A rapid and simple quantification of human apolipoprotein E-rich high-density lipoproteins in serum.

A rapid and simple method for the quantitative determination of human serum apo E-rich high-density lipoproteins is described. A sample was divided into two parts; one part was mixed with an equal volume of 13% polyethylene glycol 6000, and the other part was mixed with a solution containing dextran sulfate, sodium phosphotungstate, and Mg2+, respectively. The mixed solutions were centrifuged (2000 g; 15 min). The supernate obtained by the former procedure contained both apo E-rich HDL and apo E-poor HDL, but that obtained by the latter procedure contained solely apo E-poor HDL. The serum apo E-rich HDL concentration in terms of apo E (E) and cholesterol (C), was given by the following equations: E = EP x 2, and C = (CP - CD) x 2, where EP and CP were the concentrations of apo E and cholesterol, respectively, in the supernate obtained with 13% polyethylene glycol, and CD was the concentration of cholesterol in the supernate obtained with the mixture solution of dextran sulfate, sodium phosphotungstate, and Mg2+. Normal serum apo E-rich HDL concentrations were 2.6 +/- 1.5 and 6.7 +/- 2.3 mg/dl (means +/- SD, n = 38) in terms of apo E and cholesterol, respectively. Apo E-rich HDL was increased strikingly in the sera from three patients with hepatobiliary diseases.     

人血清载脂蛋白CII酶联免疫测定法的研究

 本文介绍一种特异、灵敏、简便人血清载脂蛋白CⅡ(apoCⅡ)竞争性酶兔疫测定法(Competitive Enzyme Immunoassay,CEIA)。单价特异抗体由免疫家兔获得。采用部分纯化的apoCs包被聚苯乙烯板。羊抗兔γ-球蛋白酶交联物用辣根过氧化物酶按简化过碘酸钠法制备。apoCⅠ、AⅠ、AⅡ以及LDL无交叉反应。本法最小检测量为25ng,标准曲线工作范围是1.5～30.0mg％,板内及板间变异系数分别为6.5～7.8％及6.6～11.0％,回收率为107.5％;185例正常人血清apoCⅡ含量,男5.1±1.9mg％(n=95),女4.8±1.7mg％(n=90)。     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.     

Effect of Feeding Xenobiotics on Serum High Density Lipoprotein and Apolipoprotein A-I in Rats

The effects on serum cholesterol level were examined in rats fed on various xenobiotics. The hypercholesterolemia induced by polychlorinated biphenyls (PCB) was characterized in rats, from which lipoproteins were isolated by ultracentrifugation. A dietary addition of 0.03% PCB, 0.3% chloretone, 0.1% aminopyrine, or 0.2% 2,6-di-tert-butyl-p-cresol (BHT) resulted in a significant increase in serum cholesterol, although the chemical structure of each of these xenobiotics was different. The serum cholesterol level was markedly increased by one month of PCB feeding, the effect of PCB on the serum phospholipid level being similar. The serum triglyceride level transiently increased within 7 days of feeding with PCB diet. PCB feeding resulted in the elevation of all lipoproteins, including VLDL, LDL, HDL₁, and HDL₂, a marked increase being observed in HDI₁. Both HDL₁ and HDL₂ isolated from PCB-treated rats contained more apolipoprotein A-I (apo A-I) and less apo E than normal. VLDL isolated from PCB-treated rats had more cholesterol and apo E, but less apo C than that of the control animals. These data demonstrate that PCB feeding resulted in increased VLDL rich in cholesterol and apo E, and increased HDL rich in apo A-I. This experimentally induced hypercholesterolemia resulting in apo A-I-rich HDL would be a useful model for investigating the metabolism of apo-A-I and HDL.     

The cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c(552) from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R(h), 16.6 A). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c(552) with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c(552) from the apo state on the addition of heme has been demonstrated using NMR techniques. The properties of the apo state of C11A/C14A cytochrome c(552) reported here contrast strongly with those of mitochondrial cytochrome c whose apo state resembles a random coil under similar conditions.     

