RESUMEN
Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.
RESUMEN
Lipoprotein classes isolated from the plasma of two patients with apolipoprotein AI (apo AI) and apolipoprotein CIII (apo CIII) deficiency were characterized and compared with those of healthy, age- and sex-matched controls. The plasma triglyceride values for patients 1 and 2 were 31 and 51 mg/dl, respectively, and their cholesterol values were 130 and 122 mg/dl, respectively; the patients, however, had no measurable high density lipoprotein (HDL)-cholesterol. Analytic ultracentrifugation showed that patients' S degrees f 0-20 lipoproteins possess a single peak with S degrees f rates of 7.4 and 7.6 for patients 1 and 2, respectively, which is similar to that of the controls. The concentration of low density lipoprotein (LDL) (S degrees f 0-12) particles, although within normal range (331 and 343 mg/dl for patients 1 and 2, respectively), was 35% greater than that of controls. Intermediate density lipoproteins (IDL) and very low density lipoproteins (VLDL) (S degrees f 20-400) were extremely low in the patients. HDL in the patients had a calculated mass of 15.4 and 11.8 mg/dl for patients 1 and 2, respectively. No HDL could be detected by analytic ultracentrifugation, but polyacrylamide gradient gel electrophoresis (gge) revealed that patients possessed two major HDL subclasses: (HDL2b)gge at 11.0 nm and (HDL3b)gge at 7.8 nm. The major peak in the controls, (HDL3a)gge, was lacking in the patients. Gradient gel analysis of LDL indicated that patients' LDL possessed two peaks: a major one at 27 nm and a minor one at 26 nm. The electron microscopic structure of patients' lipoprotein fractions was indistinguishable from controls. Patients' HDL were spherical and contained a cholesteryl ester core, which suggests that lecithin/cholesterol acyltransferase was functional in the absence of apo AI. The effects of postprandial lipemia (100-g fat meal) were studied in patient 1. The major changes were the appearance of a 33-nm particle in the LDL density region of 1.036-1.041 g/ml and the presence of discoidal particles (12% of total particles) in the HDL region. The latter suggests that transformation of discs to spheres may be delayed in the patient. The simultaneous deficiency of apo AI and apo CIII suggests a dual defect in lipoprotein metabolism: one in triglyceride-rich lipoproteins and the other in HDL. The absence of apo CIII may result in accelerated catabolism of triglyceride-rich particles and an increased rate of LDL formation. Additionally, absence of apo CIII would favor rapid uptake of apo E-containing remnants by liver and peripheral cells. Excess cellular cholesterol would not be removed by the reverse cholesterol transport mechanism since HDL levels are exceedingly low and thus premature atherosclerosis occurs.
Asunto(s)
Apolipoproteínas A/deficiencia , Apolipoproteínas C/deficiencia , Arteriosclerosis/genética , Arteriosclerosis/fisiopatología , Colesterol/sangre , Grasas de la Dieta/metabolismo , Humanos , Lipoproteínas/sangre , Persona de Mediana Edad , Triglicéridos/sangreRESUMEN
We describe a child, the issue of phenotypically normal parents, who had fat malabsorption, both intestinal and hepatic steatosis, and serum cholesterol and triglyceride concentrations of 38 and 63 mg/dl, respectively. Lipoprotein electrophoresis, Ouchterlony double diffusion, and electron microscopy demonstrated that normal low density lipoproteins (LDL: 1.006 less than rho less than 1.063 g/ml) were absent. Lipoprotein particles in the rho less than 1.006-g/ml fraction were triglyceride rich, very large (93.2 +/- 35.1 nm), and contained the B-48 but not the B-100 apoprotein; both species of apolipoprotein (apo) B were found in the parents' lipoproteins. These chylomicrons and chylomicron remnants were present even in the patient's fasting plasma, which suggested prolonged dietary fat absorption. Plasma levels of high density lipoprotein lipids and proteins were low, and the phosphatidylcholine/sphingomyelin ratio was reduced as in typical abetalipoproteinemia. The monosialylated form of apo C-III was not identified on polyacrylamide gel electrophoresis, which suggested that this protein was elaborated only with very low density lipoproteins (VLDL). A radioimmunoassay for apo B employing a polyclonal antisera to plasma LDL gave apparent plasma apo B levels of 0.6, 66, and 57 mg/dl in the patient and his father and mother, respectively. The displacement curve generated by the parents' VLDL and LDL did not did not differ from control lipoproteins. The patient's chylomicron-chylomicron remnant fraction displaced normal LDL over the entire radioimmunoassay range, but the efficiency of displacement was strikingly less than with B-100 containing lipoproteins. If the patient's B-48 protein is not qualitatively abnormal, these results confirm very limited immunochemical cross-reactivity between at least one major epitope on B-100 and the epitopes expressed on B-48. The apo B defect in this patient appears to be recessive. It abolishes B-100 production and may additionally limit the formation of B-48.
