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Journal of Lipid Research Sep 1984Four of the principle apolipoproteins of murine serum have been isolated and characterized. On the basis of their physicochemical properties, they are homologous with... (Comparative Study)
Comparative Study
Four of the principle apolipoproteins of murine serum have been isolated and characterized. On the basis of their physicochemical properties, they are homologous with the human and rat apoA-I, A-II, B, and C-III. The group of apolipoproteins of middle to low molecular weight, i.e., A-I, A-II and C-III, were separated from the protein moiety of high density lipoproteins (HDL) by gel filtration chromatography, followed by electrophoresis in alkaline-urea polyacrylamide gel with electrophoretic elution. Murine apoA-I, the major protein of HDL (60-80%) displayed an Mr of approximately 27,000, and was polymorphic (four prominent isoproteins with isoelectric points in the range of pH 5.5-5.7). The amino acid profiles of mouse, rat, and human apoA-I generally resembled each other, the former being distinguished by a content of one isoleucine residue per mole. Amino terminal sequence analysis revealed marked homology between the mouse, rat, dog, and human proteins; mouse and rat apoA-I differed at residues 9 and 18 with potential dissimilarities at residues 5 and 15, while the murine and canine sequences were distinct at residues 6, 9, 13, 15, and 30. Apolipoprotein A-II was a monomer, exhibiting an Mr approximately 11,000 in SDS gels; in addition, it was polymorphic (three apparent isoproteins with pI in the pH range 5.05-5.2), and resembled its human and rat counterparts in amino acid composition. ApoC-III, an acidic peptide of pI 4.74 and of Mr approximately 9,600, possessed an amino acid composition very like that of the homologous human and rat proteins. The homology of mouse apoC-III with the human protein was confirmed by NH2-terminal sequence analysis, which revealed identical amino acids in six positions (1, 2, 4, 8, 9, and 13). As shown earlier (Camus et al. 1983. J. Lipid Res. 24: 1210-1228), two forms of immunologically reacting apoB predominated in mouse VLDL and LDL. After isolation of these lipoproteins in the presence of 1 mM PMSF, the apparent sizes of the high and low Mr forms, apoBH and apoBL, were in the ranges approximately 400,000-530,000 and approximately 250,000-280,000, respectively, according to the SDS gel system. We observed that inclusion of 1 mM PMSF was essential to retard degradation of the high Mr form apoBH. The murine B proteins were isolated from apoVLDL and apoLDL by gel filtration chromatography on Sephadex G150 in anionic detergent, and displayed apparent Mr values of 460,000 (apoBH) and 250,000 (apoBL) in 3% SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Amino Acids; Animals; Apolipoprotein A-I; Apolipoprotein A-II; Apolipoprotein C-III; Apolipoproteins; Apolipoproteins A; Apolipoproteins B; Apolipoproteins C; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Female; Humans; Lipoproteins, HDL; Lipoproteins, LDL; Lipoproteins, VLDL; Male; Mice; Rats; Species Specificity; Ultracentrifugation
PubMed: 6436419
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1984We have isolated and characterised A-IV apolipoprotein (apo-A-IV) from human lymph and plasma by immunoabsorbance chromatography and two-dimensional electrophoresis. Two...
We have isolated and characterised A-IV apolipoprotein (apo-A-IV) from human lymph and plasma by immunoabsorbance chromatography and two-dimensional electrophoresis. Two different apo-A-IV-containing lipoproteins were isolated from four different sources, human lymph triglyceride-rich fraction (TRL), lymph lipoprotein-deficient fraction (LDF), plasma high-density lipoprotein (HDL), and plasma lipoprotein-deficient fraction (LDF). The lipoprotein complexes obtained from lymph TRL and plasma HDL were similar and contained apo-A-IV, apo-A-I, and small molecular weight peptides (apo-C or -A-II). The second lipoprotein complex was isolated from lymph LDF and plasma LDF, and contained apo-A-IV, apo-A-I, and a peptide of Mr = 59,000. The lipid composition of the lipoprotein complexes varied according to the source: triglyceride predominating in lymph TRL and phospholipid and cholesteryl ester from the other sources. Free cholesterol was conspicuously present in very small amounts. Using two-dimensional electrophoresis and immunoblotting techniques, eleven isoproteins of apo-A-IV were identified (pI-4.98, 5.06, 5.10, 5.15, 5.20, 5.22, 5.25, 5.30, 5.34, 5.42, and 5.48). The isoprotein pattern of lymph TRL and plasma HDL was similar, but that of lymph and plasma LDF were different patterns. These results suggest that apo-A-IV associated with d less than 1.21 lipoproteins and apo-A-IV present in LDF may be in metabolically separate lipoproteins and may have different physiological roles.