Quantification of apolipoprotein D in human urine by zone immunoelectrophoresis assay: a methodological and clinical study.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in unconcentrated native human urine. A standard curve, linear between 1 and 8 mg apo D/l was obtained with ZIA. The relative coefficients of variation for this method were 5-9% (n = 15 x 6) with a mean +/- SD of 7 +/- 1.4% and below 11% (n = 6 x 15) for within-run and between-run reproducibility, respectively. Equal amounts of apo D in unconcentrated and diluted urines, in serum and of the purified protein produced the same zone migration distances indicating parallelism between the immunologic reactions of apo D in different sample matrixes. Storage experiments with normal urines demonstrated good stability of apo D in both acidic and alkalinized urine over at least 2 days at +5 degrees C and during several days at -20 degrees C to -40 degrees C. Using ZIA, urine samples from 50 normal healthy men aged 23-65 years were analyzed for apo D. Mean and SD were: 2.8 +/- 2.1 mg/l, 2.6 +/- 1.8 micrograms/min and 0.24 +/- 0.13 mg/mmol for concentration, rate of excretion and mass/creatinine concentration, respectively.     

一次密度梯度超速离心分离人血清四种主要脂蛋白的某些理化性质的研究

 本文对一次密度梯度超速离心获得的四种脂蛋白(VLDL、LDL、HDL_2、HDL_3)进行了理化性质的研究。超速离心分析LDL、HDL_2,HDL_3均呈现一个单一尖锐的上浮峰,上浮速率分别为S_1 6.9和F~0_(1.21) 5.7及2.6。等电点聚焦电泳显示,VLDL主要含载脂蛋白C族,E族和少量A-I,B。HDL_2、HDL_3二者载脂蛋白的种类很相似,但量上略有差异,均以载脂蛋白A-Ⅰ,A-Ⅱ为主,Apoc’s,E次之。VLDL、LDL、HDL_2和HDL_3的化学组分分析与文献报道相似。 作者用本法初步分析了不同性别的各脂蛋白分布图,获得有意义的结果。     

Human apolipoprotein A-IV polymorphism: frequency and effect on lipid and lipoprotein levels

Summary Human apolipoprotein (apo) A-IV is genetically polymorphic, the apo A-IV polymorphism being controlled by two common alleles, A-IV¹ and A-IV². We have developed a method for typing the apo A-IV polymorphism by Western blotting using polyclonal rabbit antiapo A-IV as the first and gold-labeled antirabbit IgG as the second antibody. Apolipoprotein phenotypes were determined in plasma samples from 473 tiroleans. The frequencies of the apo A-IV alleles in this sample were f(A-IV¹)=0.919, f(A-IV²)=0.077, and f(A-IV³)=0.004. Although average triglyceride levels were lower in apo A-IV 2-1 heterozygotes, average total serum cholesterol and triglyceride levels were not significantly different among apo A-IV types. High density lipoprotein (HDL) cholesterol was significantly increased in individuals with the A-IV 2-1 phenotype. We estimate that genetic variation at the apo A-IV gene locus accounts for 11% of the total variability in HDL-cholesterol levels in Tiroleans. The effects of the apo A-IV polymorphism described here are consistant with, and may serve to enrich, our limited knowledge of the role of apo A-IV in lipid metabolism.     

Iron-sulfur cluster biosynthesis. A comparative kinetic analysis of native and Cys-substituted ISA-mediated [2Fe-2S]2+ cluster transfer to an apoferredoxin target

ISA type proteins mediate cluster transfer to apoprotein targets. Rate constants have been determined for cluster transfer from Schizosaccharomyces pombe ISA to apo Fd. Substitution of the cysteine residues of ISA produced derivative proteins (C72A, C136A, and C138A) that were found to be at least as active in cluster transfer reactions as the native form at 25 degrees C (k(2) approximately 170 M(-1) min(-1) for native, k(2) approximately 169 M(-1) min(-1) for C72A, k(2) approximately 206 M(-1) min(-1) for C136A, and k(2) approximately 242 M(-1) min(-1) for C138A), although the yield of cluster transfer was found to be lower as a consequence of the enhanced lability of clusters in the derivative proteins. Minor variations in rate constant for the ISA Cys derivatives do not reflect any change in the affinity of binding to the apo Fd since k(2) was found to be independent of the concentration of apo Fd over the range of 1-25 microM. The pH dependence of cluster transfer rates was found to be similar for native and C136A ISA, with an observed pK(a) of 7.8 determined from the pH profiles for cluster transfer activity of each protein. The temperature dependence of the rate constant defining the cluster transfer reaction for the wild type versus this C136A ISA derivative is distinct (DeltaH* approximately 6.3 kcal mol(-1) and DeltaS* approximately -27.3 cal K(-1) mol(-1) for native and DeltaH* approximately 2.7 kcal mol(-1) and DeltaS* approximately -38.9 cal K(-1) mol(-1) for C136A ISA). Instability of the protein-bound cluster precluded a comparison with data from pH and temperature dependencies for the two other Cys derivatives. Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer is rate-limiting. A comparison of cross-species rate constants for cluster transfer to apo Fd targets from Homo sapiens and S. pombe demonstrated that the identity of the Fd is less critical for promoting cluster transfer from Sp ISA (at 25 degrees C, k(2) approximately 170 M(-1) min(-1) for Sp Fd and k(2) approximately 169 M(-1) min(-1) for Hs Fd). This contrasts with an earlier observation for ISU-mediated cluster assembly [Wu, S., et al. (2002) Biochemistry 41, 8876-8885], where the rates differed for Hs and Sp target Fd's, suggesting distinct binding sites for binding of holo ISA and ISU to apo Fd.     