Asunto(s)
Apolipoproteínas B/biosíntesis , Apolipoproteínas B/deficiencia , Síndromes de Malabsorción/metabolismo , Adulto , Apolipoproteína B-100 , Apolipoproteína B-48 , Biopsia , HDL-Colesterol/sangre , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Lactante , Intestino Delgado/patología , Lipoproteínas LDL/sangre , Hígado/patología , Masculino , Microscopía ElectrónicaRESUMEN
A unique kindred with premature cardiovascular disease, tubo-eruptive xanthomas, and type III hyperlipoproteinemia (HLP) associated with familial apolipoprotein (apo) E deficiency was examined. Homozygotes (n = 4) had marked increases in cholesterol-rich very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), which could be effectively lowered with diet and medication (niacin, clofibrate). Homozygotes had only trace amounts of plasma apoE, and accumulations of apoB-48 and apoA-IV in VLDL, IDL, and low density lipoproteins. Radioiodinated VLDL apoB and apoE kinetic studies revealed that the homozygous proband had markedly retarded fractional catabolism of VLDL apoB-100, apoB-48 and plasma apoE, as well as an extremely low apoE synthesis rate as compared to normals. Obligate heterozygotes (n = 10) generally had normal plasma lipids and mean plasma apoE concentrations that were 42% of normal. The data indicate that homozygous familial apoE deficiency is a cause of type III HLP, is associated with markedly decreased apoE production, and that apoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.
Asunto(s)
Apolipoproteínas E/deficiencia , Hiperlipoproteinemia Tipo III/genética , Adolescente , Adulto , Anciano , Niño , Colesterol/sangre , Femenino , Homocigoto , Humanos , Hiperlipoproteinemia Tipo III/sangre , Hiperlipoproteinemia Tipo III/complicaciones , Lipoproteínas/sangre , Masculino , Persona de Mediana Edad , Linaje , Fosfolípidos/sangre , Valores de Referencia , Enfermedades de la Piel/etiología , Triglicéridos/sangre , Xantomatosis/etiologíaRESUMEN
The status of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP) in seven human glioma cell lines was evaluated to extend our knowledge of human glioblastoma multiforme tumor metabolism for future drug design. Cell lines SF-767, SF-763, A-172, U-87 MG, U-251 MG, U-343 MG, and SF-539 were used. Binding of 125I-labeled LDL to these cells at 4 degrees C was carried out to determine the number of LDL receptors on cells and the affinity of LDL for these receptors. The content of LRP was measured by immunoblotting. The presence of specific saturable LDL receptors was proven in six of the cell lines investigated. SF-767 cells revealed high-affinity LDL binding (equilibrium dissociation constant, Kd = 7 nM) and maximum binding capacity approximating 300,000 receptors/cell. Most of the remaining cell lines had relatively lower affinity (Kd = 38-62 nM) but also had very high numbers of receptors (128,000-950,000/cell). All cell lines exhibited LRP, but the expression was variable. The cell lines SF-539, U-87 MG, and U-343 MG were particularly rich in this protein. The data suggest that glioblastoma cells have high numbers of LDL receptors; however, there is considerable variation in binding affinity. Overall, this finding suggests that LDL receptors on glioblastoma cells could potentially be useful for targeting antitumor agents. LRP, a multifunctional receptor expressed on glioblastoma cells, also has the possibility for serving as a therapeutic target.
