Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Chemical labeling studies on bovine heart mitochondrial cytochrome c oxidase dispersed in nonionic detergents

In order to investigate the structural interactions of nonionic detergents with bovine heart mitochondrial cytochrome c oxidase (COX), a series of hydrophilic chemical modification reagents were used to map regions on COX which are not shielded by dodecyl beta-D-maltoside (DM), Triton X-100 (TX-100), and Tween 80 (TW-80). Low levels of incorporation of the chemical reagents [35S]benzenediazoniumsulfonate (DABS) and N-succinimidyl [3H]propionate (SP) into COX dispersed in TW-80 indicate that the bulky headgroup and hydrophobic moiety of this detergent effectively shield the enzyme from the aqueous environment. Subunits II and Va/Vb [nomenclature of Merle, P., & Kadenbach, B. (1982) Eur. J. Biochem. 125, 239-244] show an increased reactivity to [35S]DABS and [3H]SP in TW-80 and may reflect an increased exposure of these subunits to the aqueous phase in comparison to COX dispersed in TX-100 or DM. More [35S]DABS is incorporated into COX in DM than TX-100-dispersed enzyme; DABS heavily labels subunits III, VIa, and VIb in DM. While COX in TX-100 is more reactive with [3H]SP than DM-dispersed enzyme, there is no difference in the distribution of label (either DABS or SP) within the subunits of COX in DM or TX-100. Increased surface exposure of COX in TX-100 is indicated by an enhanced sensitivity of COX electron-transfer activity in enzyme chemically modified by the cross-linking reagent N-succinimidyl 3-[(4-azidophenyl)dithio]propionate (SADP) in TX-100 as compared to enzyme chemically cross-linked in the other detergents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2alpha receptors.

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Do ice nucleating lipoproteins protect frozen insects against toxic chemical agents?

As the body fluid of freeze-tolerant organisms freezes, solutes become concentrated in the gradually smaller unfrozen fluid fraction, and dissolved trace metals may reach toxic levels. A dialysis technique was used to investigate the metal binding capacity of the low density fraction of the hemolymph from the freeze tolerant beetle Phyto depressus. The low density fraction, assumed to contain the ice nucleating lipoproteins, showed approximately 100 times greater capacity to bind metals (Cd ²⁺, Cu ²⁺ and Zn ²⁺) than the proteins albumin, hemoglobin and similar to metallothionein. The high metal binding capacity in the low density fraction raises the question if the ice nucleating lipoproteins might assist in detoxification of potentially toxic concentrations of metals that may occur when a large fraction of the bodyfluids of freeze tolerant insects freeze. This hypotheis is consistent with the fact that the lipoprotein ice nucleators are present in far greater amounts than required for ice nucleation, and also with the fact that the lipoprotein ice nucleators have a remarkably high content of amino acids with negatively charged residues that may act as metal binding sites.     

Guggulsterone enhances glycosylated low density lipoprotein binding in rat liver

Modification of low density lipoprotein by nonenzymic glycosylation resulted in decreased receptor-mediated lipoprotein catabolism. Guggulsterone treatment caused significant increase in binding of [¹²⁵I] low density lipoprotein as well as [¹²⁵I] glycosylated low density lipoprotein. Scatchard plot analysis of the binding activity revealed that under the influence of guggulsterone, the liver membrane contains increased amounts of a functional lipoprotein receptor that binds more low density lipoprotein particles.     

Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response

When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with 100 μM H₂O₂, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE. The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited an apparent K _m of 40 mM and a V _max of 285,000 U (mg protein)^–1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases. Received: 15 October 1997 / Accepted: 2 February 1998     

Characterization and specificity of lipoprotein binding to term human placental membranes

The binding characteristics of very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoprotein fractions to a purified human term placental microvillous membrane preparation were determined. Binding of LDL was saturable with a maximal binding capacity of 270 ng LDL protein per mg of membrane protein. Scatchard analysis revealed the presence of a single population of 3.4 · 10¹¹ sites per mg of membrane protein and a mean affinity constant of 5.8 · 10⁻⁹ M. Binding of VLDL was also saturable but the maximal capacity was 4.5-times greater than that of LDL. The Scatchard analysis revealed the presence of 2.1 · 10¹¹ binding sites and an affinity constant nearly one order of magnitude greater than that of LDL. Binding of HDL showed less tendency to saturate. Scatchard analysis showed a similar number of receptor sites to that calculated for VLDL and LDL but the affinity constant for HDL was over 100-fold less than that of VLDL. Self- and cross-inhibition studies of VLDL and LDL binding revealed that VLDL was better at blocking the binding of LDL than was LDL itself. This preferential binding of VLDL suggests that this lipoprotein fraction could be an important source of cholesterol for placental progesterone production.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2α receptors

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Solution Structure of S100A1 Bound to the CapZ Peptide (TRTK12)

As is typical for S100-target protein interactions, a Ca²⁺-dependent conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin-capping protein CapZ. In addition, the Ca²⁺-binding affinity of S100A1 is found to be tightened (greater than threefold) when TRTK12 is bound. To examine the biophysical basis for these observations, we determined the solution NMR structure of TRTK12 in a complex with Ca²⁺-loaded S100A1. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca²⁺-bound state. Next, the structure of S100A1-TRTK12 was compared to that of another S100A1-target complex (i.e., S100A1-RyRP12), which illustrated how the binding pocket in Ca²⁺-S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when the structures of S100A1-TRTK12 and S100B-TRTK12 were compared, providing insights regarding how more than one S100 protein can interact with the same peptide target. Such comparisons, including those with other S100-target and S100-drug complexes, provide the basis for designing novel small-molecule inhibitors that could be specific for blocking one or more S100-target protein interactions.     

Identification and characterization of a high density lipoprotein-binding protein in cell membranes by ligand blotting

Cholesterol efflux from cultured cells can be mediated through binding of high density lipoprotein (HDL) to a cell-surface site which shows many characteristics of a biological receptor. To determine whether a specific protein forms a component of this site, cell membrane proteins were analyzed by ligand blotting using 125I-HDL3. Results demonstrated that membranes from a number of cell types possess a protein with an apparent molecular mass of 110 kDa that binds HDL and apoA-I and apoA-II proteoliposomes, but not low density lipoprotein, acetylated low density lipoprotein, or apoE proteoliposomes. The binding activity of this protein was increased by loading cells with cholesterol and was abolished by trypsin treatment of intact cell monolayers. These results suggest that HDL binds with specificity to a cell-surface protein which is regulated by intracellular cholesterol levels. Since HDL binding to intact cell monolayers shows the same characteristics, the 110-kDa binding protein may represent the proposed HDL receptor that functions to facilitate transport of cholesterol from cells to HDL particles.     

Lipoproteins and apolipoproteins in intestinal lymph of the preruminant calf, Bos spp., at peak lipid absorption

