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1.
Apolipoprotein A-IGiessen (Pro143----Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase 总被引:2,自引:0,他引:2
G Utermann J Haas A Steinmetz R Paetzold S C Rall K H Weisgraber R W Mahley 《European journal of biochemistry》1984,144(2):325-331
Apolipoprotein A-IGiessen is a variant form of apo A-I that is displaced from the corresponding normal A-I isoforms on isoelectric focusing gels by a single charge unit towards the cathode [Utermann et al. (1982) J. Biol. Chem. 257, 501-507]. Three subjects heterozygous for the variant were detected in one family. The percentage of the total A-I in plasma represented by the A-IGiessen in these subjects ranged over 25-30%. The variant and normal major A-I isoforms from the proband (Y.J.) were purified by preparative isoelectric focusing and cleaved with CNBr. Analytical focusing of CNBr fragments demonstrated a charge difference between CB3Giessen and normal CB3. Sequence analysis of CB3Giessen revealed that a proline existing in normal A-I was replaced by an arginine in the variant A-I at residue 143. The ability of the mutant A-I to activate purified lecithin:cholesterol acyltransferase was determined in vitro. The cofactor activity of [Arg143]apolipoprotein A-I was about 60-70% of that demonstrated by control A-I. Residue 143 is in a putative beta-turn between two of the repeating amphiphilic helices in apolipoprotein A-I and may be a critical determinant of the protein's structure and function. 相似文献
2.
A mutation in apolipoprotein A-I in the Iowa type of familial amyloidotic polyneuropathy 总被引:15,自引:0,他引:15
Immunoblotting of isoelectric focusing gels of plasma and direct genomic DNA sequencing have been used to characterize a mutation in apolipoprotein A-I associated with the familial amyloidotic polyneuropathy originally described by Van Allen in an Iowa kindred. An arginine for glycine substitution in apolipoprotein A-I identified in the proband's amyloid fibrils was determined to be the result of a mutation of guanine to cytosine in the apolipoprotein A-I gene at the position corresponding to the first base of codon 26. Direct sequencing of genomic DNA of three affected individuals who died in the 1960s confirmed the inheritance of the disorder. Immunoblot analysis detected the variant apolipoprotein A-I in the proband's plasma and in several at-risk members of the kindred. In addition, allele-specific amplification by the polymerase chain reaction was used to detect carriers of the variant gene. 相似文献
3.
A sensitive and rapid immunological detection method was used to screen for apolipoprotein A-IV variants. Antibodies to human lymph chylomicron or plasma apolipoprotein A-IV, and plasma apolipoprotein A-I were raised in rabbits. Antibodies to apolipoprotein A-I or apolipoprotein A-IV were shown to be monospecific to their respective antigens by reactivity against human chylomicron apolipoproteins by immunoblot analysis. Plasma samples were obtained from dyslipidemic subjects from the Lipid Research Clinic of Columbia University. The plasma samples were isoelectrically focused (pH 4-6) on slab gels. Plasma proteins were then transferred to nitrocellulose paper for immunoblotting. Apolipoprotein A-IV polymorphism was determined by specific immunological detection of apolipoprotein A-IV. Identical apolipoprotein A-IV isoprotein patterns were observed when either antibodies to lymph or plasma apolipoprotein A-IV were used for immunoblotting. All the dyslipidemic plasma samples screened contained the two major and one or two minor isoproteins of normal plasma. In two instances, new apolipoprotein A-IV variants having an additional isoform were detected. One subject was hypertriglyceridemic (triacylglycerols = 342 mg/dl, cholesterol = 251 mg/dl) and had an additional major acidic apolipoprotein A-IV isoform. Another subject with mild hypocholesterolemia (triacylglycerols = 209 mg/dl, cholesterol = 120 mg/dl) was found to have additional major and minor basic apolipoprotein A-IV isoforms. The specificity of this technique allows detection of polymorphism of apolipoproteins of similar isoelectric points by use of a single dimension isoelectric focusing gel. This technique also demonstrated the presence of altered apolipoprotein A-I isoforms in the plasma of a patient with Tangier disease. These isoforms were previously identified as isoforms 2 and 4 of normal plasma by use of two-dimensional gel electrophoresis. However, by use of this new technique and careful evaluation of previously published two-dimensional gels, we now identify these apolipoprotein A-I isoforms as being more acidic than those of normal plasma. 相似文献
4.
