver. One procedure yields a light Golgi fraction (GF1 + 2) and the other "intact" stacks of cisternae. Triglyceride fatty acids in nascent very low density lipoproteins (VLDL) were labeled by injection of [3H]palmitate intravenously, and radiolabeled lipoproteins were injected as markers of potentially contaminating endosomes. GF1 + 2 fractions were enriched manyfold in the endosomal markers, indicative of substantial endosomal contamination, whereas intact Golgi fractions from the same livers were about 7% as contaminated. By electron microscopy, GF1 + 2 fractions contained mainly multivesicular bodies (MVBs), together with some Golgi-derived secretory vesicles. The small endosomal contamination of intact Golgi fractions was further reduced by a simple modification of the procedure, which removed most entrained endosomes. The surface constituents of Golgi VLDL (d less than 1.010 g/ml) released from these highly purified intact Golgi fractions differed from those of plasma VLDL. Golgi VLDL contained fivefold less unesterified cholesterol than plasma VLDL, but twofold more phospholipids. Golgi VLDL and plasma VLDL contained similar amounts of cholesteryl esters and triglycerides. The protein content of Golgi VLDL was substantially lower than that of plasma VLDL. ApoB-100 and apoB-48 were similarly represented, but nascent VLDL contained less of the C apolipoproteins. ApoA-I was present mainly as the proprotein in Golgi VLDL, but was virtually lacking in plasma VLDL. ApoE comprised about 22% of the protein mass of Golgi VLDL as well as plasma VLDL; the distribution of apoE isoforms was also similar. Apolipoproteins E and pro A-I released from ruptured Golgi cisternae were largely bound to the Golgi VLDL or were associated with Golgi membranes. Particles resembling low density lipoproteins (LDL) and high density lipoproteins (HDL) were not seen by electron microscopy in contents of intact Golgi fractions. These observations indicate that nascent Golgi VLDL are the primary particulate precursors of rat plasma lipoproteins of hepatocytic origin, and suggest that particles with the density of plasma HDL and LDL do not exist within the secretory pathway of normal hepatocytes. Thus, the results of this research on the properties of nascent plasma lipoprotein precursors contained within uncontaminated hepatocytic Golgi fractions differ substantially from previous published work.     

Lipoprotein secretion in lean and obese Zucker female rats in vivo and in a single-pass-perfused liver preparation

The plasma lipoprotein composition as well as lipoprotein synthesis and secretion were studied in vivo and in a single-pass-perfused liver preparation in lean and obese Zucker rats. Compared with their lean littermates the levels in the plasma of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) + low density lipoprotein (LDL) and high density lipoprotein (HDL) were increased 4-, 2- and 2.5 fold, respectively, in obese rats. In these rats both VLDL and IDL + LDL were enriched in triglycerides, while the HDL were enriched in cholesterol. Although the VLDL and IDL + LDL protein concentrations were the same in lean and obese rats, the HDL protein concentration was 3-fold greater in the obese rats. Both the lean and obese rats incorporated similar amounts of [14C]leucine into total liver protein. However, obese rats incorporated 2.5-fold and 6-fold more [14C]leucine into VLDL and HDL in vivo, 2.7-fold and 1.7 fold more [35S]methionine in VLDL and HDL present in the perfusate, than did lean rats. The perfusate [35S]S-labelled apoproteins (apo-B100, B48; apo-E, apo-AI, apo-AIV and apo-C) were separated by gel electrophoresis and identified by autoradiography. Incorporation of [3H]glycerol into liver, VLDL, IDL + LDL and HDL triglycerides was 2-, 48-, 13- and 1.5-fold higher in obese than in lean rats, respectively. The [3H]-labelled triglycerides in VLDL and IDL + LDL present in the perfusate was 5.4-fold and 4.4-fold more in obese rat. There was no difference in the incorporation of [3H]glycerol into triglycerides of perfusate HDL between the two genotypes of rats. Thus, the hypertriglyceridaemia observed in obese Zucker rats results from very high synthetic rates of both the lipid and protein moieties of plasma lipoproteins. Before this study, no report of the simultaneous triglycerides and protein synthesis in vivo and in a single-pass-perfused liver preparations had been reported.     

