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1.
E M Manevich M A Martynova G I Muzya E L Vodovozova J G Molotkovsky L D Bergelson 《Biochimica et biophysica acta》1988,963(2):302-310
The interaction of human serum low-density lipoproteins (LDL) with various types of prostaglandins (PG) was studied using equilibrium dialysis, steady-state fluorescence polarization spectroscopy and photolabeling methods. Low concentrations (10(-13)-10(-9) M) of PGE1 and PGF2 alpha were shown to induce specific rearrangements of the lipids on the LDL surface, whereas the closely related PGE2 and PGF1 alpha had no effect. With fluorescent labeled LDL, the PGE1-induced changes of the steady-state fluorescence polarization (P) were shown to be time- and concentration-dependent, saturable and reversible. However, equilibrium dialysis revealed a very low binding capacity of LDL for PGE1 (approx. 1 prostaglandin molecule per 600 LDL particles). Approximately the same PGE1 concentration was sufficient to cause maximal changes of P, to enhance the binding to apolipoprotein B of a photoreactive sphingomyelin analogue inserted into the LDL surface and to alter the thermal phase behavior of the LDL surface lipids. It is proposed that the LDL surface rearrangement caused by prostaglandins is due to the interaction of prostaglandins with apolipoprotein B, resulting in formation of short-lived complexes. The mechanism of this interaction is discussed in terms of the non-equilibrium ligand-receptor interaction model proposed earlier to explain the interaction of prostaglandins with high-density lipoproteins (Bergelson, L.D. et al. (1987) Biochim. Biophys. Acta 921, 182-190). It is suggested that direct prostaglandin-lipoprotein interactions may play a role in the homeostasis of cholesterol. 相似文献
2.
Differential uptake of proteoglycan-selected subfractions of low density lipoprotein by human macrophages 总被引:7,自引:0,他引:7
E Hurt-Camejo G Camejo B Rosengren F Lopez O Wiklund G Bondjers 《Journal of lipid research》1990,31(8):1387-1398
Macrophages and arterial chondroitin sulfate proteoglycans (CSPG) are probably associated with extracellular and intracellular lipoprotein deposition during atherogenesis. We found that human arterial CSPG can be used to select subclasses from low density lipoprotein (LDL) with different structural properties and capacities to interact with human monocyte-derived macrophages (HMDM). Four subclasses, LDL(PG)1 to LDL(PG)4, in order of decreasing CSPG-complexing capacity, were prepared and characterized in terms of their ability to interact with HMDM. The LDL subclasses with highest avidity for CSPG, LDL(PG)1 and LDL(PG)2, were bound, internalized, and degraded more efficiently than those of lower avidity for CSPG. From LDL(PG)1 to LDL(PG)4, the gradual decrease in uptake by HMDM and decreasing avidity for CSPG were associated with a gradual decrease in isoelectric point (from 5.93 to 5.68) and an augmented ratio of surface polar lipid to core nonpolar components (from 0.35 to 0.54). Competition experiments indicated that the proteoglycan-selected subfractions shared the binding sites and uptake mechanisms of native LDL. The results suggest the existence of a structurally related gradation in the avidity of LDL subpopulations for cells and matrix components. The presence within LDL subpopulations of a differential capacity to interact with intimal extracellular and cellular elements could be associated with a similar heterogeneity in their atherogenic potential. 相似文献
3.
Bancells C Benítez S Jauhiainen M Ordóñez-Llanos J Kovanen PT Villegas S Sánchez-Quesada JL Oörni K 《Journal of lipid research》2009,50(3):446-455
Electronegative LDL [LDL(-)] is an atherogenic subfraction of plasma LDL that has increased apolipoprotein E (apoE) and apoC-III content, high density, and increased susceptibility to aggregation. These characteristics suggest that LDL(-) could bind to proteoglycans (PGs); therefore, our aim was to evaluate its affinity to PGs. Binding of LDL(-) and native LDL [LDL(+)] to human aortic PGs was determined by precipitation of LDL-glycosaminoglycan complexes, LDL incubation in PG-coated microtiter wells, and affinity chromatography on PG column. All methods showed that LDL(-) had higher binding affinity to PGs than did LDL(+). PG capacity to bind LDL(-) was increased approximately 4-fold compared with LDL(+) in precipitation and microtiter assays. Chromatography on PG column showed LDL(-) to consist of two subpopulations, one with higher and one with lower PG binding affinity than LDL(+). Unexpectedly, the lower PG affinity subpopulation had increased apoE and apoC-III content. In contrast, the high PG affinity subpopulation presented phospholipase C (PLC)-like activity and increased aggregation. These results suggest that PLC-like activity could alter LDL lipid composition, thereby promoting particle aggregation and binding to PGs. This propensity of a subpopulation of LDL(-) to bind to PGs could facilitate its retention in the extracellular matrix of arterial intima and contribute to atherosclerosis progression. 相似文献
4.