Topics: Amino Acids; Apolipoproteins A; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Humans; Lymph; Molecular Weight; Structure-Activity Relationship; Triglycerides
PubMed: 6501321
DOI: No ID Found -
Journal of Lipid Research May 2006Apolipoprotein [a] (apo[a]) gene size is a major predictor of lipoprotein [a] level. To determine genetic predictors of allele-specific apo[a] levels beyond gene size,...
Apolipoprotein [a] (apo[a]) gene size is a major predictor of lipoprotein [a] level. To determine genetic predictors of allele-specific apo[a] levels beyond gene size, we evaluated the upstream C/T and pentanucleotide repeat (PNR) polymorphisms. We determined apo[a] sizes, allele-specific apo[a] levels, and C/T and PNR in 215 Caucasians and 139 African Americans. For Caucasians, apo[a] size affected allele-specific levels substantially greater in subjects with apo[a] < 24 K4; for African Americans, the size effect was smaller than in Caucasians, <24 K4, but did not decrease at higher repeats. In both groups, the level decreased with increasing size of the other allele. Controlling for apo[a] sizes, PNR decreased allele-specific apo[a] levels in Caucasians with increasing PNR > 8. In a multiple regression model, apo[a] allele size and size and expression of the other apo[a] allele (and PNR > 8 for Caucasians) significantly predicted allele-specific apo[a] levels. For a common PNR 8 allele, predicted values were similar in the two ethnicities for small size apo[a]. Allele-specific apo[a] levels were influenced by the other allele size and expression. Observed differences between Caucasians and African Americans in allele-specific apo[a] levels were explained for small apo[a] sizes by the other allele size and PNR; the ethnicity differences remain unexplained for larger sizes.
Topics: Black or African American; Aged; Alleles; Apolipoproteins A; Female; Humans; Male; Microsatellite Repeats; Polymorphism, Genetic; White People
PubMed: 16495513
DOI: 10.1194/jlr.M500359-JLR200 -
American Journal of Human Genetics Dec 1988Genetic variation at various human apolipoprotein gene loci plays a vital role in modulating lipid metabolism. However, information regarding genetic variation at...
Genetic variation at various human apolipoprotein gene loci plays a vital role in modulating lipid metabolism. However, information regarding genetic variation at apolipoprotein loci is scanty, and the available data are largely restricted to Caucasian populations. Using recently developed isoelectric focusing-immunoblotting techniques, we have screened a large number of serum samples from Nigerian blacks to investigate structural variation at six apolipoprotein loci: A-I, A-II, A-IV, C-II, E, and H. With the exception of a single example of a putative APO A-I variant, the APO A-I and A-II loci were found to be monomorphic. Several new variants have been identified at the APO A-IV locus, which are apparently restricted to the black gene pool. By comparison with Caucasians, in whom APO C-II is invariant, four allelic variants have been identified in the Nigerians at the APO C-II structural locus. A common three-allele polymorphism has been observed for APO E, with a striking high frequency of the APO E*4 allele. In addition to three common alleles, the APO H locus is characterized by having an allele marker unique to blacks. The mean heterozygosity at these apolipoprotein loci is higher in Nigerian blacks as compared with the Caucasians.
Topics: Alleles; Apolipoprotein A-I; Apolipoprotein A-II; Apolipoprotein C-II; Apolipoproteins; Apolipoproteins A; Apolipoproteins C; Apolipoproteins E; Black People; Gene Frequency; Glycoproteins; Humans; Nigeria; Phenotype; Polymorphism, Genetic; beta 2-Glycoprotein I
PubMed: 3143263
DOI: No ID Found -
The American Journal of Pathology Oct 2001Extravascular coagulation and diminished fibrinolysis are processes that contribute to the pathology of both inflammatory arthritis and atherosclerosis. We hypothesized...
Extravascular coagulation and diminished fibrinolysis are processes that contribute to the pathology of both inflammatory arthritis and atherosclerosis. We hypothesized that, given its homology with plasminogen, apolipoprotein (apo) (a), the distinctive glycoprotein of the atherogenic lipoprotein (Lp) (a), may be equally implicated in inflammatory arthritis. We detected the presence of apo(a) as part of Lp(a) in human arthritic synovial fluid. The abundance of apo(a) in synovial fluid rose in proportion to plasma apo(a) levels and was higher in inflammatory arthritides than in osteoarthritis. In addition, apo(a) immunoreactive material, but not apo(a) transcripts, was detected in inflammatory arthritic synovial tissues. These data indicated that synovial fluid apo(a) originates from circulating Lp(a) and that diffusion of Lp(a) through synovial tissue is facilitated in inflammatory types of arthritis. In synovial tissues, apo(a) co-localized with fibrin. These observations could be reproduced in a model of antigen-induced arthritis, using transgenic mice expressing human Lp(a). Although in this mouse model the presence of apo(a) did not change the severity of arthritis, the co-localization of apo(a) with fibrin in synovial tissue suggests that, in humans, apo(a) may modulate locally the fibrinolytic activity and may thus contribute to the persistence of intra-articular fibrin in inflammatory arthritis.
