Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated the effect of Cu2+ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation of HDL with Cu2+ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (lambda max). LDL incubated with Cu2+ also showed a rapid decrease in Trp fluorescence intensity with time, with no associated increase in lambda max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in lambda max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in lambda max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in lambda max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.
 ...
PMID:Apolipoprotein-lipid association in oxidatively modified HDL and LDL. 830 91

Apolipoprotein E (apoE), a plasma apolipoprotein that plays a central role in lipoprotein metabolism, is localized in the senile plaques, congophilic angiopathy, and neurofibrillary tangles of Alzheimer disease. Late-onset familial and sporadic Alzheimer disease patients have an increased frequency of one of the three common apoE alleles, epsilon 4, suggesting apoE4 is associated with increased susceptibility to disease. To follow up on this suggestion, we compared the binding of synthetic amyloid beta (beta/A4) peptide to purified apoE4 and apoE3, the most common isoform. Both isoforms bound synthetic beta/A4 peptide, the primary constituent of the plaque and angiopathy, forming a complex that resisted dissociation by boiling in SDS. Oxygen-mediated complex formation was implicated because binding was increased in oxygenated buffer, reduced in nitrogen-purged buffer, and prevented by reduction with dithiothreitol or 2-mercaptoethanol. Binding of beta/A4 peptide was saturable at 10(-4) M peptide and required residues 12-28. Examination of apoE fragments revealed that residues 244-272 are critical for complex formation. Both oxidized apoE4 and apoE3 bound beta/A4 peptide; however, binding to apoE4 was observed in minutes, whereas binding to apoE3 required hours. In addition, apoE4 did not bind beta/A4 peptide at pH < 6.6, whereas apoE3 bound beta/A4 peptide from pH 7.6 to 4.6. Together these results indicate differences in the two isoforms in complexing with the beta/A4 peptide. Binding of beta/A4 peptide by oxidized apoE may determine the sequestration or targeting of either apoE or beta/A4 peptide, and isoform-specific differences in apoE binding or oxidation may be involved in the pathogenesis of the intra- and extracellular lesions of Alzheimer disease.
 ...
PMID:Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease. 836 70

Apolipoproteins share a common structural feature, their interaction with phospholipids. It is believed that amphipathic helical sequences enable apolipoproteins to bind to lipid bilayer and to form discoidal particles of defined dimensions. While the knowledge of the apo A-I sequence and secondary structure has been used to make predictions about its mode of association with lipids, the available experimental data necessary to propose a precise model of these discoidal structures are still limited. An important step in our understanding of these structures would be to identify the apolipoprotein lipid-associated domains. Proteolysis of apo A-I-DMPC reconstituted HDL (rHDL) and free apo A-I is used here to identify lipid-protected domains of apo A-I. Free cleaved peptides were separated from rHDL associated peptides by density gradient centrifugation. The lipid-associated peptides were further analyzed by SDS-PAGE and transferred by Western blot to a ProBlott membrane for sequencing. Cleavage occurred at residue 43 with proteinase K, 46 with trypsin and residue 47 or 48 with pronase. A large domain from about residue 45 to the C-terminal remains highly protected against hydrolysis eventhough it contains several bonds susceptible to proteolytic cleavage. No protected fragments were detected by SDS-PAGE after enzymatic cleavage of free apo A-I in identical experimental conditions.
 ...
PMID:Enzymatic hydrolysis of reconstituted dimyristoylphosphatidylcholine-apo A-I complexes. 837 88

A series of mutant apolipoprotein (apoA-I) constructs were designed and then expressed in cell culture to identify structural domains within the mature native apoA-I protein that participate in the activation of the plasma enzyme, lecithin-cholesterol acyltransferase (LCAT). Evolutionary conservation analysis has shown previously that apoA-I contains eight repeats containing 22 amino acids and two repeats containing 11 amino acids that are highly conserved among species as well as within the apolipoprotein supergene family. These tandem repeats begin at residue 44 and are usually marked by a proline residue, with six of the 22-mer repeats showing high amphipathic alpha-helical character. To determine if specific 11- or 22-amino acid domains are essential for maximal LCAT activation within the entire native protein, each of the 10 repeats was sequentially deleted using a polymerase chain reaction based method of mutagenesis. The wild-type and mutant apoA-I gene constructs were expressed in Chinese hamster ovary (CHO) cells and stable lines established. Wild-type and mutant apoA-I protein were purified from 48-96-h conditioned serum-free medium and characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Wild-type apoA-I showed a single migrating band of 28,000 daltons that corresponded to the mobility of human plasma apoA-I, whereas apoA-I deletion mutants (lacking 22- or 11-mer repeats) showed the corresponding shift to lower molecular size. To measure the relative LCAT activation of all deletion mutant apoA-I proteins relative to wild-type apoA-I, an assay system utilizing small unilamellar vesicles as the lipid substrate was used. The results of these studies suggest that several central amphipathic alpha-helical regions within the mature protein are critical in LCAT activation.
 ...
PMID:Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships. 840 82