Bancells C Benítez S Ordóñez-Llanos J Öörni K Kovanen PT Milne RW Sánchez-Quesada JL 《The Journal of biological chemistry》2011,286(2):1125-1133
Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-). 相似文献
5.
Previous studies using cynomolgus monkeys have shown that isocaloric substitution of dietary fish oil for lard reduced the in vitro binding of plasma low density lipoproteins (LDL) to arterial proteoglycans (PG) (Edwards, I.J., A.K. Gebre, W. D. Wagner, and J. S. Parks. 1991. Arterioscler. Thromb., 11: 1778-1785). The purpose of the present study was to determine whether all LDL subfractions were equally affected by the type of dietary fat with regard to PG binding and to identify compositional changes in LDL subfractions that might relate to the differential in PG binding. Two groups of cynomolgus monkeys (n = 5 each) were fed atherogenic diets (40% calories as fat; 0.26 mg cholesterol/kcal) containing 20% of calories as egg yolk and 20% as either lard or menhaden fish oil. LDL were isolated from plasma by ultracentrifugation and size exclusion chromatography and subfractionated by density gradient centrifugation. Three density ranges of LDL subfractions were collected from the gradients for determination of chemical composition, apoE and apoB content by ELISA, and binding to arterial PG in vitro. The d 1.015-1.025 g/ml subfraction contained 39 +/- 8% of the LDL cholesterol in the lard group but only 7 +/- 3% for the fish oil group. Values for cholesterol distribution were opposite for the d 1.035-1.045 g/ml subfraction, 8 +/- 1% versus 41 +/- 8%, respectively. Similar trends were noted for the distribution of apoB. For the lard group, LDL binding to arterial PG increased with decreasing density (i.e., increasing size) of the subfractions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Three novel partial deletions of the low-density lipoprotein (LDL) receptor gene in familial hypercholesterolemia 总被引:2,自引:2,他引:0
Kimiko Yamakawa Kohki Takada Hisako Yanagi Shigeru Tsuchiya Koichi Kawai Soichi Nakagawa Goro Kajiyama Hideo Hamaguchi 《Human genetics》1989,82(4):317-321
Summary The low-density lipoprotein (LDL) receptor genes from 18 unrelated Japanese heterozygotes and 1 homozygote with classical familial hypercholesterolemia were analyzed by Southern blot hybridization using fragments of the human LDL receptor cDNA as probes. Four different deletion mutations were detected among 20 mutant LDL receptor genes (20%); they were characterized by restriction mapping. None of these mutations has previously been reported in Caucasian patients with FH: three of the mutations were novel and one was similar to the detetion mutation of FH-Tonami described previously in Japanese patients. In three of the four deletion mutations, the rearrangements were related to intron 15 of the LDL receptor gene, in which many Alu sequences exist. The data suggest that a wide range of molecular heterogeneity exists even in major rearrangements resulting in deletions in the LDL receptor gene. The data also support the hypothesis that there are preferential sites within the LDL receptor gene for major rearrangements resulting in deletions. The possibility that a higher frequency of deletion mutations occurs in classical FH than previously suspected is discussed. 相似文献
7.