Apolipoprotein A-IV was isolated from the d less than 1.21 g/ml fraction of rat serum by gel filtration followed by heparin-Sepharose affinity chromatography; this method also facilitated the preparation of apolipoprotein A-I and apolipoprotein E. The apolipoprotein A-IV preparation was characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and immunodiffusion. The lipid-binding properties of this protein were studied. Apolipoprotein A-IV associated with dimyristoylphosphatidylcholine (DMPC) to form recombinants which contained two molecules of apolipoprotein A-IV and had a lipid/protein molar ratio of 110. The density of the DMPC/apolipoprotein A-IV particles was determined to be 1.08 g/ml and the particles were visualized by electron microscopy as discs which were 5.8 nm thick and 18.0 nm in diameter. The stability of the DMPC/apolipoprotein A-IV recombinants, as determined by resistance to denaturation, was comparable to the stability of DMPC/apolipoprotein A-I complexes. However, by competition studies it was found that apolipoprotein A-I competed for the binding to DMPC more effectively than did apolipoprotein A-IV. It is concluded that, while rat apolipoprotein A-IV resembles other apolipoproteins in its lipid-binding characteristics, it may be displaced from lipid complexes by apolipoprotein A-I. 相似文献
5.
Familial apolipoprotein A-I and C-III deficiency, variant II 总被引:8,自引:0,他引:8
E J Schaefer J M Ordovas S W Law G Ghiselli M L Kashyap L S Srivastava W H Heaton J J Albers W E Connor F T Lindgren 《Journal of lipid research》1985,26(9):1089-1101
The biochemical, clinical, and genetic features were examined in the proband (homozygote) and heterozygotes (n = 17) affected with familial apolipoprotein A-I and C-III deficiency, variant II (previously described as apolipoprotein A-I absence). The proband was a 45-year-old white female with mild corneal opacification and significant three-vessel coronary artery disease (CAD), who died shortly after bypass surgery. Autopsy findings included significant atherosclerosis in the coronary and pulmonary arteries and the abdominal aorta as well as extracellular stromal lipid deposition in the cornea. No reticuloendothelial lipid deposits in the liver, bone marrow, or spleen were noted (unlike Tangier disease). Laboratory features included marked high density lipoprotein (HDL) deficiency and undetectable plasma apolipoproteins (apo) A-I and C-III. The percentage of plasma cholesterol in the unesterified form was normal at 30%. The activity and mass of lecithin:cholesterol acyltransferase (LCAT) were 42% and 36% of normal, respectively, and the cholesterol esterification rate was 43% of normal. Deficiencies of plasma vitamin E and essential fatty acid (linoleic, C18:2) were also noted. Evaluation of plasma lipoproteins and apolipoproteins in 37 kindred members revealed 17 heterozygotes with HDL cholesterol values below the 10th percentile of normal. Of these, all had apoA-I levels more than one standard deviation below the normal mean, and 37.5% had a similar decrease in apoC-III values. Mean (+/- SD) plasma HDL cholesterol, apoA-I, and apoC-III values (mg/dl) in heterozygotes were 54.0%, 62.4%, and 79.2% of normal, respectively. No evidence of CAD was observed in 10 heterozygotes 40 years of age or less; however, CAD was detected in 3 of 7 heterozygotes over 40 years of age, one of whom died at age 56 years of complications of myocardial infarction and stroke. The inheritance pattern in this kindred was autosomal codominant. ApoA-I isolated from a heterozygote had an isoelectric focusing pattern and amino acid composition similar to normal. Utilizing DNA isolated from two obligate heterozygotes, no abnormalities in the apoA-I or apoC-III genes were detected by Southern blot analysis utilizing specific probes following restriction enzyme digestion. The data indicate that familial apolipoprotein A-I and C-III deficiency, variant II, is similar to variant I (described by Norum et al. 1982. N. Engl. J. Med. 306: 1513-1519), but differs at the clinical level (lack of xanthomas), the biochemical level (lack of detectable apoA-I, lower apoA-II level), and at the gene level.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
6.