Asunto(s)
Glioblastoma/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de LDL/metabolismo , Sitios de Unión , Western Blotting , Diseño de Fármacos , Expresión Génica , Glioblastoma/patología , Humanos , Lipoproteínas LDL/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Termodinámica , Células Tumorales CultivadasRESUMEN
The kinetics and specificity of the interaction of nascent HepG2 LDL with the HepG2 LDL receptor were examined. 125I-Labeled HepG2 LDL and plasma LDL were bound by HepG2 cells in a specific and saturable manner at 4 degrees C. Competition studies with HepG2 LDL and plasma LDL indicated that both ligands bound to the same receptor. Scatchard analyses of the specific 4 degrees C-binding data revealed a Kd of 75 nM for HepG2 LDL and a Kd of 30 nM for plasma LDL suggesting that HepG2 LDL bind less efficiently to the HepG2 LDL receptor than plasma LDL. Binding, internalization and degradation studies carried out at 37 degrees C indicated that HepG2 cells are capable of catabolizing their own nascent LDL; however, under normal experimental conditions re-uptake of nascent LDL is quantitatively insignificant.
Asunto(s)
Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Unión Competitiva , Línea Celular/metabolismo , Humanos , Radioisótopos de Yodo , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Tamaño de la PartículaRESUMEN
A quantitative assessment of the distribution and state of microfilament-related proteins in the heterocellular fundic gastric epithelium was carried out. Actin content, as determined by the DNAase inhibition assay, ranged from 29 to 42 micrograms/mg of tissue protein, depending upon the tissue source. About 60% of the total actin existed in fresh tissue in the polymeric form (F-actin). The distribution of fluorescent-labelled phallicidin demonstrated that F-actin was concentrated predominantly in the acid-secreting oxyntic cells. The patterns of distribution corresponded to the location of the numerous elongated apical surface microvilli seen within oxyntic cell canaliculi. In the isolated apical membrane, actin represented about 10% of the total protein and was present entirely as F-actin. After mild treatment of membranes with Triton X-100, filaments could be readily visualized by negative staining. More extensive Triton X-100 extraction solubilized intrinsic membrane protein and yielded an insoluble residue highly enriched in actin and containing several additional polypeptides. Homogenization and fractionation of the gastric epithelium in low ionic strength media led to the depolymerization of a significant proportion of the tissue actin which was recovered in the homogenate supernatant. When purified by DNAase affinity chromatography, this gastric actin displayed structural and functional properties similar to muscle actin. Incubation of the homogenate supernatant in KCl-Mg2+ induced the formation of actin-rich gels. The gels contained myosin as well as several other peptides that may be actin-binding proteins.
Asunto(s)
Actinas/análisis , Mucosa Gástrica/análisis , Actinas/aislamiento & purificación , Adenosina Trifosfatasas/análisis , Animales , Citoesqueleto/ultraestructura , Geles , Miosinas/análisis , Conejos , PorcinosRESUMEN
Gradient gel electrophoresis in conjunction with automated densitometry was applied to the identification and estimation of subpopulations of high-density lipoproteins (HDL) in the ultracentrifugal d less than or equal to 1.200 fraction from human plasma. The frequency distribution of relative migration distances (RF values) of subpopulation peaks in HDL patterns of a group (n = 194) of human subjects showed five apparent maxima: two in the RF range associated with the HDL2 subclass, and three in the RF range of the HDL3 subclass. HDL within RF intervals bounding these maxima were designated (HDL2b)gge, (HDL2a)gge, (HDL3a)gge, (HDL3b)gge and (HDL3c)gge and were shown to correspond approximately to material determined by analytic ultracentrifugation within the HDL2b, HDL2a and HDL3 components. Material represented by the HDL2a component, as resolved by three-component analysis of the ultracentrifugal Schlieren pattern, was found by gradient gel electrophoresis to be polydisperse in particle size. Mean hydrated densities and particle sizes of HDL corresponding to those with RF values of the frequency maxima were: 1.085 g/ml and 10.57 nm in the (HDL2b)gge; 1.115 g/ml and 9.16 nm in the (HDL2a)gge; 1.136 g/ml and 8.44 nm in the (HDL3a)gge; 1.154 g/ml and 7.97 nm in the (HDL3b)gge; and 1.171 g/ml and 7.62 nm in the (HDL3c)gge. The mean hydrated density values of the subpopulations within the (HDL3a)gge and (HDL3b)gge were comparable to those of the HDL3L and HDL3D components recently characterized by zonal ultracentrifugation. High order and statistically significant correlations between densitometric scans of the (HDL2b)gge, (HDL2a)gge and (HDL3)gge material, as obtained from gradient gels, and plasma concentrations of the HDL2b, HDL2a and HDL3 components, as obtained from analytic ultracentrifugation, were demonstrated.