Effects of apolipoproteins on the kinetics of cholesterol exchange

The effects of apolipoproteins on the kinetics of cholesterol exchange have been investigated by monitoring the transfer of [14C]cholesterol from donor phospholipid/cholesterol complexes containing human apolipoproteins A, B, or C. Negatively charged discoidal and vesicular particles containing purified apolipoproteins complexed with lipid (75 mol % egg PC, 15 mol % dicetyl phosphate, and 10 mol % cholesterol) and a trace of [14C]cholesterol were incubated with a 10-fold excess of neural, acceptor, small unilamellar vesicles (SUV; 90 mol % egg PC and 10 mol % cholesterol). The donor and acceptor particles were separated by chromatography on DEAE-Sepharose, and the rate of movement of labeled cholesterol was analyzed as a first-order exchange process. The kinetics of exchange of cholesterol from both vesicular and discoidal complexes that contain apoproteins are consistent with an aqueous diffusion mechanism, as has been established previously for PC/cholesterol SUV. The addition of 2-3 molecules of apo A-I to a donor SUV does not significantly alter the half-time (t1/2), which is 80 +/- 9 min at 37 degrees C. However, addition of 5-12 apo A-I molecules progressively decreases t1/2 from 65 +/- 2 to 45 +/- 4 min. This enhancement in the rate of desorption of cholesterol molecules is presumed to arise from the creation of packing defects at boundaries around the apoprotein molecules, which are intercalated among the phospholipid and cholesterol molecules in the surface of the donor SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of apolipoprotein A-II with recombinant HDL containing egg phosphatidylcholine, unesterified cholesterol and apolipoprotein A-I

The preparation of discoidal, recombinant HDL (r-HDL) containing various phospholipids, apolipoproteins and a range of concentrations of unesterified cholesterol has been reported by several investigators. The present study describes the preparation of r-HDL containing both apolipoprotein (apo) A-I and apo A-II. r-HDL with 100:1 (mol:mol) egg PC.apo A-I and 0 (Series I), 5 (Series II) or 10 (Series III) mol% unesterified cholesterol were prepared by the cholate dialysis method. The resulting complexes had a Stokes' radius of 4.7 nm and contained two molecules of apo A-I per particle. When the r-HDL (2.0 mg apo A-I) were supplemented with 1.0 mg of apo A-II, one of the apo A-I molecules was replaced by two molecules of apo A-II. This modification was not accompanied by a loss of phospholipid, nor by major change in particle size. The addition of 2.5 or 4.0 mg of apo A-II resulted in the displacement of both apo A-I molecules from a proportion of the r-HDL and the formation of smaller particles (Stokes' radius 3.9 nm), which contained half the original number of egg PC molecules and three molecules of apo A-II. The amount of apo A-I displaced was dependent on the concentration of unesterified cholesterol in the r-HDL: when 2.5 mg of apo A-II was added to the Series I, II and III r-HDL, 44, 60 and 70%, respectively, of the apo A-I was displaced. Addition of 4.0 mg of apo A-II did not promote further displacement of apo A-I from any of the r-HDL. By contrast, the association of apo A-II with r-HDL was independent of the concentration of unesterified cholesterol and was a linear function of the amount of apo A-II which had been added. It is concluded that (1), the structural integrity of egg PC.unesterified cholesterol.apo A-I r-HDL, which contain two molecules of apo A-I, is not affected when one of the apo A-I molecules is replaced by two molecules of apo A-II; (2), when both apo A-I molecules are replaced by apo A-II, small particles which contain three molecules of apo A-II are formed; and (3), the displacement of apo A-I from r-HDL is facilitated by the presence of unesterified cholesterol in the particles.     