Isoelectric focusing (IEF) of apolipoproteins is an important procedure for the isolation of apolipoprotein isoforms. In this paper we describe the use of agarose/urea as a focusing medium for IEF of apolipoproteins. This technique, which offers several advantages over the traditional polyacrylamide gel focusing, yields a further microheterogeneity of the known isoforms of apolipoproteins A-I and E. In addition, by subsequent immunoprecipitation, both specificity and sensitivity are enhanced. Using a monospecific antiserum against Apo A-I, a new minor isoform was detected at the basic end of the Apo A-I spectrum (pI 6.02) which has not yet been observed in normal plasma samples. Another advantage of this technique in combination with immunoprecipitation is the specific detection of Apo E phenotypes which is essential for the diagnosis of hyperlipoproteinemia type III. 相似文献
7.
Studies on the polymorphism of human apolipoprotein A-I 总被引:3,自引:0,他引:3
Upon preparative isoelectric focussing of human apo-HDL, four major forms of apolipoprotein A-I have been isolated. As identified by the following nomenclature and pI, they comprise: apolipoprotein A-I1, pI 5.62; apolipoprotein A-I2, pI 5.53; apolipoprotein A-I3, pI 5.45; apolipoprotein A-I4 pI 5.36. These forms of apolipoprotein A-I were shown to have identical migration on polyacrylamide gel electrophoresis, molecular weights of 26 000 on sodium dodecyl sulfate gel electrophoresis and a common antigenicity with antisera against apolipoprotein A-I or A-I1. Each form had very similar amino acid compositions with the exception of form apolipoprotein A-I4 which contained one isoleucine residue per mol. All forms but apolipoprotein A-I4 were activators of lecithin:cholesterol acyltransferase, the latter was inhibitory to the reaction. From these results, it was concluded that apolipoprotein A-I1, A-I2 and A-I3 are equivalent forms of apolipoprotein A-I whereas apolipoprotein A-I4 is different or heterogeneous. Upon refocussing, the polymorphs were shown to be stable at their pI and not affected by changes in concentration and by the presence of urea or ampholytes. Exposure of a form of apolipoprotein A-I to alkaline pH partially regenerated the original heterogeneity; however, apolipoprotein A-I4 regenerated from apolipoprotein A-I1 did not contain isoleucine, which further demonstrates form apolipoprotein A-I4 heterogeneity. 相似文献
8.
Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reaction-amplified DNA. 总被引:2,自引:0,他引:2
Y Takada J Sasaki M Seki S Ogata Y Teranishi K Arakawa 《Journal of lipid research》1991,32(8):1275-1280
A new genetic variant apolipoprotein (apo), A-I Yame, was discovered during screening for apoA-I genetic variants with isoelectric focusing gel electrophoresis. To investigate the structural abnormality of apoA-I Yame, we amplified the apoA-I gene isolated from the proband's peripheral blood leukocytes with the polymerase chain reaction (PCR) method and directly sequenced these PCR fragments. ApoA-I Yame was found to have aspartic acid (GAC) substituted by tyrosine (TAC) at residue 13. We also identified this substitution by an automated DNA sequencer. This substitution was confirmed with amino acid sequencing of the isolated apoA-I Yame by Immobiline gel electrophoresis. This combined method, direct PCR from genomic DNA-derived individual peripheral blood leukocytes and subsequent direct sequencing, can be used to identify the entire sequence of apoA-I in a short period of time. Furthermore, with this method, it is possible to identify both alleles in heterozygous individuals. 相似文献
9.