Topics: Animals; Antigens; Apolipoproteins A; Arthritis; Arthritis, Rheumatoid; Fibrin; Humans; Joints; Lipoprotein(a); Mice; Mice, Transgenic; Osteoarthritis; Particle Size; Synovial Fluid; Synovial Membrane
PubMed: 11583972
DOI: 10.1016/S0002-9440(10)62531-X -
Journal of Lipid Research Jan 1996Amino acid analysis was performed on four Lp[a] preparations to evaluate whether or not the amino acid data was consistent with Lp[a] containing one molecule of...
Amino acid analysis was performed on four Lp[a] preparations to evaluate whether or not the amino acid data was consistent with Lp[a] containing one molecule of apolipoprotein[a] [apo(a)] linked to one molecule of apoB-100. Amino acid analysis was carried out in duplicate on a Beckman model 121 amino acid analyzer. Apo[a] size was determined by a high-resolution agarose gel electrophoretic method that provides an estimate of apo[a] kringle 4 repeats. When Lp[a] was assumed to contain one apo[a] and one apoB molecule per particle, the average absolute bias between the expected molar percentage of each amino acid, as based on the known sequence of apo[a] and apoB, and the obtained molar percentage ranged from 2 to 3.5%. In contrast, by assuming two molecules of apo[a] and one of apoB per Lp[a] particle, the bias between the expected and observed molar percentage ranged from 8.5% to 10%, and by assuming one apo[a] and two apoB the bias ranged from 8.8% to 11.4%. Comparison of Lp[a] concentrations, calculated from six stable amino acids and the Lp[a] composition predicted from the known sequence, was in excellent agreement (bias ranging from 0.3% to 0.9%) with the Lp[a] concentration calculated from the sum of the amino acid concentrations, when Lp[a] was assumed to contain one molecule of apo[a] and one molecule of apoB. However, there was poor agreement (7.4% to 8.4% bias) when it was assumed that Lp[a] contains two molecules of apo[a] and one molecule of apoB. These results indicate that the evaluated Lp[a] preparations contain one apo[a] per Lp[a] particle. Evaluation of amino acid analysis data provides a relatively simple approach to determine the molar ratio of apoB to apo[a] in Lp[a] and provides evidence that Lp[a] contains one molecule of apo[a] and one molecule of apoB.
Topics: Amino Acids; Apolipoproteins A; Apolipoproteins B; Humans; Lipoprotein(a)
PubMed: 8820114
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1989The defect in a kindred with marked plasma high density lipoprotein (HDL) deficiency and premature atherosclerosis was examined. The homozygous proband died of coronary...
The defect in a kindred with marked plasma high density lipoprotein (HDL) deficiency and premature atherosclerosis was examined. The homozygous proband died of coronary artery atherosclerosis at age 45 and had undetectable levels of plasma apolipoproteins A-I and C-III, proteins of HDL. In family studies 10 heterozygotes were identified whose mean apoA-I, apoC-III, apoA-IV, and HDL cholesterol levels were 67, 57, 65, and 62% of normal. These subjects were noted to have restriction fragment length polymorphisms following DNA digestion with a number of enzymes including BamHI, EcoRI, HindIII, XmnI, PstI, and PvuII, following hybridization with a probe spanning 1.1 kilobases approximately 2.5 kilobases 5' to the apoA-I gene. Cloning and sequence analysis of the abnormal allele indicated that the defect is due to the complete deletion of the apoA-I, -C-III, and -A-IV gene complex on chromosome 11, with both ends of the deletion being located in areas of highly repetitive DNA. The data support the concept of an independent role for HDL in the pathogenesis of atherosclerosis.
Topics: Apolipoprotein A-I; Apolipoprotein C-III; Apolipoproteins A; Apolipoproteins C; Arteriosclerosis; Base Sequence; Chromosome Deletion; Chromosomes, Human, Pair 11; Genes; Genetic Carrier Screening; Homozygote; Humans; Lipoproteins, HDL; Molecular Sequence Data; Multigene Family; Restriction Mapping
PubMed: 2506176
DOI: No ID Found -
American Journal of Physiology.... Jun 2013Apolipoprotein A-IV (apoA-IV) is synthesized by the intestine and secreted when dietary fat is absorbed and transported into lymph associated with chylomicrons. We have...