Asunto(s)
Lipoproteínas HDL/sangre , Adulto , Autoanálisis , Centrifugación por Gradiente de Densidad/métodos , Densitometría , Electroforesis/métodos , Humanos , Lipoproteínas HDL/aislamiento & purificación , Microscopía Electrónica , Persona de Mediana Edad , Peso MolecularRESUMEN
The major bovine HDL subfraction, fraction I-HDL, was incubated with increasing amounts of dimyristoylphosphatidylcholine (DMPC). HDL size, as determined by gradient gel electrophoresis and electron microscopy, increased with increasing HDL-phospholipid to DMPC mole ratios. Control fraction I-HDL were spherical, hexagonally-packing particles with a peak on gradient gel electrophoresis at 12.3 +/- 0.1 nm; at a ratio of 1:0.5, larger, mainly spherical particles with a peak at 12.9 +/- 0.08 nm were formed. At a ratio of 1:1, occasional square-shaped particles were seen by electron microscopy; by gradient gel analysis, the mean diameter of the HDL-product increased to 13.7 +/- 0.1 nm. At the 1:2 ratio, extensive domains of square-packing particles were noted; the major size peak of this product was 14.6 +/- 0.08 nm. In all incubations with DMPC, a small 9.4 +/- 0.08 nm product was formed; it was most pronounced at the 1:2 ratio. The large, less dense particles generated by incubation contained apolipoprotein A-I and small molecular weight proteins. The 9.4 nm product contained only apolipoprotein A-I. The less dense product formed during incubation at the 1:2 ratio had a decreased protein-to-lipid ratio relative to control HDL and a 2-fold increase in percent phospholipid. At a 1:2 ratio, incorporation of DMPC into fraction I-HDL results in the loss of one molecule of apolipoprotein A-I; the resultant particle is a stable phospholipid-rich and protein-poor HDL which has a square-packing geometry. These phospholipid-laden HDL are morphologically similar to lipoproteins isolated from interstitial fluid or from plasma of abetalipoproteinemic patients. Our data suggest that the unusual morphological properties of the latter biologically formed particles may be due to increases in the polar lipid contents, and concomitant decreases in surface protein.
Asunto(s)
Abetalipoproteinemia/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Lipoproteínas HDL/metabolismo , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Humanos , Linfa/metabolismo , Sustancias Macromoleculares , Microscopía Electrónica , Peso MolecularRESUMEN
Cigarette smoking is associated with an increased risk of premature atherosclerosis. The underlying mechanisms responsible for this association are unknown. Recent work from this laboratory has shown that ex vivo exposure to plasma to gas-phase cigarette smoke (CS) produces a rapid inhibition of lecithin-cholesterol acyltransferase (LCAT) activity and crosslinking of HDL-apolipoproteins. The goal of the present study was to investigate the mechanism(s) by which CS inhibited LCAT and modified HDL. When dialyzed human plasma (12 ml) was exposed to the gas-phase of an equivalent of 1/8 of a cigarette (one 'puff') at 15 min intervals for 3 h, LCAT activity was reduced by 76 +/- 1% compared to controls; supplementation of plasma with glutathione produced a dose-dependent protection of LCAT activity where at the highest concentration (1 mM) 78% protection was observed. A similar protection was obtained with N-acetyl cysteine (1 mM). In addition to LCAT inhibition, HDL-apolipoproteins were crosslinked after 3 h exposure of plasma to CS; crosslinking was reduced by the addition of either glutathione or N-acetyl cysteine to plasma. The amino compounds N-acetyl lysine, N-acetyl arginine, and aminoguanidine failed to protect LCAT and HDL indicating a specificity with regard to the ability of free thiols to buffer the deleterious components of CS which inhibited LCAT and crosslinked HDL-apolipoproteins. Since LCAT contains two free cysteine residues (Cys-31 and -184) near the active site of the enzyme, we tested whether pretreatment of plasma with the reversible sulfhydryl modifying compound, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), could protect LCAT from CS-induced inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Nicotiana , Fosfatidilcolina-Esterol O-Aciltransferasa/antagonistas & inhibidores , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Plantas Tóxicas , Humo , Compuestos de Sulfhidrilo/química , Acetilcisteína/farmacología , Sitios de Unión , Reactivos de Enlaces Cruzados , Ácido Ditionitrobenzoico/farmacología , Glutatión/farmacología , Humanos , Lipoproteínas HDL/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fumar/efectos adversosRESUMEN