A method has been developed for quantitative analysis of 'free' apolipoprotein A-I and apolipoprotein A-I associated with high-density lipoprotein (HDL) in serum. The method utilizes the difference between the rate of electrophoretic migration of apolipoprotein A-I associated with HDL (alpha) and 'free' apolipoprotein A-I (pre-beta) in agarose gel. Apolipoprotein A-I is subsequently quantitated by electrophoresis in a second dimensional gel containing anti-apolipoprotein A-I antibodies. Using this method all apolipoprotein A-I of normal fasting serum was found associated with HDL (n = 16). By contrast, 'free' apolipoprotein A-I accounted for up to 12% of the total in the serum of patients with isolated hypertriglyceridemia (n = 8) or mixed hyperlipoproteinemia (n = 8). Between 30 and 35% of 'free' apolipoprotein A-I was found in one patient afflicted with the apolipoprotein C-II deficiency syndrome. Also, 'free' apolipoprotein A-I could be detected in normal postabsorptive serum. 30 and 90 min following heparin-enhanced lipolysis 'free' apolipoprotein A-I accounted for 23 and 20%, respectively, of the total apolipoprotein A-I of serum. Apolipoprotein A-I associated with HDL remained unaltered. It appears, therefore, that 'free' apolipoprotein A-I is liberated from triglyceride-rich lipoproteins during lipolysis. 相似文献
10.
Human apolipoprotein A-I polymorphism. Identification of amino acid substitutions in three electrophoretic variants of the Münster-3 type 总被引:3,自引:0,他引:3
H J Menzel G Assmann S C Rall K H Weisgraber R W Mahley 《The Journal of biological chemistry》1984,259(5):3070-3076
Variant forms of apolipoprotein A-I (apo-A-I) have been shown to exist in the human population. One mutant form, referred to as apo-A-I-Münster-3, is one charge unit more basic than normal apo-A-I on isoelectric focusing gels. This variant has the same immunologic characteristics and molecular weight as normal apo-A-I. The apo-A-I-Münster-3 from subjects in three unrelated families (in two of which the trait has been shown to be transmitted as an autosomal co-dominant) has been analyzed by partial amino acid sequencing to define the cause of the electrophoretic abnormality. In the apo-A-I of family A, the abnormality was shown to occur in the smallest cyanogen bromide fragment, CB-2 (residues 87-112), and amino acid sequencing revealed asparagine instead of the usual aspartic acid at residue 103. Subjects with this mutant form have shown no signs of dyslipoproteinemia. The NH2-terminal cyanogen bromide fragment (CB-1, residues 1-86) from the apo-A-I of family B was shown to differ electrophoretically from normal CB-1, and amino acid sequencing revealed that a substitution of arginine for proline at residue 4 was responsible for this variant form. Analysis of the plasma lipids of one affected family B member demonstrated that the percentage of the total cholesterol that was esterified was somewhat lower than that normally observed. In a third family, family C, a variant having the same electrophoretic abnormality as the other two was determined to have an amino acid substitution at yet a different position. In this variant, histidine was found at residue 3 in the apo-A-I sequence, rather than the usual proline. In all three cases, the substitution could account for the electrophoretic abnormality. It is proposed that these three apo-A-I-Münster-3 variants be designated apo-A-I(Asp103----Asn), apo-A-I(Pro4----Arg), and apo-A-I(Pro3----His), respectively, to indicate the substitution that accounts for the abnormality in isoelectric focusing gels. 相似文献
11.
The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes 总被引:1,自引:0,他引:1
Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I. 相似文献
12.
A purification method for apolipoprotein A-I and A-II 总被引:1,自引:0,他引:1
M C Peitsch A Kress P G Lerch J J Morgenthaler H Isliker H J Heiniger 《Analytical biochemistry》1989,178(2):301-305
Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I. 相似文献
13.