Human plasma in vitro inhibits the growth of coagulase negative staphylococci, S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity against S. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated with S. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation. To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition. These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.
 ...
PMID:Antimicrobial activity of lipoprotein particles containing apolipoprotein Al. 845 79

The glycoprotein apolipoprotein[a] (apo[a]) is present in plasma at highly variable concentrations and appears as a number of genetically determined size isoforms (400-800 kDa), disulfide linked to apoB-100 in low density lipoprotein to produce lipoprotein [a](Lp[a]). Apo[a] is synthesized by the liver, but the site of association of apo[a] and apoB and factors that regulate its production are unknown. To examine the morphogenesis of the Lp[a] particle, baboon hepatocytes expressing a single, low molecular weight isoform of apo[a] were labeled with [35S]cysteine and methionine, and apo[a] was analyzed by immunoprecipitation and SDS-PAGE. Steady-state labeling revealed two molecular weight forms of apo[a] inside the cell. Only the large form was recovered from the culture medium. Pulse-chase studies and endoglycosidase treatment revealed that the lower molecular weight form of apo[a] represented a precursor with a prolonged residence time in the endoplasmic reticulum or an early Golgi compartment, after which it was processed to the mature form. A proportion of the mature form of apo[a] was rapidly secreted after synthesis, whereas the remainder had a prolonged residence time in a late Golgi compartment. In all experiments, apoB co-precipitated with apo[a] from the culture medium, but not from cell lysates. Density gradient ultracentrifugation and immunoblot analysis revealed that the majority of apo[a] was secreted into the medium in a free form, suggesting that the association between apo[a] and apoB occurred after secretion. Regulation of the movement of apo[a] between intracellular compartments may be one mechanism by which the plasma levels of Lp[a] are influenced.
 ...
PMID:Intracellular maturation of apolipoprotein[a] and assembly of lipoprotein[a] in primary baboon hepatocytes. 846 34

We have studied the rat ethanol-liquid diet model for chronic ethanol modulation of lipid homeostasis, apolipoprotein (apo) B production and apoB mRNA editing. Male Wistar rats were fed one of three diets: i) regular chow, ii) an isocaloric liquid diet, or iii) isocaloric ethanol-liquid diet where ethanol accounts for 35.5% of the total calories, for up to 40 days. There was no difference in body weight or liver/body weight ratio among the three groups of animals at the end of the feeding period. Hepatic and plasma triglycerides were elevated in the ethanol-treated animals only, correlated with an accumulation of lipid particles in the liver of these animals. By DNA excess hybridization, the steady state mRNA levels of apoB and apoB mRNA-editing protein relative to actin were not significantly altered. The proportion of edited apoB mRNA; i.e., apoB-48 mRNA/(apoB-48 + B-100) mRNA, increased in a time-dependent manner from approximately 50% to 100% in the ethanol-treated group. It remained unchanged in the chow- and liquid diet-fed animals. The proportion of apoB-48/apoB-100 protein synthesis was determined by [35S]methionine labeling followed by specific immunoprecipitation and SDS-polyacrylamide gel electrophoresis. The amount of newly synthesized apoB-48 increased from 30-50% to > 99% of the total apoB (apoB-48 + apoB-100). This increase in apoB-48 biosynthesis is reflected by an increase in circulating plasma apoB-48 from barely detectable to approximately 50% of total plasma apoB. Fractionation of plasma lipoproteins by fast protein liquid chromatography (FPLC) indicates that the ethanol-induced hypertriglyceridemia is completely accounted for by an increase in plasma very low density lipoprotein (VLDL). The proportion of apoB-48 as a percent of total apoB in the VLDL fraction increased from approximately 50% in controls to > 90% in ethanol-treated animals. Furthermore, there is a strong correlation between plasma triglyceride concentration and proportion of edited apoB-mRNA in the liver of ethanol-treated rats, but no direct correlation of the latter with intrahepatic triglyceride content. Ethanol-treated rats represent a new model for studying the regulation of apoB mRNA editing by dietary factors in vivo.
 ...
PMID:Ethanol modulates apolipoprotein B mRNA editing in the rat. 857 34

The apolipoprotein E4 (apoE4) gene dose is a major risk factor for late-onset and sporadic Alzheimer's disease with 50% of homozygous patients developing the disease by age 70. Previous studies have shown localization of apoE to the cytoplasm of certain neurons within the brain. In addition, apoE3, but not apoE4, forms SDS-stable complexes with the microtubule-associated proteins tau and MAP-2. To extend these studies and quantitate the association of apoE with other proteins, the association of apoE3 and apoE4 with several cytoskeletal proteins was examined using both gel shift and overlay assays. In the gel shift assay, apoE3 formed SDS-stable complexes with the longest isoform of human recombinant tau (T4L), the shortest isoform of human recombinant tau (T3), and the 160-kDa neurofilament protein (NFM). ApoE4 did not bind T3, T4L, or NFM in this assay. The association of apoE3 and apoE4 with T4L, actin, or tubulin was further examined in an overlay assay with known amounts of the cytoskeletal proteins slot-blotted onto nitrocellulose and incubated in 0.15 microM (5 microg/ml) apoE3 or apoE4. In this assay, apoE3 and apoE4 bound T4L and tubulin equally well. In contrast, apoE3 bound actin with a significantly greater affinity than did apoE4. These results indicate that apoE isoforms interact with cytoskeletal proteins with at least two different binding affinities. The more avid interaction results in the formation of complexes which are SDS stable and occurs almost exclusively with apoE3, while the other interactions between apoE and cytoskeletal proteins are specific for apoE3.
 ...
PMID:Differential binding of apolipoprotein E isoforms to tau and other cytoskeletal proteins. 862 Sep 24