Membrane-associated cathepsin L: a role in metastasis of melanomas

Subcellular distribution of cathepsin L, the major protein released by transformed or ras transfected fibroblasts, was examined in murine liver, murine B16 amelanotic melanoma and human A2058 melanoma after sequential differential and Percoll density gradient centrifugation. In both murine and human melanomas, cathepsin L activity was found to be enriched in plasma membrane fractions; cathepsin L in these fractions was in both native and acid activatable forms. Plasma membrane fractions from B16 melanoma subpopulations of "low" and "high" metastatic potential were assayed for activity of cathepsin L and of heat stable endogenous inhibitors. The relative specific activity of cathepsin L was 7-fold greater in the subpopulation of "high" metastatic potential, whereas cysteine proteinase inhibitory activity was 5-fold less. Since cathepsin L can degrade intact basement membrane, this membrane-associated cathepsin L may well contribute to metastatic spread of melanomas.     

The effects of liposome-reconstituted apolipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

Rat intermediate density lipoproteins (IDL) bind specifically to high and low affinity binding sites on rat liver membranes. In a recent paper (Brissette, L., and No?l, S.-P. (1986) J. Biol. Chem. 261, 6847-6852), we have demonstrated that human low density lipoproteins and high density lipoproteins-3 can totally prevent the specific binding of rat IDL to the low affinity binding sites. The aim of the present studies was to determine the effects of apoA-I, apoC, and apoE, reconstituted into liposomes, on the binding of rat iodinated IDL to rat liver membranes. We found that a 50-, 100-, or 300-fold excess of liposome-reconstituted apoE, apoC, or apoA-I, respectively, abolished the specific binding of IDL to the low affinity binding sites. Only apoE liposomes had an effect on the high affinity component; at a 100-fold excess no specific binding of IDL could be detected. Liposomes by themselves or associated with erythrocyte membrane proteins had virtually no effect on the binding of IDL. Taken together our results suggest that apoE is the only ligand that can compete efficiently for the sites that bind rat IDL with a high affinity. These sites may be the expression of both the remnant and the LDL receptors. The binding to the low affinity component probably represents weak interactions between IDL and "unspecified-lipoprotein binding sites," which can be entirely masked by human low density lipoproteins, high density lipoproteins-3, or liposome-reconstituted apoA-I, apoE, or apoC at appropriate concentrations.     

Changes in lipoprotein binding and uptake by hepatocytes during rat liver regeneration

The binding and uptake of cholesterol enriched lipoproteins by isolated hepatocytes was decreased at 16 hours after partial hepatectomy, with a tendency to return to control values as the regeneration proceeds. The number of lipoprotein binding sites of total cellular membranes remained similar to control at 16 and 24 hours. The plasma lipoprotein pattern, determined by electrophoretic analysis, showed a lower per cent of very low density lipoproteins (VLDL) and a higher per cent of low density lipoproteins (LDL) at 16 and 24 hours post-partial hepatectomy. At these times, plasma lecithin: cholesterol acyltransferase (LCAT) activity was decreased. It is intriguing to suggest that the regenerating liver could regulated the blood lipoprotein pattern and the uptake of lipoproteins by modulating the surface expression of the receptors.     

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.     

The inositol 1,4,5-trisphosphate-binding site in adrenal cortical cells is distinct from the endoplasmic reticulum