Complexes of apolipoprotein A-II and egg yolk phosphatidylcholine were prepared in mixtures of different composition in the absence and presence of sodium cholate. By gradient gel electrophoresis, complex preparations were polydisperse and particle size distributions were influenced by the composition of the reconstitution mixture. Complexes generally exhibited a discoidal morphology by electron microscopy, but showed increased formation of vesicular complexes at elevated levels of egg yolk PC in the mixtures. By chemical crosslinking, complexes formed in the absence of cholate were shown to consist primarily of discoidal species with three apolipoprotein A-II molecules per particle in the mixtures investigated; complexes formed in the presence of cholate included species ranging from three to five apolipoprotein A-II per particle. The number of apolipoprotein A-II per particle and the sizes of the complexes, prepared in cholate, increased with increase of egg yolk PC in the reconstitution mixture. Relative to the particle size distribution of discoidal complexes formed in the absence of cholate, those prepared in cholate showed a distribution shifted to larger particle sizes. Complexes of similar particle size distribution formed in the presence or absence of cholate showed similar physical-chemical properties. Discoidal complexes with the same number of apolipoprotein A-II per particle but of different size and composition were observed, suggesting the possibility of some conformational adaptation of apolipoprotein A-II leading to stabilization of egg yolk PC bilayers of different diameter. Properties of particle size distributions of discoidal complexes prepared in cholate of apolipoprotein A-II and egg yolk PC were compared with those of complexes of apolipoprotein A-I previously reported (Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) Biochim. Biophys. Acta 750, 353-364).
Asunto(s)
Apolipoproteínas A/aislamiento & purificación , Ácidos Cólicos , Yema de Huevo/análisis , Fosfatidilcolinas/aislamiento & purificación , Apolipoproteína A-II , Ácido Cólico , Cromatografía en Gel , Densitometría , Electroforesis en Gel de Poliacrilamida , Tamaño de la Partícula , Unión ProteicaRESUMEN
This study shows that, in control and transgenic mice, there is a parallel increase in LCAT activity and plasma apo A-I concentrations during postnatal development. We also demonstrate that human apo A-I is a much more efficient activator (1.6-fold) of mouse LCAT activity than mouse apo A-I. We propose that the differences in amino acid sequence between human and mouse apo A-I may account for the higher LCAT activity with human apo A-I.
Asunto(s)
Apolipoproteína A-I/farmacología , Esterol O-Aciltransferasa/metabolismo , Animales , Activación Enzimática , Humanos , Cinética , Ratones , Ratones Transgénicos , Especificidad de la EspecieRESUMEN
The influence of sodium oleate (oleate) on complexing of apolipoprotein A-I (apo A-I) with egg yolk phosphatidylcholine (EYPC) was evaluated. Without the use of additional detergent such as sodium cholate, oleate facilitates formation of a single complex of unique stoichiometry, approx. 76:2:20, molar ratio EYPC/apo A-I/oleate, and mean size 7.4 nm with round to ellipsoidal morphology. Near complete reassembly of apo A-I into the complex occurs when the stoichiometry of the mixture approximates that of the complex itself. With increasing content of EYPC in the mixture, the same complex is formed but in decreasing yield; larger complexes are not formed. The rate of complex formation decreases with increase of EYPC in the mixture. Reduction of pH in the reassembly mixture from 8.0 to 5.4 results in a marked reduction in complex formation indicating that ionized oleate facilitates lipidation. Removal of oleate by interaction of the complex with fatty acid-free human serum albumin does not degrade the complex. Incorporation of increasing amounts of unesterified cholesterol into the EYPC-sonicate progressively inhibits oleate-facilitated complex formation. This study shows that oleate, a physiologically relevant lipolysis-derived product, facilitates reassembly of apo A-I with EYPC and promotes formation of a small lipid-poor particle similar to that observed in nascent HDL and during in vivo or in vitro lipolysis of triacylglycerol-rich lipoproteins in the presence of HDL.
Asunto(s)
Apolipoproteína A-I/química , Ácido Oléico , Ácidos Oléicos/farmacología , Fosfatidilcolinas/química , Yema de Huevo , Lipólisis , Desnaturalización ProteicaRESUMEN
Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.