Apolipoprotein A-IV (apoA-IV) is synthesized by the intestine and secreted when dietary fat is absorbed and transported into lymph associated with chylomicrons. We have recently demonstrated that loss of apoA-IV increases chylomicron size and delays its clearance from the blood. There is still uncertainty, however, about the precise role of apoA-IV on the transport of dietary fat from the intestine into the lymph. ApoA-IV knockout (KO) mice do not have a gross defect in dietary lipid absorption, as measured by oral fat tolerance and fecal fat measurements. Here, using the in vivo lymph fistula mouse model, we show that the cumulative secretion of triglyceride (TG) into lymph in apoA-IV KO mice is very similar to that of wild-type (WT) mice. However, the apoA-IV KO mice do have subtle changes in TG accumulation in the intestinal mucosa during a 6-h continuous, but not bolus, infusion of lipid. There are no changes in the ratio of esterified to free fatty acids in the intestinal mucosa of the apoA-IV KO, however. When we extended these findings, by giving a higher dose of lipid (6 μmol/h) and for a longer infusion period (8 h), we found no effect of apoA-IV KO on intestinal TG absorption. This higher lipid infusion most certainly stresses the intestine, as we see a drastically lower absorption of TG (in both WT and KO mice); however, the loss of A-IV does not exacerbate this effect. This supports our hypothesis that apoA-IV is not required for TG absorption in the intestine. Our data suggest that the mechanisms by which the apoA-IV KO intestine responds to intestinal lipid may not be different from their WT counterparts. We conclude that apoA-IV is not required for normal lymphatic transport of TG.
Topics: Animals; Apolipoproteins A; Dietary Fats; Fatty Acids, Nonesterified; Fistula; Infusions, Parenteral; Intestinal Absorption; Intestinal Mucosa; Kinetics; Lipids; Lymph; Mice; Mice, Inbred C57BL; Mice, Knockout; Triglycerides
PubMed: 23599044
DOI: 10.1152/ajpgi.00409.2012 -
Proceedings of the National Academy of... Nov 1986The genes coding for three proteins of the plasma lipid transport system--apolipoproteins A1 (APOA1), C3 (APOC3), and A4 (APOA4)--are closely linked and tandemly...
The genes coding for three proteins of the plasma lipid transport system--apolipoproteins A1 (APOA1), C3 (APOC3), and A4 (APOA4)--are closely linked and tandemly organized on the long arm of human chromosome 11. In this study the human APOA4 gene has been isolated and characterized. In contrast to APOA1 and APOC3 genes, which contain three introns, the APOA4 gene contains only two. An intron interrupting the 5' noncoding region of the APOA1 and APOC3 mRNAs is absent from the corresponding position of the APOA4 mRNA. However, similar to APOA1 and APOC3 genes, the introns of the APOA4 gene separate nucleotide sequences coding for the signal peptide and the amphipathic domains in APOA4. These results suggest that the APOA1, APOC3, and APOA4 genes were derived from a common evolutionary ancestor and indicate that during evolution the APOA4 gene lost one of its ancestral introns. Two restriction endonuclease sites, an Xba I located in the second intron of the APOA4 gene and a different Xba I located 9 kilobases 3' to the APOA4 gene, are polymorphic in Mediterranean and Northern European populations. Haplotype analysis indicated that even though these polymorphic sites are located within 9 kilobases they do not display significant nonrandom association. Finally, restriction mapping analysis of DNA from a patient with combined APOA1-APOC3 deficiency and premature coronary artery disease indicated that this patient has a structurally normal APOA4 gene.
Topics: Apolipoprotein A-I; Apolipoprotein C-III; Apolipoproteins A; Apolipoproteins C; Base Sequence; Biological Evolution; Coronary Disease; DNA; Humans; Polymorphism, Genetic; RNA, Messenger; Recombination, Genetic
PubMed: 3095836
DOI: 10.1073/pnas.83.22.8457 -
Journal of Lipid Research Aug 1986As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins,...
As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day. The results are consistent with the conclusions that: apoA-IV is present in human plasma in three distinct metabolic pools; apoA-IV associated with the triglyceride-rich lipoproteins is a precursor to the apoA-IV HDL and LFF pools; apoA-IV in LFF is not a free protein and its turnover rate is faster than that of apoA-IV in HDL; since no transfer of apoA-IV from the HDL or the LFF occurs, these pools may represent a terminal pathway for the catabolism of apoA-IV; and the catabolism of apoA-IV in HDL is dissociated from that of apoA-I although both apoproteins may reside on the same lipoprotein particles.
Topics: Apolipoprotein A-I; Apolipoproteins A; Chromatography, Gel; Chylomicrons; Humans; Iodine Radioisotopes; Kidney Transplantation; Kinetics; Radioimmunoassay; Reference Values
PubMed: 3095477
DOI: No ID Found