The distribution of binding sites for the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was investigated in subcellular fractions of bovine adrenal cortex. The [3H]Ins(1,4,5)P3-binding capacity was enriched in the microsomal fraction, which contained a single class of high affinity binding sites with a Kd of 21.6 +/- 3.0 nM. The specific [3H]Ins(1,4,5)P3 binding appeared to be sharply pH dependent and was inhibited by millimolar concentrations of ATP. Upon fractionation of microsomes on sucrose density gradient there was a clearcut separation of the Ins(1,4,5)P3 receptor-containing fractions from those enriched in specific endoplasmic reticulum markers such as sulfatase C activity or RNA content. The microsomes enriched in Ins(1,4,5)P3-binding sites were of lower density than the endoplasmic reticulum and co-purified partly with the plasma membrane. In addition, Ins(1,4,5)P3-sensitive 45Ca2+ uptake into the microsomes was maximal in the lighter fractions. This distinction between Ins(1,4,5)P3-binding sites and endoplasmic reticulum-derived microsomes was confirmed upon fractionation according to their electrophoretic mobilities by free flow electrophoresis. These results indicate that in adrenal cortical cells, the source of Ca2+ mobilized by Ins(1,4,5)P3 upon stimulation with an agonist is not located in the endoplasmic reticulum. Our data support the hypothesis that a specialized vesicular organelle, distinct from endoplasmic reticulum and in close apposition with the plasma membrane, is involved in intracellular Ca2+ homeostasis.     

Processing of lipoproteins in human monocyte-macrophages

Subcellular fractionation of human monocyte-macrophages (HMM) yielded a fraction rich in endosomes, lysosomes, and mitochondria. This pellet was further fractionated in a metrizamide gradient and the subcellular organelles were distributed among seven distinct bands. All of the bands contained lysosomal enzymes in similar amounts. However one band, poor in mitochondria, was markedly enriched in cathepsin D and cholesteryl ester hydrolase activities. A number of different ligands (low density lipoproteins (LDL), malondialdehyde-altered LDL, beta-migrating very low density lipoprotein, high density lipoprotein, reductively methylated LDL, mannose-bovine serum albumin, and transferrin) were presented to HMM at a concentration of 20 micrograms/ml at 4 degrees C. Three minutes after warming the cells at 37 degrees C all ligands except two were found predominantly in the cathepsin D- and cholesteryl ester hydrolase-rich fraction. Unlike the other ligands, LDL had distributed to other more dense fractions and reductively methylated LDL was found mainly in less dense fractions. At a lower concentration, 2 micrograms/ml, the distribution of LDL was identical to the other ligands. In vitro incubation of the fractions obtained from the gradient suggested that cathepsin D was largely responsible for the hydrolysis of the lipoproteins. We conclude that studies of LDL metabolism in HMM must take into account the different processing of this ligand at commonly used concentrations.     

The binding of low density lipoprotein by liver membranes in the pig.

Porcine liver membranes are capable of high affinity binding of homologous low density lipoproteins (LDL). Binding is time and temperature dependant and substrate saturable. High affinity binding sites are half saturated at 11 μg/ml lipoprotein-protein. The binding of ¹²⁵I-LDL is inhibited by unlabelled homologous LDL, very low density lipoproteins (VLDL) and high density lipoproteins (HDL) and also be human LDL and HDL, but not by unrelated proteins tested. The binding and displacement patterns with membranes from several other porcine tissues are similar to those of liver membranes. These results suggest the presence of “lipoprotein binding sites” in liver membranes which recognize structural features common to the lipoproteins and further indicate that liver membranes are not unique in their ability to bind LDL.     

Low density lipoprotein heterogeneity in the cebus monkey

In studies of cebus monkey plasma lipoproteins, we have used an ultracentrifugally generated density gradient to isolate two distinct species of low density lipoproteins (LDL). Compositional analyses revealed that each of the ultracentrifugally isolated fractions was enriched in cholesteryl esters and contained a single apolipoprotein which in terms of its mobility on SDS gels corresponded to apolipoprotein B-100, the major apolipoprotein of human LDL. Hydrodynamic measurements carried out in the analytical ultracentrifuge showed that F1.20 values were 30.0 for LDL1 and 23.5 for LDL2. In a solution of density 1.0069 g/ml, the sedimentation rates were 5.9 and 7.2 S for LDL1 and LDL2, respectively. In addition to sedimentation velocity data, we describe a new approach for using these same data to obtain calculated values for molecular weight. The hydrated densities calculated for the two fractions were 1.033 and 1.045 g/ml and calculated molecular weights were 3.08 million for LDL1 and 2.42 million for LDL2. Hydrated density values were in excellent agreement with those calculated from compositional data. Electron microscopy data showed that LDL1 had a larger mean diameter of 26.7 nm than LDL2 which had a diameter of 19.3 nm. Native gel electrophoretic analyses of the two LDL fractions in 3.5% acrylamide showed that, consistent with its size, LDL1 had slower mobility than LDL2.     