Study of the interaction between plasma proteoglycans and LDL by means of fluorescence spectroscopy]
F Siddi A Senes R Coinu M Formato G M Cherchi 《Bollettino della Società italiana di biologia sperimentale》1992,68(11):655-661
The relevance of the interaction between LDL and PGs in the development of atherosclerotic processes is well known. However, the exact nature of the interaction and the consequent structural and/or conformational modifications of the lipoprotein remain to be clarified. It has been demonstrated that after this interaction the LDL particle is not recognized by specific cellular receptors and enters the scavenger pathway operating in different cell types. These effects have been shown by using aortic PGs, but PGs are also present in the plasma compartment and may interact constantly with LDL, taking part in the regulation of lipid metabolism. In order to assess the capability of plasma PGs to induce LDL modifications, we investigated their interactions by studying the changes in the organizational parameters of LDL by fluorescence spectroscopy. Plasma PGs were isolated by DEAE Sephacel ion exchange chromatography and Sephacryl S300 gel filtration in two different families: a low-charge PG and a high-charge PG. Human LDL was prepared from plasma of normolipemic donors by ultracentrifugal flotation between 1.025-1.045 g/ml. Steady-state anisotropy measures were obtained by analyzing the rotational diffusion rate of DPH after incubation of LDL with plasma PGs in a physiological ratio. In our experimental conditions, LDL incubation with plasma low-charge PG did not modify DPH fluorescence anisotropy, whereas LDL treatment with highly charged PGs induced a marked decrease of this parameter, suggesting a significant effect on LDL microviscosity. The data show that both the charge and the GAG composition of PGs appear to be critical factors in LDL-PG interaction.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages 总被引:2,自引:0,他引:2
The aim of this work was to investigate the possible mechanisms for uptake by human monocyte-derived macrophages (HMDM) of low density lipoprotein (LDL) pretreated with human arterial chondroitin-6-SO4-rich proteoglycan (LDL-PG). HMDM were incubated with 125I-labeled tyramine cellobiose-labeled LDL-PG, native LDL, and acetylated LDL (Ac-LDL). The results showed that two to four times more LDL-PG than LDL was bound and internalized by the HMDM. Competition experiments showed that LDL-PG competed with native LDL for the apoB,E (LDL) receptor, but not for the Ac-LDL scavenger receptor. Both the LDL and LDL-PG uptake were reduced after preincubation of the macrophages with unlabeled native LDL, though to a lesser extent with LDL-PG. The specific binding of 125I-labeled LDL and 125I-labeled LDL-PG at 4 degrees C was both saturable and concentration-dependent. The dissociation constant (Kd) for binding was 8.6 x 10(-9) M for LDL and 9.4 x 10(-9) M for LDL-PG, but the maximum binding (Bmax) was 1.5-times higher for LDL-PG. Cholesterol derived from LDL-PG was less effective than native LDL in suppressing HMG-CoA reductase activity. The results indicate that the uptake of LDL-PG is mediated not only by the LDL-receptor, but also by another unspecific pathway, which may not be subjected to regulation. These results provide further support for the hypothesis that LDL modifications induced by arterial PG may contribute to the formation of foam cells. 相似文献
9.
Prostaglandin (PG) E1 was demonstrated to stimulate the transfer of phosphatidylcholine and cholesterol esters from human high density lipoproteins (HDL3) to low density lipoproteins (LDL). The enhancement effect of PGE1, on the interlipoprotein lipid transfer was seen at low PG concentrations under conditions of spontaneous exchange as well as in the presence of lipoprotein-depleted plasma, or partly purified plasma lipid exchange protein. PGE2 and PGF2 showed no significant influence on the interlipoprotein lipid transfer. Evidence is presented suggesting that the PGE1-induced stimulation of interlipoprotein lipid exchange results in enhancement of LCAT-catalyzed cholesterol esterification in plasma. It is proposed that the effect of PGE1 is due to the previously described PGE1-induced reorganization of the HDL surface [(1984) FEBS Lett. 173, 291-293] and that PG-lipoprotein interaction may be a factor regulating cholesterol homeostasis. 相似文献
10.
Characterization of six partial deletions in the low-density-lipoprotein (LDL) receptor gene causing familial hypercholesterolemia (FH). 总被引:13,自引:3,他引:10
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Two hundred thirty-four unrelated heterozygotes for familial hypercholesterolemia (FH) were screened to detect major rearrangements in the low-density-lipoprotein (LDL) receptor gene. Total genomic DNA was analyzed by Southern blot hybridization to probes encompassing exons 1-18 of the LDL receptor gene. Six different mutations were detected and characterized by the use of exon-specific probes and detailed restriction mapping. Each mutation is unique and suggests that molecular heterogeneity underlies the molecular pathology of FH. There appear to be preferential sites within the LDL receptor gene for major rearrangements resulting in deletions. 相似文献
11.