The concentration of high density lipoproteins (HDL) is inversely related to the risk of atherosclerosis. The two major protein components of HDL are apolipoprotein (apo) A-I and apoA-II. To study the role of apoA-II in lipoprotein metabolism and atherosclerosis, we have developed three lines of C57BL/6 transgenic mice expressing human apoA-II (lines 25.3, 21.5, and 11.1). Northern blot experiments showed that human apoA-II mRNA was present only in the liver of transgenic mice. SDS-polyacrylamide gel electrophoresis and Western blot analysis demonstrated a 17.4-kDa human apoA-II in the HDL fraction of the plasma of transgenic mice. After 3 months on a regular chow, the plasma concentrations of human apoA-II were 21 +/- 4 mg/dl in the 25.3 line, 51 +/- 6 mg/dl in the 21.5 line, and 74 +/- 4 mg/dl in the 11.1 line. The concentration of cholesterol in plasma was significantly lower in transgenic mice than in control mice because of a decrease in HDL cholesterol that was greatest in the line that expressed the most apoA-II (23 mg/dl in the 11.1 line versus 63 mg/dl in control mice). There was also a reduction in the plasma concentration of mouse apoA-I (32 +/- 2, 56 +/- 9, 91 +/- 7, and 111 +/- 2 mg/dl for lines 11.1, 21.5, 25.3, and control mice, respectively) that was inversely correlated with the amount of human apoA-II expressed. Additional changes in plasma lipid/lipoprotein profile noted in line 11.1 that expressed the highest level of human apoA-II include elevated triglyceride, increased proportion of total plasma, and HDL free cholesterol and a marked (>10-fold) reduction in mouse apoA-II. Total endogenous plasma lecithin:cholesterol acyltransferase (LCAT) activity was reduced to a level directly correlated with the degree of increased plasma human apoA-II in the transgenic lines. LCAT activity toward exogenous substrate was, however, only slightly decreased. The biochemical changes in the 11.1 line, which is markedly deficient in plasma apoA-I, an activator for LCAT, are reminiscent of those in patients with partial LCAT deficiency. Feeding the transgenic mice a high fat, high cholesterol diet maintained the mouse apoA-I concentration at a normal level (69 +/- 14 mg/dl in line 11.1 compared with 71 +/- 6 mg/dl in nontransgenic controls) and prevented the appearance of HDL deficiency. All this happened in the presence of a persistently high plasma human apoA-II (96 +/- 14 mg/dl). Paradoxical HDL elevation by high fat diets has been observed in humans and is reproduced in human apoA-II overexpressing transgenic mice but not in control mice. Finally, HDL size and morphology varied substantially in the three transgenic lines, indicating the importance of apoA-II concentration in the modulation of HDL formation. The LCAT and HDL deficiencies observed in this study indicate that apoA-II plays a dynamic role in the regulation of plasma HDL metabolism.
 ...
PMID:Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II. 863 92

This paper describes the use of an antiserum, specific for apolipoprotein (apo) B-48, in a competitive, enzyme-linked immunosorbent assay (ELISA) for apo B-48 in triacylglycerol-rich lipoprotein (TRL) fractions prepared from fasting and post-prandial plasma samples. Previously we showed the antiserum to act as an effective immunoblotting agent following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its use in this ELISA indicates that the antiserum recognises the C-terminal region of the protein on the surface of lipoprotein particles. The ELISA had a sensitivity of less than 37 ng/ml and intra- and inter-assay coefficients of variation of 3.8% and 8.6%, respectively. There was no cross-reaction in the ELISA against serum albumin, ovalbumin, thyroglobulin, or apo B-100 (purified by immunoaffinity chromatography), and high lipid concentrations (as Intralipid) did not interfere. A low density lipoprotein fraction reacted in the ELISA but SDS-PAGE-Western blot analysis confirmed the presence, in the fraction, of a small amount of apo B-48, indicating the existence of low density dietary-derived lipoprotein particles. ELISA and SDS-PAGE-Western blot analysis were used to measure apo B-48 in 12 series of postprandial samples collected from 4 diabetic and 8 normal subjects, following test meals of varying fat content. The mean correlation between the two methods was r = 0.74. The mean fasting concentration of apo B-48 in the TRL fractions from 15 healthy men was 0.46 microgram/ml of plasma.
 ...
PMID:Quantitation of apolipoprotein B-48 in triacylglycerol-rich lipoproteins by a specific enzyme-linked immunosorbent assay. 866 32


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>