We have recently evaluated the in vivo role of the liver in lipoprotein homeostasis in the preruminant calf (Bauchart, D., D. Durand, P. M. Laplaud, P. Forgez, S. Goulinet, and M. J. Chapman, 1989. J. Lipid Res. 30: 1499-1514). We now present the partial characterization of lipoprotein particles in postprandial intestinal lymph at peak lipid absorption (i.e., 10 h after a meal) in the preruminant calf fed a curdled milk replacer. Intestinal lymph from four male preruminant calves was analyzed for its content of lipids and fractionated by sequential and density gradient ultracentrifugation into chylomicrons (Sf greater than 400), very low density lipoproteins (VLDL) (Sf less than 400; d less than 1.006 g/ml), and a series of lipoprotein subfractions with d greater than 1.006 g/ml. Postprandial lymph contained predominantly triglycerides (1099 +/- 611 mg/100 ml), with lesser amounts of phospholipids (197 +/- 107 mg/100 ml) and cholesterol (52 +/- 30 mg/100 ml). The most abundant particles were triglyceride-rich chylomicrons and VLDL which accounted for approximately 76% and approximately 19%, respectively, of total d less than 1.21 g/ml lipoproteins. As judged by negative stain electron microscopy, chylomicron particle diameters ranged from 650 to 2400 A, while VLDL were smaller and distributed over a distinct size range (340-860 A). These two lipoprotein classes each presented protein components with Mr comparable to those of human apoB-48, apoA-I, and C apoproteins, together with an Mr 52,000 protein resembling human beta 2-glycoprotein-I. In addition, VLDL exhibited a polypeptide with Mr approximately 61,000. Lymph lipoproteins with d greater than 1.006 g/ml consisted primarily (approximately 81% of total) of particles distributed over the 1.053-1.119 g/ml density range. Electrophoretic analysis of the latter lipoprotein fraction showed it to be heterogeneous, including particles with the migration characteristics of low and of high density lipoproteins, respectively. Subfractions in the d 1.053-1.076 g/ml range were dominated by particles with Stokes diameters typical of high density lipoproteins (HDL), but also contained three different populations of low density lipoprotein-like particles. The high molecular weight apolipoproteins in these same cholesteryl ester-rich (greater than 30% of lipoprotein mass) subfractions comprised components with Mr resembling those of human apoB-100 and apoB-48, respectively, and with the latter protein predominating to a varying degree. A counterpart to human apoA-I was the major protein component over the entire density range from d 1.053 to 1.119 g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions

The enzyme catalase (EC 1.11.1.6) is light sensitive and subject to a rapid turnover in light, similar to the D1 reaction center protein of photosystem II. After 3 h of preadaptation to darkness or to different light intensities (90 and 520 μmol m⁻² s⁻¹ photosynthetic photon flux density), sections of rye leaves (Secale cereale L.) were labeled for 4 h with l-[³⁵S]methionine. From leaf extracts, catalase was immunoprecipitated with an antiserum prepared against the purified enzyme from rye leaves. Both incorporation into catalase and degradation of the enzyme polypeptide during a subsequent 16-h chase period increased with light intensity. At a photon flux density of 520 μmol m⁻² s⁻¹, the apparent half-time of catalase in rye leaves was 3 to 4 h, whereas that of the D1 protein was much shorter, about 1.5 h. Exposure to stress conditions, such as 0.6 m NaCl or a heat-shock temperature of 40°C, greatly suppressed both total protein synthesis and incorporation of the label into catalase and into the D1 protein. Immunoblotting assays indicated that in light, but not in darkness, steady-state levels of catalase and of the D1 protein strongly declined during treatments with salt, heat shock, or translation inhibitors that block repair synthesis. Because of the common property of rapid photodegradation and the resulting dependence on continuous repair, declines in catalase as well as of the D1 protein represent specific and sensitive indicators for stress conditions that suppress the translational activities of leaves.     

Function of hepatic triglyceride lipase in lipoprotein metabolism

Rat hepatic triglyceride lipase (H-TGL) was purified from liver tissue extracts by affinity chromatography on Sepharose 4B with covalently linked heparin. The purified rat H-TGL exhibited the properties previously described for this enzyme. Enzyme protein was injected into rabbits for anti-H-TGL antibody production. Antirat-H-TGL did not react against rat lipoprotein lipase (LPL) but inhibited H-TGL-activity both in vitro and in vivo greater than 90%. These antibodies were injected into rats and lipoprotein analyses were performed over a 36-hr period. It could be shown that inactivation of H-TGL by anti-H-TGL gamma-globulins in vivo led to an increase in total triglyceride concentration from 70 mg/dl to 230 mg/dl due to an increase in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) triglycerides 4 hr after antibody injection; a marked increase in high density lipoprotein (HDL) phospholipid concentration was observed with almost no change in HDL-cholesterol and HDL-triglycerides. This study documents the ability of antirat-H-TGL-gamma-globulins to inhibit H-TGL in vitro and in vivo. Furthermore, the inhibition of triglyceride removal in vivo demonstrated that this enzyme together with LPL is responsible for the catabolism of VLDL-triglyceride.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Proteomic analysis of human low‐density lipoprotein reveals the presence of prenylcysteine lyase,a hydrogen peroxide‐generating enzyme

The molecular mechanisms underlying the relationship between low‐density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid‐phase IEF has been used to resolve LDL proteins into well‐defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL‐associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol‐binding protein, and paraoxonase‐1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time‐dependent generation of H₂O₂ which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H₂O₂ generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.     

