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N Domingo

Publications and source records attributed to N Domingo.

At least 37 records · Page 2Linked to original sources
PubMed1992

Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Evidence for the synthesis and secretion of APF--a bile lipid associated protein--by isolated rat hepatocytes.

Bile lipids are thought to be secreted in a lipoprotein complex in which they are associated with cholesterol and a protein called the anionic polypeptidic fraction (APF). APF is present in both bile and serum HDL. The association of APF with both bile and lipoprotein strongly suggests that hepatocytes may be responsible for the synthesis and secretion of this protein. In the present work we attempted to verify this by studying the incorporation of [14C]leucine into APF in isolated rat hepatocytes and by immunolocalization in cell cultures. Results obtained showed that synthesis of APF by cells follows the same kinetic pattern as albumin and that it was the third most abundant protein in the bile secretion. Immunolocalization confirmed that APF is synthesized in the endoplasmic reticulum of hepatocytes. This protein which appears to be rapidly secreted could be of great value for the specific detection of the lipids destined for bile secretion.

Albumins↗
PubMed1989

Detection and characterization of anionic polypeptide fraction binding sites in rat liver plasma membranes and cultured hepatocytes.

The binding of human 125I-labeled 'anionic polypeptidic fraction' (APF) to purified rat liver plasma membranes was studied. The dissociation constant for this binding was 3.0 micrograms protein/mg membrane protein. Binding was competitively inhibited by unlabeled human APF, but not by human LDL (low density lipoproteins). When unlabeled HDL3 was added, binding of labeled APF was competitively reduced to a level between that of unlabeled APF and unlabeled LDL. Experiments with cultured rat hepatocytes confirmed those obtained with liver membranes and suggested the presence in rat liver of saturable APF-binding sites which seem to be specific for APF. The physiologic significance of these APF binding sites is discussed in relation to the fate of cholesterol in the liver.

Animals↗
PubMed1988

Identification and localization of the apoprotein fraction of the bile lipoprotein complex in human gallstones.

The anionic polypeptidic fraction is the protein constituent of the bile lipoprotein complex. Double immunodiffusion and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies show that the anionic polypeptidic fraction is present in human gallstones. In terms of weight percentage, this protein accounts for 0.1% +/- 0.087 (n = 6) of the total weight of gallstones. Immunolocalization studies confirm the presence of the anionic polypeptidic fraction in human gallstones and suggest that this protein is preferentially associated with pigmented layers in gallstones. A speculative role for the anionic polypeptidic fraction in cholesterol nucleation is discussed.

Amino Acids↗
PubMed1988

Effect of bile anionic polypeptidic fraction on the fate of cholesterol carried by liposomes in the rat.

[14C]Cholesterol associated with liposomes with or without anionic polypeptidic fraction was administered intravenously to the rat. The cholesterol originated from liposomes including anionic polypeptidic fraction is secreted in bile much later, is stored in liver in higher quantity, and is metabolized into bile salts in lesser quantity during the 4 hr of experimentation than the cholesterol issued from liposomes exempt of anionic polypeptidic fraction. From these results it can be postulated that the cholesterol associated with liposomes containing anionic polypeptidic fraction might be directed in a particular liver pathway.

Animals↗
PubMed1987

Bile lipid secretion in isolated perfused rat liver. A model for metabolic studies.

Isolated perfused rat liver was used to study the effects of constant taurocholate perfusion, with or without the addition of phosphatidylcholine unilamellar vesicles, upon both the bile salt-dependent and bile salt-independent secretion of bile. Taurocholate introduction increased bile flow and normalized the bile lipid secretion by restoring the bile salt-dependent secretion. At a flow rate of 30 ml/min, the liver was perfused by a single-pass method. The perfusion medium contained 17.5 microM taurocholate with or without 5.83 microM phosphatidylcholine. In light of a recent quantitative dynamic concept on the interphase partition of lipids, it was calculated that more than 99% of the taurocholate reaches the liver as monomers and/or dimers. It was also deduced that the lipids were secreted in bile as small discoidal lipoprotein structures rather than unilamellar lipoproteic vesicles. During the course of the experiments (2 hr), the excellent criteria of viability of this model make it highly suitable for the investigation of hepatic metabolism. Furthermore, the addition of phosphatidylcholine unilamellar vesicles to the perfusate constitutes a potential vector for various liposoluble molecular species.

Animals↗
PubMed1987

Combined effects of chlorpromazine and high fat diet on lipid levels and enzyme activities in bile, liver and plasma of Wistar rats.

The effects of high fat diet and injection of chlorpromazine on bile lipid secretion were studied in the rats fed a control diet (C), a saturated fat, high cholesterol diet (S) and a polyunsaturated fat, high cholesterol diet (PU). As compared to controls, injection of chlorpromazine in the S and PU diet groups caused no appreciable change in the level of bile salts and bile phospholipids. Chlorpromazine did however enhance bile cholesterol, especially in the PU group, and lower secretion of lysosomal enzyme (beta-glucuronidase) into bile. Impairment of lysosomal enzyme secretion but not of bile lipid secretion suggests that the lysosomal activity is not directly involved in the bile secretion mechanism. These data point up the risks of using chlorpromazine therapy in association with a diet high in fat and cholesterol.