Kimiko Yamakawa Hisako Yanagi Keijiro Saku Jun Sasaki Takaaki Okafuji Yae Shimakura Koichi Kawai Shigeru Tsuchiya Kohki Takada Setsuya Naito Kikuo Arakawa Hideo Hamaguchi 《Human genetics》1991,86(5):445-449
Summary To assess the relationship between relatively severe hereditary hypercholesterolemia with Achilles tendon xanthomas and the defect of the low density lipoprotein (LDL) receptor gene, family studies were carried out in 17 hypercholesterolemic families. In 16 out of the 17 families, hypercholesterolemia co-segregated with four different gross rearrangements, six different restriction fragment length polymorphism (RFLP) haplotypes, or an abnormal TaqI band of the LDL receptor gene. These findings are compatible with the interpretation that hypercholesterolemia is caused by defective LDL receptor genes, and that the origin of the mutant LDL receptor genes in Japanese generally differs among different pedigrees. In the remaining family, the proband and his sibling, both having relatively severe hypercholesterolemia and Achilles tendon xanthomas, shared an RFLP haplotype, although the proband's other sibling with moderate hypercholesterolemia but without Achilles tendon xanthomas did not. The mutant gene for familial defective apolipoprotein B-100 was not detected in the 17 probands. These data suggest that most, if not all, of the relatively severe hereditary hypercholesterolemia associated with Achilles tendon xanthomas is caused by a defect of the LDL receptor gene. 相似文献
12.
A novel spectrophotometric assay for monitoring structural rearrangements of native low-density lipoproteins (LDL) is proposed. The approach is based on the analysis of the visible light absorbance maximum of lipoproteins at approximately 461 nm assigned to beta-carotene situated in the hydrophobic parts of LDL. It offers a direct method to study the surface-interior coupling of the lipoprotein particle under physiological conditions. The detected signal is intrinsic to LDL and responsible for the most of the beta-carotene signal from the whole plasma. The negligible interference of beta-carotene absorbance due to the high-density lipoproteins is experimentally verified. Since beta-carotene absorbance belongs to the visible spectral region, no spectral overlapping/artifacts in plasma are expected. The signal sensitivity has been studied through conformational changes of LDL induced by ionic strength, by temperature, and by ligand binding. The results of caffeine binding to LDL indicate that there could be only one dominant type of binding site for caffeine on LDL particles. It can be concluded that visible spectrum characteristics of beta-carotene molecules offer advantages in LDL ligand binding studies which can possibly be extended to monitor the interactions of LDL directly in plasma. 相似文献
13.
Procedure of polydispersity determinations of low density lipoproteins (LDL) on low concentration scale (0.04%) using analytical ultracentrifuge with absorption optics was produced. No corrections for Johnston-Ogston effect and hydrostatic compressibility effect are required. Isothermal compressibility of LDL particles was estimated to be equal to 1.9 X 10(-5) Bar-1. An equation was obtained relating the flotation coefficients of LDL from different sources with solvent density and buoyant density of their particles. It was revealed that LDL particles from individual human plasma are divided into three-four subgroups having specific flotation characteristics and particular quantities of the material in these subgroups. 相似文献
14.