Murine macrophage tumors are a source of a 260,000-dalton acetyl-low density lipoprotein receptor

Subcutaneous injection of murine macrophage cell line P388D1 into syngeneic DBA/2 produced tumors, which upon solubilization with 40 mM octyl glucoside contained acetylated low density lipoprotein binding activity. The tumor-derived receptor specifically bound acetylated low density lipoprotein with an affinity of approximately 3 X 10(-8) M but did not bind low density lipoprotein or high density lipoprotein. It was identical in binding specificity, affinity, and Pronase sensitivity to the receptor in intact cells or that obtained from solubilized cultured cell membranes. Partial purification of the receptor was achieved by solubilizing tumors with 1% Triton X-100 followed by chromatography on polyethyleneimine cellulose. After elution with a NaCl gradient in the presence of octyl glucoside and association with liposomes, a 287-fold purification of the receptor was achieved. The receptor was identified by specific ligand blotting as a 260,000-dalton protein having a pI of approximately 6.0. Binding to the receptor by acetylated low density lipoprotein, malondialdehyde-modified low density lipoprotein, and maleic anhydride-modified serum albumin was demonstrated by ligand blotting. A single receptor protein can, therefore, account for the binding of multiple types of charge-modified lipoprotein and nonlipoprotein ligands to the macrophage cell surface.     

Binding of unmodified low-density lipoproteins to human fibroblasts. An investigation by immunoelectron microscopy

The binding of unmodified low density lipoproteins to the plasma membrane of fibroblasts was studied at the ultrastructural level. The bound low density lipoprotein was visualized by an indirect immunoperoxidase technique, with the use of an antiserum against apoprotein B. Immunoreactive regions representing bound apoprotein B were found on the plasma membrane, in indented regions with a diameter of 0.15–0.30 μm and a fuzzy coat on the cytoplasmic side. Fibroblasts from a patient homozygous for hyperlipoproteinaemia type IIa showed no immunoreactive material in the indented regions. The specific ¹²⁵I-labelled low density lipoprotein binding to these homozygous fibroblasts was 7% compared to control fibroblasts.     

In vivo Se binding to human plasma proteins after administration of SeO3

Binding of ^{7 5} Se to plasma proteins was studied in four cancer patients who received 200–250 μCi of ^{7 5} SeO₃²⁻ (1.25 μg of selenium) intravenously for tumor scans. During the hour after injection, the ^{7 5} Se disappeared rapidly from the plasma. Gel filtration chromatography and dialysis experiments indicated that a large amount of the ^{7 5} Se returned to the plasma bound to protein between 1 and 6 h after injection. Up to 16% of the plasma ^{7 5} Se was found in very-low-density lipoproteins and low-density lipoproteins as early as 3 mikn after injection but very little ^{7 5} Se was found in high density lipoproteins. The very low density lipoprotein ^{7 5} Se activity declined very rapidly whereas low density lipoprotein ^{7 5} Se activity fell more slowly. Zonal ultracentrifugal studies of one subject's lipoproteins revealed a continuum of ^{7 5} Se-binding proteins from the very low density lipoprotein peak to the low density lipoprotein peak. Most of the ^{7 5} Se could be removed from the lipoproteins by denaturation with 8 M urea or treatment with 0.5 M mercaptoethanol. These treatments removed very little of the ^{7 5} Se from plasma collected 48 h after injection indicating a different type of binding of selenium in lipoproteins than in other plasma proteins.