Partial purification of a high density lipoprotein-binding protein from rat liver and kidney membranes

The existence of a cell receptor which recognises plasma high density lipoprotein (HDL) has been suggested from studies which demonstrate specific binding of HDL3 to cultured cells derived from various tissues in the body. This study provides evidence of a specific HDL-binding protein in crude plasma membranes prepared from rat kidney and liver. Following separation of solubilised membrane proteins on polyacrylamide gel slabs and 'Western' blotting, one major band was identified which bound HDL3, or apo AI or apo AII. The protein, which was present in both liver and kidney membranes, was partially purified by repetitive preparative SDS-polyacrylamide gel electrophoresis and although accompanied by considerable loss of binding activity, could still be detected by the ligand-blotting procedure used initially to detect its presence in cell membranes.     

Identification of a high density lipoprotein binding protein from adrenocortical membranes

A protein band with an apparent molecular weight of 78,000 daltons has been identified in the solubilised plasma membrane extract of sheep adrenal cortex which binds HDL3 devoid of E apolipoprotein. Following 'Western' blotting, and development of the nitrocellulose strips with appropriate antisera and color reagent, the same band, unlike other cortical membrane proteins or albumin, bound AI and AII apolipoproteins. Human LDL bound weakly to the same band but more strongly to another two proteins of higher molecular weight. These studies confirm the same degree of specificity of HDL3 binding found with cultured adrenal cells and strengthen the suggested existence of a specific HDL receptor.     

THE METABOLISM OF CHYLOMICRON CHOLESTERYL ESTER IN RAT LIVER : A Combined Radioautographic-Electron Microscopic and Biochemical Study

Chylomicrons containing labeled cholesterol, mainly (70%) present as cholesteryl ester, were injected intravenously into intact rats, and samples of liver were obtained 27–210 min later. Most (58–75%) of the injected label was recovered in the liver after 27–75 min. Hepatic uptake occurred without hydrolysis of the labeled cholesteryl ester. In separate experiments, in vitro perfusion of livers of similarly treated rats for 30–35 min washed out only 3–9% of the labeled sterol. Samples of liver and small intestine were prepared for electron microscopy with Aquon as the dehydrating agent. Good retention (70% or more) of labeled cholesterol and satisfactory preservation of ultrastructure were obtained. After 30 min, the radioautographic reaction was localized mainly over the region of the cell boundary of the parenchymal liver cells, with fewer grains being present over intracellular organelles. At later time intervals, when considerable hydrolysis of the labeled cholesteryl ester had occurred, the radioautographic reaction was more evenly distributed. Phagocytosed labeled lipid was seen in Kupffer cells after the larger lipid load; phagocytosis by parenchymal cells was not seen. In other experiments, cholesteryl ester hydrolase activity was found in all subcellular fractions, the microsome and plasma membrane fractions showing the highest activity per mg protein. The mechanism of cholesteryl ester transport into the liver cell may involve: (1) hydrolysis at the cell surface; or (2) slow entry of intact molecules followed by intracellular hydrolysis of the ester bond.     

Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay.

The epitopes for two monoclonal antibodies (MAbs) directed towards human apolipoprotein A-I (apoA-I), designated AI-1 and AI-3, have been more precisely defined. Previous work in our laboratory demonstrated that AI-1 and AI-3 recognize antigenic determinants located within cyanogen bromide (CNBr) fragments 1 (CF1) and 3 (CF3), respectively. Using peptides generated from endoproteinase cleavage of CF1 and CF3, we now report that both MAbs are specific for two previously unreported epitopes along the apoA-I molecule. The ability of whole endoproteinase digest mixtures to bind the MAbs, as determined by means of a competitive enzyme-linked immunosorbent assay (ELISA), indicated regions of CF1 and CF3 that were likely to form the epitopes. Purified peptides derived from the digests were then used to localize the epitopes recognized by MAbs AI-1 and AI-3 to within residues 28-47 and 140-147 of apoA-I, respectively. We have previously reported that the epitopes for both MAbs are exposed on HDL2, HDL3, and free apoA-I. Thus, the precise mapping of the binding sites recognized by AI-1 and AI-3 has enabled the identification of regions along apoA-I that are exposed on the surface of lipoprotein particles.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Purification, properties and kinetics of sheep and human renin substrates.

Sheep plasma renin substrate was purified 1,200-fold by using nephrectomised sheep plasma, followed by DEAE-Sephadex chromatography and gel filtration. The purified substrate contained 8 mu-g angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7.5, K-m of the human renin-sheep substrate reaction was 0.29 mu-M and for sheep renin-sheep substrate, 2.0 mu-M. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin. Human renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0.9 mu-g angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. K-m for the human renin-human substrate reaction was high and could not be accurately determined (range 3-8 mu-M, mean 5.7 mu-M). The presence of human substrate in a human renin-sheep substrate system did not alter the measured initial velocity. In both sheep and man, the normal concentration of renin substrate is considerably less than K-m and must therefore be considered a determinant of angiotensin production rate in vivo.     

Studies on the metabolism of rat serum very low density apolipoprotein.