L. D. Bergelson 《Bioscience reports》1989,9(1):27-40
Using high density and low density lipoproteins (HDL and LDL) labeled with fluorescent analogues of phosphatidylcholine or sphingomyelin it was found that low amounts (10–12 M) of prostaglandins E1 and F2 induced different structural rearrangements of the lipoprotein surface, whereas prostaglandins E2 and F1 had no effect. The effects of prostaglandin E1 on HDL were largely paralled by those of this prostaglandin on synthetic recombinants prepared from pure apolipoprotein A1, phospholipids and cholesterol and were demonstrated to be caused by prostaglandin-apolipoprotein interaction. The interaction resembled that of a ligand with a specific receptor protein because it was specific, reversible, concentration and temperature dependent and saturable. However the retaining capacity of HDL or LDL for prostaglandin E1 as determined by equilibrium dialysis was very low and a single prostaglandin E1 molecule was able to induce structural changes in large numbers of discrete lipoprotein particles. To explain this remarkable fact a non-equilibrium model of ligand-receptor interaction is proposed. According to that model in open systems characterized by weak ligand-receptor binding, high diffusion rate of the ligand and long relaxation times which exceed the interval between two successive receptor occupations, the ligand-induced changes will accumulate, resulting in transformation of the system into a new state which may be far away from equilibrium. It is emphasized that the low mobility of lipids constituting the environment of the receptor protein plays a critcal role in this type of signal amplification.It was further demonstrated that the PGE1-induced changes of the lipoprotein surface resulted in an enhancement of LDL-to-HDL transfer of cholesterol esters and phosphatidylcholine especially in the presence of serum lipid transfer proteins. The acceleration of the interlipoprotein transfer caused by prostaglandin E1 in turn increases the rate of cholesterol esterification in serum. It is suggested that in such a way prostaglandin E1 may influence the homeostasis of cholesterol.Abbreviations LDL
low density lioproteins
- HDL
high density lipoproteins
- PG
prostaglandin
- ASM
anthrylvinyl-labeled sphingomyelin (N-12-(9-anthryl)-11-trans-dodecanoylsphingosin-1-phosphocholine
- APC
anthrylvinylphosphatidylcholine (1-radyl-2-[(9-anthryl)-11-transdodecanoyl)-sn-glycerophosphocholine
- NAP-SM
nitroazidophenyl labeled sphingomyelin (N-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sphingosin-1-phosphocholine)
- NAP-PC
adizophenyl labeled phosphatidylcholine (1-radyl-2-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sn-glycero-3-phosphocholine
- DPPC
dipalmitoylphosphatidylcholine
- P
fluorescence polarization
- E
parameter of tryptophanyl to ASM resonance energy transfer
- LEP
lipid-exchange protein 相似文献
15.
Oxidized low density lipoproteins (LDL) are now considered to be one of the atherogenic lipoproteins in vivo and to play an important role in the pathogenesis of atherosclerosis. We previously demonstrated in mouse peritoneal macrophages that oxidized LDL stimulated prostaglandin (PG) E2 synthesis when incorporated into the cells [Yokode, M. et al. (1988) J. Clin. Invest. 81, 720-729]. In this study, we investigated arachidonate metabolism in macrophages after foam cell transformation. The cells were incubated with 100 micrograms/ml of oxidized LDL for 18 h, then stimulated with zymosan. Lipid-enriched macrophages which had taken up oxidized LDL produced much less eicosanoids, such as PGE2, 6-keto-PGF1 alpha, and leukotriene C4 than control cells. After labeling of the cells with [14C]arachidonic acid, they were stimulated with zymosan and the phospholipase activity was determined. The activity of lipid-enriched cells was about two-thirds of that of control cells. Then we investigated the fatty acid composition of their phospholipid fraction to clarify arachidonic acid content and mobilization. Percent of arachidonic acid of lipid-enriched cells decreased and less arachidonic acid mobilization was observed after stimulation with zymosan. These data suggest that impaired arachidonate metabolism in lipid-enriched macrophages can be explained by their decreased phospholipase activity and changes in their fatty acid composition. 相似文献
16.
R G Salomon W Sha C Brame K Kaur G Subbanagounder J O'Neil H F Hoff L J Roberts 《The Journal of biological chemistry》1999,274(29):20271-20280
Levuglandin (LG) E2, a cytotoxic seco prostanoic acid co-generated with prostaglandins by nonenzymatic rearrangements of the cyclooxygenase-derived endoperoxide, prostaglandin H2, avidly binds to proteins. That LGE2-protein adducts can also be generated nonenzymatically is demonstrated by their production during free radical-induced oxidation of low density lipoprotein (LDL). Like oxidized LDL, LGE2-LDL, but not native LDL, undergoes receptor-mediated uptake and impaired processing by macrophage cells. Since radical-induced lipid oxidation produces isomers of prostaglandins, isoprostanes (isoPs), via endoperoxide intermediates, we postulated previously that a similar family of LG isomers, isoLGs, is cogenerated with isoPs. Now iso[4]LGE2-protein epitopes produced by radical-induced oxidation of arachidonic acid in the presence of protein were detected with an enzyme-linked immunosorbent assay. Iso[4]LGE2-protein epitopes are also generated during free radical-induced oxidation of LDL. All of the LGE2 isomers generated upon oxidation of LDL are efficiently sequestered by covalent adduction with LDL-based amino groups. The potent electrophilic reactivity of iso-LGs can be anticipated to have biological consequences beyond their obvious potential as markers for specific arachidonate-derived protein modifications that may be of value for the quantitative assessment of oxidative injury. 相似文献
17.