Animals↗
PubMed1986

A quantitative dynamic concept of the interphase partition of lipids: application to bile salt-lecithin-cholesterol mixed micelles.

A system is proposed for a quantitative classification of lipids, based on interphase partition coefficients. This system enables calculation of exchanges of lipid molecules between phases. The mass/volume chemical unit mol X cm-3, strictly derived from the CGS system, is used, thus simplifying mathematical relations. Applied to bile salt-lecithin-cholesterol mixed micelles, this dynamic concept gives new insight into the variations of physico-chemical parameters. Experimental results obtained with the glycodesoxycholate and the taurocholate show a striking difference in partition coefficients between aqueous and mixed bile salt-lecithin interfacial phases. A new model applying triangular co-ordinates to a bile salt-lecithin-cholesterol mixed lipid phase is described.

Bile Acids and Salts↗
PubMed1985

Purification of the human anionic polypeptide fraction of the apo-bile lipoprotein complex by zonal ultracentrifugation.

The two main proteic constituents of the human Apo-bile lipoprotein complex (BLC), i.e., the anionic polypeptide fraction (APF) and the IgA fragments, were separated by preparative zonal ultracentrifugation using a sucrose gradient containing 1.5 mM glycodesoxycholate. The purification of the APF was verified by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and immunology, and its amino acid composition then was determined. This procedure was used to obtain a polyclonal antiserum directed solely against the APF.

Amino Acids↗
PubMed1981

Interaction of immunoglobulins and lipids in human gallbladder bile.

The lipoprotein complex from human gallbladder bile was challenged with anti-IgA and anti-IgG antisera in order to determine whether the apolipoprotein complex isolated from the detergent-free form of bile lipoprotein contains IgA and IgG. The apolipoprotein complex indeed crossreacts with anti-IgA and anti-IgG. In addition, the interactions of IgA and IgG with lipids were studied by ultracentrifugation and gel chromatography to determine whether these interactions occur in human bile.

Bile↗
PubMed1979

The apoprotein fraction of the bile lipoprotein complex: isolation, partial characterization and phospholipid binding properties.

A bile apoprotein fraction (Apo BLC) was isolated by preparative isoelectric focusing (I.E.F.) from the detergent-free form of the bile lipoprotein complex (BLC). Analytical I.E.F. of Apo BLC yields a characteristic and reproducible pattern of two narrow acidic bands (pI 4,8-5,0). This apoprotein presents a strong tendency to undergo self-aggregation in aqueous buffer. A low molecular weight constituent of Apo BLC has been isolated after gel filtration, its mean Mw is estimated by SDS-PAGE at 7,500 daltons. The binding capacity of Apo BLC for phospholipids was investigated on dimyristoylphosphatidylcholine liposomes by gel filtration and zone electrophoresis. The resulting structures, larger than the original single-shelled vesicles, acquire and anodic electrophoretic mobility. Apo BLC has a weaker affinity for lysophosphatidylcholines: these phospholipids decrease the degree of aggregation of the apoprotein. These studies contribute additional data concerning the high affinity of Apo BLC for phosphatidylcholines, which are the major phospholipid constituents of bile. The discussion deals with the fact that association of Apo BLC with bile phosphatidylcholines may present some implications in the pathogeny of LpX and in the process of intestinal fat absorption.

Apolipoproteins↗
PubMed1978

Possible roles of bile lipids and colipase in lipase adsorption.

The adsorption isotherms of bile salts, phospholipids, and cholesterol were determined with siliconized glass beads. It was observed that the molar fractions of cholesterol, phospholipid, and bile polypeptide fractions increased simultaneously and considerably on the surface of the beads in comparison to the corresponding fractions found in bile. The composition of the adsorbed film is approximately 1 cholesterol: 2 phospholipid: 3 bile salt molecules. The performed complex of lipase, colipase, and bile lipids behaves as an entity which determines lipase adsorption. The modification of the interface quality of a lipid substrate by a detergent is not perse the reason for the lack of lipase adsorption. A model is proposed according to which lipolysis under physiological conditions would occur in two steps requiring two cofactors. Colipase would be necessary for the formation of the lipase-bile lipoprotein complex, and bile lipids would be required to direct the adsorption of this lipolytic entity toward the emulsified substrate.

Adsorption↗
PubMed1978

Inhibition of lipase adsorption at interfaces. Role of bile salt micelles and colipase.

The effects of bile salts and colipase on the adsorption of lipase at an interface were studied by hydrophobic affinity chromatography on phenyl- and octyl-Sepharose. In the absence of bile salts, lipase or colipase binds separately to the gel. This is unchanged in the presence of adsorbed bile salts, when one bile salt molecule is associated per hydrophobic ligand. The same data are obtained in the presence of monomeric bile salt solutions. In contrast, lipase adsorption is totally prevented in a micellar bile salt solution. These results favor the idea that the formation of a lipase-bile salt complex in solution is responsible for the lack of interfacial lipase adsorption.

Adsorption↗