Asunto(s)
Apolipoproteínas B/química , Carcinoma Hepatocelular/metabolismo , Lipoproteínas LDL/sangre , Apolipoproteínas B/sangre , Carbohidratos/sangre , Electroforesis en Gel de Poliacrilamida , Endopeptidasas , Glicosilación , Humanos , Hidrólisis , Mapeo Peptídico , Receptores de LDL/metabolismo , Células Tumorales CultivadasRESUMEN
The effects of lipid transfers and hepatic lipase (HL) on the concentration, composition, particle size distribution and morphology of high density lipoproteins (HDL) have been investigated. Human plasma supplemented with additional very low density lipoproteins (VLDL), cholesteryl ester transfer protein (CETP) and HL has been incubated at 37 degrees C for up to 8 h. The HDL became depleted of cholesteryl esters and reduced in particle size. Within 2 h of such incubation they had also lost about 30% of their apo A-I. However, with extension of the incubations beyond 2 h, the apo A-I returned progressively to the HDL fraction until, after 8 h, the concentration of apo A-I in HDL was identical to that in non-incubated samples. This return of apo A-I to the HDL density range was accompanied by a progressive appearance in electron micrographs of discoidal HDL particles. Thus, the depletion of the core lipid content and the reduction in particle size of HDL promoted by lipid transfers and HL activity in vitro is accompanied by a shedding of apo A-I which forms the nucleus of new discoidal HDL particles. The potential physiological importance of such a process is considerable.
Asunto(s)
Apolipoproteína A-I/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas , Lipasa/metabolismo , Lipoproteínas HDL/metabolismo , Hígado/enzimología , Adulto , Proteínas de Transferencia de Ésteres de Colesterol , Humanos , Cinética , Lipoproteínas HDL/ultraestructura , Masculino , Microscopía Electrónica , Persona de Mediana EdadRESUMEN
The interaction of HDL2b, a major subclass (d = 1.063 - 1.100 g/ml) of human plasma high-density lipoproteins, with discoidal complexes composed of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (weight ratio, DMPC/apolipoprotein A-I (2.1 - 2.5:1); dimensions, 10.0 x 4.4 nm) was investigated. Incubation at 37 degrees C for 4.5 h of HDL2b with discoidal complexes resulted in a transfer of DMPC from the discoidal complexes to the HDL2b, a release of lipid-free apolipoprotein A-I from the discoidal complexes during such transfer, and a dissociation of some apolipoprotein A-I from the HDL2b surface. The number of discoidal complexes degraded during interaction with HDL2b depended on the initial molar ratio of HDL2b to discoidal complexes. Approximately one molecule of HDL2b was required for the degradation of one discoidal complex particle, and the degradation process appeared limited by the capacity of the HDL2b for uptake of DMPC. Degradation of discoidal complexes was also observed when human plasma LDL (d = 1.006-1.063 g/ml) was substituted for HDL2b in the interaction mixture.
Asunto(s)
Apolipoproteínas , Lipoproteínas HDL/sangre , Fosfatidilcolinas , Adulto , Apolipoproteína A-I , Dimiristoilfosfatidilcolina , Electroforesis , Humanos , Microscopía Electrónica , UltracentrifugaciónRESUMEN
Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.
Asunto(s)
Apolipoproteínas , Colesterol , Fosfatidilcolinas , Apolipoproteína A-I , Cromatografía en Gel , Yema de Huevo , Femenino , Humanos , Microscopía Electrónica , Unión ProteicaRESUMEN
Density gradient ultracentrifugation of human serum high density lipoproteins (HDL) from both normolipemic males and females results in a distribution of HDL concentration versus subfraction hydrated density which has three maxima. Gradient gel electrophoresis of total HDL is characterized by three banding maxima, the positions of which suggest the presence of three particle size ranges: I. 10.8-12.0 nm, II. 9.7-10.7 nm, and III. 8.5-9.6 nm. Gradient gel electrophoresis of density gradient subfractions established an inverse relationship between particle size and particle hydrated density which was corroborated by electron microscopy and analytic ultracentrifugation. Comparison of male HDL from size ranges I, II, and III with female HDL from the same size ranges showed only small differences in the mean value of the peak F degrees 1.20 rate, size, molecular weight, protein weight percent, and weight protein/weight phospholipid. Major differences between males and females were seen in the relative amounts of HDL in density gradient subfractions 1-3 (size range I material) and 11-12 (size range III material); the percent total HDL in the group of subfractions 1-3 was greatly increased in female HDL while that of the group of subfractions 11-12 was increased in the male HDL. These studies indicate the presence of at least three major components in HDL instead of two (HDL2 and HDL3) and that peak F degrees 1.20 rate differences in HDL schlieren patterns between males and females are a function of the relative levels of these three components.