Bovine luteal cells can utilize low density lipoprotein (LDL) or high density lipoprotein (HDL) as a source of cholesterol for steroidogenesis, and administration of PGF-2 alpha in vitro suppresses lipoprotein utilization. The objective of this study was to examine the mechanism by which PGF-2 alpha exerts this effect. Cultured bovine luteal cells received 0.25 microCi[14C]acetate/ml, to assess rates of de-novo sterol and steroid synthesis, with or without lipoproteins. Both LDL and HDL enhanced progesterone production (P less than 0.01), but caused a significant reduction in the amount of radioactivity in the cholesterol fraction. PGF-2 alpha treatment inhibited the increase in lipoprotein-induced progesterone synthesis (P less than 0.01), but did not prevent the reduction in de-novo cholesterol synthesis brought about by LDL or HDL. PGF-2 alpha alone reduced cholesterol synthesis (P less than 0.01), but it was not as effective as either LDL or HDL. Both lipoproteins and PGF-2 alpha also decreased the amount of radioactivity in the progesterone fraction (P less than 0.01), and the effect of PGF-2 alpha was similar to that of the lipoproteins. It is concluded that lipoproteins can enhance progesterone production and also suppress de-novo cholesterol synthesis in bovine luteal cells, but only the former effect of lipoproteins is inhibited by PGF-2 alpha. Therefore, it is suggested that PGF-2 alpha allows entry of lipoprotein cholesterol into the cell, but prevents utilization for steroidogenesis. In addition, PGF-2 alpha alone can suppress cholesterol synthesis, as well as decrease conversion of cholesterol to progesterone. 相似文献
18.
Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts 总被引:16,自引:7,他引:9
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《The Journal of cell biology》1981,90(3):595-604
The visible wavelength excited fluorophore 3,3'- dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)- LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory. 相似文献
19.
J D Belcher L L Rudel M Waite 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,81(1):87-96
Intact rats removed more radiolabelled triacylglycerol, cholesterol, and cholesterol ester but not phosphatidylcholine (PC) in the first 6 min than hepatectomized rats. There was no difference between intact and hepatectomized rats in the transfer of radiolabelled chylomicron lipids to other lipoproteins. Specific radioactivity measurements demonstrated a net transfer of PC (intact and hepatectomized rats) and unesterified cholesterol (intact rats only) onto both the low density lipoprotein/high density lipoprotein-1 (LDL/HDL1) and HDL2 fractions. [3H]Fatty acids were rapidly incorporated into blood cell phospholipids and into HDL and LDL cholesterol esters of both intact and hepatectomized rats. Substantial rearrangements of [3H]palmitate occurred during lipid uptake by liver. 相似文献
20.
Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts
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The effect of cell density on low density lipoprotein (LDL) binding by cultured human skin fibroblasts was investigated. Bound LDL was visualized by indirect immunofluorescence. Cellular lipid and cholesterol were monitored by fluorescence in cells stained with phosphine 3R and filipin, respectively. LDL binding and lipid accumulation were compared in cells in stationary and exponentially growing cultures, in sparsely and densely plated cultures, in wounded and non-wounded areas of stationary cultures, and in stationary cultures with and without the addition of lipoprotein-deficient serum. We conclude that LDL binding and cholesterol accumulation induced by LDL are influenced by cell density. It appears that, compared to rapidly growing cells, quiescent (noncycling) human fibroblasts exhibit fewer functional LDL receptors. 相似文献