Intrahepatic assembly of very low density lipoproteins: immunologic characterization of apolipoprotein B in lipoproteins and hepatic membrane fractions and its intracellular distribution

Monoclonal antibodies, prepared against rat apoB, were used to examine apoB structure in serum lipoproteins and characterize the forms and localization of apoB in liver membrane fractions and cultured hepatocytes. Of the several antibodies obtained, four, having separate epitopes, were characterized. Western blot analysis showed that three (DB11, F4, and LB14) antibodies recognized both apoBL and apoBS. One antibody (HB41) recognized only apoBL. This antibody showed unusual properties. Competition ELISA assays showed that the epitope recognized by HB41 was more effectively expressed on low density lipoproteins (LDL) compared to very low density lipoproteins (VLDL). In addition, treatment of lipoproteins with detergents and sulfhydryl reducing agents also increased the expression of the HB41 epitope. Since HB41 has been found to inhibit LDL binding to hepatocyte receptors, these data indicate that the HB41 epitope is located on the carboxy-terminal side of the apoBS junction (probably within the LDL receptor binding domain). Western blotting hepatic microsomal subfractions showed that in the rough and smooth microsomes, HB41 recognized only apoBL, while in the Golgi it recognized both apoBL and a protein having a molecular weight slightly smaller. In contrast, Western blotting with a polyclonal antibody known to recognize both apoBL and apoBS showed that, in rough and smooth microsomes, proteins in addition to apoBL and apoBS having molecular weights between 120,000 and 30,000 were recognized. These proteins, likely to be proteolytic fragments of apoB, were barely detectable in the Golgi. Additional biosynthetic studies show that the [35S]methionine-labeled proteins smaller than apoB were immunoprecipitated from the rough microsome subfraction. Pulse-chase experiments show that these are produced with the same kinetics as full-size apoBL and apoBS, indicating that they are not incomplete nascent chains. Finally, immunofluorescence microscopy was used to determine the localization of monoclonal epitopes. ApoB monoclonal antibodies that recognized exclusively apoBL (HB41) and apoBL and apoBS (DB11) produced an immunofluorescence pattern characteristic of the endoplasmic reticulum, but not the Golgi. These data suggest that, in cultured rat hepatocytes, the majority of both molecular weight forms of apoB are localized in the endoplasmic reticulum, the initial site of VLDL assembly. The additional finding that proteolytic fragments of apoB are enriched in the microsomal fraction suggests that if the proteolysis occurs during subcellular fractionation, immature apoB is susceptible to proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.     

Identification and properties of complexes formed by myeloperoxidase with lipoproteins and ceruloplasmin

The first evidence of multi-component complexes formed by myeloperoxidase (MPO), ceruloplasmin (CP), and very low/low density lipoproteins (VLDL/LDL) obtained by electrophoresis, gel filtration, and photon-correlation spectroscopy (PCS) is presented in this paper. Complexes were observed when isolated MPO, CP, and VLDL/LDL were mixed and/or when MPO was added to the blood plasma. Complex LDL–MPO–CP was detected in 44 of 100 plasma samples taken from patients with atherosclerosis, and 33 of 44 samples also contained the VLDL–MPO–CP complex. MPO concentration in these patients’ plasma exceeded 800 ng/ml. Interaction of MPO with high density lipoproteins (HDL) was not revealed, as well as binding of CP to lipoproteins in the absence of MPO. Adding antibodies against apoB-100 to VLDL–MPO–CP and LDL–MPO–CP complexes results in release of lipoproteins. Using PCS the diameters of complexes under study were evaluated. By comparing concentrations of the components in complexes formed by MPO, CP, and lipoproteins their stoichiometry was assessed as 2VLDL:1MPO:2CP and 1LDL:1MPO:2CP. Lipoproteins affected the inhibition of MPO peroxidase activity by CP. The affinity of lipoproteins to MPO–CP complex was assessed using apparent dissociation constants determined as ～0.3 nM for VLDL and ～0.14 nM for LDL.     