Three monoclonal mouse hybridoma antibodies, designated 2AI, 4AI, and 5AI, specific for human plasma apolipoprotein A-I (apoA-I) were characterized. In an enzyme-linked immunosorbent assay (ELISA) each of the antibodies reacted with purified apoA-I and with A-I in normal human serum. Immunoblotting of apoA-I subjected to isoelectric focusing revealed that the three antibodies reacted with all the charge isomorphs of apoA-I and with proapoA-I. Using a solid phase competitive displacement assay, the antigenic determinant for antibody 5AI could be localized to cyanogen bromide fragment 3 of apoA-I (residues 113-148), while the epitope for antibody 4AI resided in cyanogen bromide fragment 4. Dot blot experiments and data obtained by the competitive displacement assay revealed that antibody 2AI reacts with high affinity with CNBr fragment 2 but that it also reacts with lower affinity with fragments 1 and 4. The antibody 5AI did not bind to a genetic variant of apoA-I (Glu----136 Lys), demonstrating that the substitution of a single amino acid in human apoA-I can cause the loss of an antigenic determinant. 相似文献
14.
H Ying K Saku R Harada N Takami N Sasaki Y Saito K Arakawa 《Biochimica et biophysica acta》1990,1047(3):247-254
Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I. 相似文献
15.
Genetic studies of human apolipoproteins. XVII: Population genetics of apolipoprotein polymorphisms in American Samoa 总被引:1,自引:0,他引:1
D E Crews M I Kamboh J R Bindon R E Ferrell 《American journal of physical anthropology》1991,84(2):165-170
Variation in human apolipoprotein genes is a major source of phenotypic differences in human lipid metabolism. Data regarding genetic variation at apolipoprotein loci in various populations are only beginning to accumulate, and they suggest that different populations vary widely in distribution of apolipoprotein alleles. Using isoelectric focusing-immunoblotting techniques, we screened 67 serum samples from self-identified Samoan residents of American Samoa to investigate structural variation at six apolipoprotein loci: A-I, A-II, A-IV, C-II, E, and H. The APO A-I, A-II, and C-II loci were found to be monomorphic by isoelectrical focusing. In Samoans, the common three-allele polymorphism was observed for APO E, with no striking differences in frequencies from Caucasian populations. The three common alleles of the APO H locus also were identified; however, frequencies of the less common alleles (APO H*I and APO H*3) were different from those observed elsewhere for Caucasians. 相似文献
16.
17.
Marta MarchesiCinzia Parolini Caterina ValettiPalma Mangione Laura ObiciSofia Giorgetti Sara RaimondiSimona Donadei Gina GregoriniGiampaolo Merlini Monica StoppiniGiulia Chiesa Vittorio Bellotti 《生物化学与生物物理学报:疾病的分子基础》2011,1812(1):87-93
Hereditary systemic amyloidosis caused by apolipoprotein A-I variants is a dominantly inherited disease characterised by fibrillar deposits mainly localized in the kidneys, liver, testis and heart. We have previously shown that the apolipoprotein A-I variant circulates in plasma at lower levels than the wild-type form (Mangione et al., 2001; Obici et al., 2004) thus raising the possibility that the amyloid deposits could sequester the circulating amyloidogenic chain or that the intracellular quality control can catch and capture the misfolded amyloidogenic chain before the secretion. In this study we have measured plasma levels of the wild-type and the variant Leu75Pro apolipoprotein A-I in two young heterozygous carriers in which tissue amyloid deposition was still absent. In both cases, the mutant was present at significantly lower levels than the wild-type form, thus indicating that the low plasma concentration of the apolipoprotein A-I variant is not a consequence of the protein entrapment in the amyloid deposits. In order to explore the cell secretion of amyloidogenic apolipoprotein A-I variants, we have studied COS-7 cells expressing either wild-type apolipoprotein A-I or two amyloidogenic mutants: Leu75Pro and Leu174Ser. Quantification of intracellular and extracellular apolipoprotein A-I alongside the intra-cytoplasmatic localization indicates that, unlike the wild-type protein, both variants are retained within the cells and mainly accumulate in the endoplasmic reticulum. The low plasma concentration of amyloidogenic apolipoprotein A-I may therefore be ascribed to the activity of the intracellular quality control that represents a first line of defence against the secretion of pathogenic variants. 相似文献
18.