There was a rapid transfer of radioactive peptides to other lipoprotein fractions during the first 30 min after the intravenous injection of 125I-labeled rat very low density lipoprotein (VLDL) into rats. After this initial redistribution of radioactivity, label disappeared slowly from all lipoprotein fractions. The disappearance of 125I-labeled human VLDL injected into rats was the same as that of rat VLDL. Most of the radioactivity transferred from VLDL to low density (LDL) and high density (HDL) lipoproteins was associated with two peptides, identified in these studies by polyacrylamide gel electrophoresis as zone IVa and IVb peptides (fast-migrating peptides, possibly analogous to some human C apolipoproteins), although radioactivity initially associated with zone I (analogous to human apolipoprotein B) and zone III (not characterized) was also transferred to LDL and HDL. That the transfer of label from VLDL to LDL and HDL primarily involved small molecular weight peptides was confirmed in studies using VLDL predominantly labeled in these peptides by in vitro transfer from 125I-labeled HDL. Both zone I and zone IV radioactivity was rapidly removed from VLDL during the first 5 min after injection. However, although most of the zone IV radioactivity was recovered in LDL and HDL, only 12% of the label lost from zone I of VLDL was recovered in other lipoproteins, with the remainder presumably having been cleared from the plasma compartment. We have concluded that, during catabolism of rat VLDL apoprotein, there is a rapid transfer of small molecular weight peptides to both LDL and HDL. During the catabolic process, most of the VLDL is rapidly removed from the circulation, with only a small portion being transformed into LDL molecules.     

Deletion of the propeptide of apolipoprotein A-I reduces protein expression but stimulates effective conversion of prebeta-high density lipoprotein to alpha-high density lipoprotein

The properties of the mature and pro-forms of recombinant apolipoprotein A-I (apoA-I) were compared with those of apoA-I isolated from human plasma. When the synthesis and secretion of pro- and mature forms of apoA-I from a baculovirus/insect cell expression system were compared in parallel experiments, the amount of the pro-form of apoA-I synthesized and secreted was severalfold higher than that of the mature form of apoA-I. A comparison of the properties of the pro- and mature forms of recombinant apoA-I and human plasma apoA-I showed no difference between all three in their secondary structure, their ability to self-associate, lipid-binding capacity, lecithin: cholesterol acyltransferase activation, and binding to the phospholipid transfer protein. The properties of reconstituted high density lipoprotein (HDL) particles formed from the proteins and their ability to promote cholesterol and phospholipid efflux from human skin fibroblasts were also similar. However, their ability to bind to plasma HDL subfractions differed, because twice as much proapoA-I associated with prebeta(1)-HDL and prebeta(2)-HDL subfractions compared with both mature recombinant and plasma apoA-I. Correspondingly, the amount of proapoA-I in alpha-HDL subfractions, especially in alpha(1)-HDL and alpha(2)-HDL, was decreased. We conclude that while the propeptide of apoA-I is required for the effective synthesis and secretion of apoA-I, cleavage of this peptide is a requisite for the effective interconversion of HDL subfractions.     

Multifaceted Modelling of Complex Business Enterprises

We formalise and present a new generic multifaceted complex system approach for modelling complex business enterprises. Our method has a strong focus on integrating the various data types available in an enterprise which represent the diverse perspectives of various stakeholders. We explain the challenges faced and define a novel approach to converting diverse data types into usable Bayesian probability forms. The data types that can be integrated include historic data, survey data, and management planning data, expert knowledge and incomplete data. The structural complexities of the complex system modelling process, based on various decision contexts, are also explained along with a solution. This new application of complex system models as a management tool for decision making is demonstrated using a railway transport case study. The case study demonstrates how the new approach can be utilised to develop a customised decision support model for a specific enterprise. Various decision scenarios are also provided to illustrate the versatility of the decision model at different phases of enterprise operations such as planning and control.     

Pathways of absorption of retinal and retinoic acid in the rat

The chemical and anatomical pathways of absorption of dietary retinal, retinoic acid, and retinol were examined in rats containing lymph, bile, and duodenal cannulae. The experiments were designed to maintain physiological conditions to the greatest possible extent. In each rat an uninterrupted flow of bile into the duodenum was maintained by connecting the duodenal cannula to the bile duct of a second rat. Labeled vitamin A compounds were introduced into the duodenum in very small amounts (7-14 micrograms) in the form of a bile-lipid mixture resembling normal intestinal contents. Under these conditions, most (70-80%) of the radioactivity recovered after the feeding of labeled retinol or retinal was found in the lymph, predominantly in saturated retinyl esters. In contrast, 92-95% of the radioactivity recovered after the feeding of labeled retinoic acid was found in the bile, and was contained in a mixture of polar metabolites, most of them more polar than free retinoic acid. Two-thirds of the small amount of radioactivity found in lymph after retinoic acid-(14)C feeding was in the form of free retinoic acid. The results indicate that under normal conditions the major pathway of retinal absorption involves its reduction to retinol, which is then esterified and transported via the lymphatics in a manner similar to that of dietary retinol. A small proportion of retinal is apparently normally oxidized, and is then transported via the portal vein and excreted in the bile in a manner similar to that of dietary retinoic acid. The relative importance, in quantitative terms, of these two pathways of retinal metabolism can vary, depending on the status of the animal.