Asunto(s)
Lipoproteínas HDL , Centrifugación por Gradiente de Densidad , Femenino , Humanos , Lipoproteínas HDL/sangre , Masculino , Microscopía Electrónica , Peso Molecular , Conformación Proteica , Factores Sexuales , UltracentrifugaciónRESUMEN
Incubation (24 h, 37 degrees C) of discoidal complexes of phosphatidylcholine and apolipoprotein A-I (molar ratio 95 +/- 10 egg yolk phosphatidylcholine-apolipoprotein A-I; 10.5 X 4.0 nm, long X short dimension; designated, class 3 complexes) with the ultracentrifugal d greater than 1.21 g/ml fraction transformed the discoidal complexes to a small product with apparent mean hydrated and nonhydrated diameter of 7.8 and 6.6 nm, respectively. Formation of the small product was associated with marked reduction in phosphatidylcholine-apolipoprotein AI molar ratio of the complexes (on average from 95:1 to 45:1). Phospholipase A2 activity of lecithin:cholesterol acyltransferase participated in the depletion process, as evidenced by production of unesterified fatty acids. In the presence of the d greater than 1.21 g/ml fraction or partially purified lecithin:cholesterol acyltransferase and a source of unesterified cholesterol, the small product could be transformed to a core-containing (cholesteryl ester) round product with a hydrated and nonhydrated diameter of 8.6 and 7.5 nm, respectively. By means of cross-linking with dimethylsuberimidate, the protein moiety of the small product was shown to contain primarily two apolipoprotein A-I molecules per particle, while the large product contained three apolipoprotein A-I molecules per particle. The increase in number of apolipoprotein A-I molecules per particle during transformation of the small to the large product appeared to result from fusion of the small particles during core build-up and release of excess apolipoprotein A-I from the fusion product. The results obtained with the model complexes were consistent for the most part with recent observations (Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984) J. Lipid Res. 25, 269-282) on the transformation, by lecithin:cholesterol acyltransferase, of the small spherical high-density lipoproteins of patients with familial lecithin:cholesterol acyltransferase deficiency.
Asunto(s)
Apolipoproteínas A/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolinas/metabolismo , Esterol O-Aciltransferasa/metabolismo , Sustancias Macromoleculares , Peso MolecularRESUMEN
Apolipoprotein A-I is a major secretory product of the human hepatoma cell line, Hep G2; approx. 70% of apolipoprotein A-I was separated from the medium as lipid-poor apolipoprotein A-I in the d greater than 1.21 g/ml fraction while 30% was associated with high-density lipoproteins (HDL) of d 1.063-1.21 g/ml. The lipid-poor apolipoprotein A-I contains 50% proapolipoprotein A-I which is similar to the isoform distribution in Hep G2 preformed HDL. We tested the ability of lipid-poor apolipoprotein A-I from Hep G2 to form complexes with dimyristoylphosphatidylcholine (DMPC) vesicles at DMPC/apolipoprotein A-I molar ratios of 100:1 and 300:1. Lipid-poor apolipoprotein A-I was recovered in complex form while at a 300:1 ratio, 68.8 +/- 6.3% was recovered. On electron microscopy, the former complexes were small discs 16.9 nm +/- 4.5 S.D. in diameter while the latter were larger discs 21.4 +/- 4.4 nm diameter. Non-denaturing gradient gel electrophoresis of complexes formed at a 100:1 ratio had a peak in the region corresponding to 9.64 +/- 0.08 nm; these particles possessed two apolipoprotein A-I molecules. At the higher ratio, 300:1, two distinct complexes were identifiable, one which banded in the 9.7 nm region and the other in the 16.9-18.7 nm region. The former particles contained two molecules of apolipoprotein A-I and the latter, three molecules. This study demonstrates that lipid-poor apolipoprotein A-I which is rich in more basic isoforms forms discrete lipoprotein complexes similar to those formed by mature apolipoprotein A-I. It is further suggested that, under the appropriate conditions, precursor or nascent HDL may be assembled extracellularly.