A rapid micromethod for apolipoprotein E phenotyping directly in serum 总被引:15,自引:0,他引:15
L M Havekes P de Knijff U Beisiegel J Havinga M Smit E Klasen 《Journal of lipid research》1987,28(4):455-463
A new method for the apolipoprotein E phenotyping has been developed. The method is based on isoelectric focusing of either delipidated or guanidine-HC1-treated serum or plasma in a horizontal slab gel system followed by immunoblotting using either polyclonal or monoclonal anti-apolipoprotein E antibodies as first antibody. Apolipoprotein E phenotyping with this method in 200 serum samples that had been stored at -20 degrees C for more than one year gave exactly the same results as obtained with the conventional method based on isoelectric focusing of delipidated very low density lipoproteins isolated from fresh serum followed by protein staining. Compared with the conventional method, the present method is less laborious because ultracentrifugation to isolate VLDL is not needed; it is suitable for large scale screening purposes; it needs only a few microliters of serum or plasma, and can easily be performed with samples with low concentrations of apolipoprotein E. 相似文献
19.
Rapid apolipoprotein E phenotyping by immunofixation in agarose 总被引:2,自引:0,他引:2
Conventional determination of apolipoprotein E isomorphs comprises ultracentrifugation of 1-5 ml serum, delipidation of very low density lipoproteins (VLDL), and isoelectric focusing (IEF) in polyacrylamide gels. In order to reduce the sample volume and to avoid nonspecific protein bands, immunoblotting was proposed. Now we describe a methodological variant that uses 25 microliters serum, replaces ultracentrifugation by precipitation of apoE-containing lipoproteins with polyethylene glycol, and delipidation by dissolution in detergent. IEF is carried out in agarose. This allows specific immunofixation of apoE-containing bands with 10 microliters antiserum per sample. This method yields apoE patterns that are specific and well resolved. Also, it offers considerable savings of time and equipment involved. 相似文献
20.
The reaction catalysed by phosphatidylcholine-sterol acyltransferase (EC 2.3.1.43) is believed to be the major source of cholesteryl ester in human plasma; the enzyme requires a protein activator. Several human apolipoproteins were found to exhibit an activator function, the major one being apolipoprotein A-I. Human apolipoprotein E exists in the population mainly in three different genetic isoforms; apolipoprotein E-2, E-3 and E-4. These isopeptides were isolated from subjects homozygous for one of the isoforms, incorporated into phospholipid/cholesterol/[14C]cholesterol complexes by the cholate dialysis procedure and used to measure capacity to activate phosphatidylcholine-sterol acyltransferase in comparison to apolipoprotein A-I lipid substrate particles prepared by the same procedure. Acyltransferase activity was measured by the formation of [14C]cholesteryl ester from [14C]cholesterol using purified enzyme. With egg yolk phosphatidylcholine as acyl donor, apo E was 15-19% as efficient as apolipoprotein A-I for activation of the acyltransferase. Apo-E-stimulated cholesteryl ester formation by the enzyme was enhanced when 1-oleoyl-2-palmitoyl-glycerophosphocholine was used as a substrate phospholipid (45% of apo A-I/phosphatidylcholine control) and most pronounced with dimyristoylglycerophosphocholine (75% of apo A-I/phosphatidylcholine control). No significant difference in activation was found between apo E isoforms. It is concluded that apolipoprotein E activates phosphatidylcholine-sterol acyltransferase in vitro and that apolipoprotein E isoforms are similarly effective. 相似文献