Binding of plasma low density lipoproteins to erythrocytes

Low density lipoproteins (LDL) containing apolipoprotein B bind to intact, freshly isolated erythrocytes. The LDL-erythrocyte interaction is of low affinity, with a Kd of 1.1 x 10(-6) M. Binding is noncooperative. There are about 200 binding sites per cell and, within the limits of experimental uncertainty, these sites comprise a homogeneous class. Binding of LDL is a temperature-independent process. The maximum amount of LDL blood increases following proteolytic digestion of the cells with trypsin or chymotrypsin. The specificity of the binding sites for LDL is not absolute: high density lipoproteins and lipid vesicles composed of phosphatidylcholine or phosphatidylcholine/cholesterol (equimolar) complete with LDL for occupancy of 60% of the binding sites. Modification of 5--6 of the 9 apolipoprotein B arginine residues with 1,2-cyclohexanedione/borate or of 10--15 of the 20 lysine residues by reductive methylation does not alter the ability of LDL to bind to erythrocytes. Native LDL and methylated-LDL alter erythrocyte morphology. However, LDL in which the arginine residues are derivatized with 1,2-cyclohexanedione/borate do not induce the discocyte leads to echinocyte transformation. Chemically modified and native LDL exchange cholesterol with erythrocytes at equal rates and to nearly equal extents. Taken together, the data suggest that the binding sites for LDL on the erythrocyte membrane are distinct from the LDL receptors at the surface of other cells--e.g., fibroblasts and lymphocytes--which do not bind HDL and which do not recognize LDL with derivatized arginine or lysine residues. It is proposed that the biological function of the erythrocyte binding sites is to mediate the exchange of cholesterol between the cell membrane and lipoproteins.     

Identification of Synapse Specific Components: Synaptic Glycoproteins, Proteins, and Transmitter Binding Sites

Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X-100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for L-glutamate, L-aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ-like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for L-glutamate (L-Glu) and L-aspartate (L-Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ-like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.     

Identification of lipoprotein-binding proteins in rat liver Golgi apparatus membranes

The low density lipoprotein (LDL) receptor has been shown to be a plasma membrane glycoprotein responsible for the cellular binding and endocytosis of plasma lipoproteins. Inasmuch as the Golgi apparatus has been shown to participate in glycoprotein processing and in the assembly of plasma lipoproteins by hepatic and intestinal epithelial cells, the present studies were designed to test the hypothesis that lipoprotein receptors are present within Golgi membranes. Utilizing ligand blotting with a variety of iodinated lipoproteins, several lipoprotein-binding proteins were identified in rat liver Golgi membranes at apparent molecular weights (Mr) 200,000, 160,000, 130,000, 120,000, 100,000, 80,000, and 70,000. The 130,000 protein was the most prominent and was identified as the mature LDL receptor by its binding characteristics and an Mr characteristic of the plasma membrane receptor. Enzymatic deglycosylation studies suggested that the 120,000 and 100,000 proteins were LDL receptor precursors lacking sialic acid. Antibody to the LDL receptor recognized all the bands on immunoblots except the 70,000 protein, with the 130,000 protein being the most prominent. Isolation of the Golgi fractions in the presence of protease inhibitors did not eliminate any of the proteins recognized by the antibody but did result in sharper bands on the blots. Additionally, we investigated the hypothesis that conditions that regulate plasma membrane LDL receptors also cause detectable changes in receptors in Golgi membranes. All the binding proteins were increased in Golgi membranes from rats treated with 17-alpha-ethynylestradiol. Colchicine caused an accumulation of 120,000 Mr protein, suggesting blockage of final sialylation in the trans Golgi. When protein synthesis was inhibited by cycloheximide, there was no reduction of mature LDL receptors in Golgi membranes, consistent with recycling of receptors through this organelle.