NMR solution structures of the apo and peptide-inhibited human rhinovirus 3C protease (Serotype 14): structural and dynamic comparison

The human rhinovirus (HRV) is a positive sense RNA virus responsible for about 30% of "common colds". It relies on a 182 residue cysteine protease (3C) to proteolytically process its single gene product. Inhibition of this enzyme in vitro and in vivo has consistently demonstrated cessation of viral replication. This suggests that 3C protease inhibitors could serve as good drug candidates. However, significant proteolytic substrate diversity exists within the 110+ known rhinovirus serotypes. To investigate this variability we used NMR to solve the structure of the rhinovirus serotype 14 3C protease (subgenus B) covalently bound to a peptide (acetyl-LEALFQ-ethylpropionate) inhibitor. The inhibitor-bound structure was determined to an overall rmsd of 0.82 A (backbone atoms) and 1.49 A (all heavy atoms). Comparison with the X-ray structure of the serotype 2 HRV 3C protease from subgenus A (51% sequence identity) bound to the inhibitor ruprintrivir allowed the identification of conserved intermolecular interactions involved in proximal substrate binding as well as subgenus differences that might account for the variability observed in SAR studies. To better characterize the 3C protease and investigate the structural and dynamic differences between the apo and bound states we also solved the solution structure of the apo form. The apo structure has an overall rmsd of 1.07 +/- 0.17 A over backbone atoms, which is greater by 0.25 A than what is seen for the inhibited enzyme (2B0F.pdb). This increase is localized to the enzyme's C-terminal beta-barrel domain, which is responsible for recognizing and binding proteolytic substrates. Amide hydrogen exchange dynamics revealed dramatic differences between the two enzyme states. Furthermore, a number of residues exhibited exchange-broadened amide NMR signals in the apo state compared to the inhibited state. The majority of these residues are associated with proteolytic substrate interaction.     

Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem.

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.     

Inhibition of apolipoprotein A-I gene expression by obesity-associated endocannabinoids

Obesity is associated with increased serum endocannabinoid (EC) levels and decreased high-density lipoprotein cholesterol (HDLc). Apolipoprotein A-I (apo A-I), the primary protein component of HDL is expressed primarily in the liver and small intestine. To determine whether ECs regulate apo A-I gene expression directly, the effect of the obesity-associated ECs anandamide and 2-arachidonylglycerol on apo A-I gene expression was examined in the hepatocyte cell line HepG2 and the intestinal cell line Caco-2. Apo A-I protein secretion was suppressed nearly 50% by anandamide and 2-arachidonoylglycerol in a dose-dependent manner in both cell lines. Anandamide treatment suppressed both apo A-I mRNA and apo A-I gene promoter activity in both cell lines. Studies using apo A-I promoter deletion constructs indicated that repression of apo A-I promoter activity by anandamide requires a previously identified nuclear receptor binding site designated as site A. Furthermore, anandamide-treatment inhibited protein-DNA complex formation with the site A probe. Exogenous over expression of cannabinoid receptor 1 (CBR1) in HepG2 cells suppressed apo A-I promoter activity, while in Caco-2 cells, exogenous expression of both CBR1 and CBR2 could repress apo A-I promoter activity. The suppressive effect of anandamide on apo A-I promoter activity in Hep G2 cells could be inhibited by CBR1 antagonist AM251 but not by AM630, a selective and potent CBR2 inhibitor. These results indicate that ECs directly suppress apo A-I gene expression in both hepatocytes and intestinal cells, contributing to the decrease in serum HDLc in obese individuals.     

地塞米松对HepG2细胞分泌载脂蛋白的影响

以培养的人肝癌细胞系HepG2为研究对象,采用“冻干浓缩培养液载脂蛋白测定法”,考察了地塞米松对HepG2细胞载脂蛋白(apolipoprotein,apo)AⅠ、AⅡ、CⅢ、B100及E分泌的影响.结果表明：地塞米松对HepG2细胞apoAⅠ和apoE的分泌有促进作用,对apoAⅡ、apoB100和apoCⅢ的分泌有抑制作用,且这种作用随地塞米松浓度的增加而增强.当培养液中地塞米松的浓度为5.5×10^－5mol/L时,apoAⅠ和apoE的分泌分别增加36.6%和49.4%（P＜0.01）,apoAⅡ、apoB100和apoCⅢ的分泌分别减少38.9%、31.9%和29.8%（P＜0.01）.     

Combined interaction of phospholipase C and apolipoprotein A-I with small unilamellar lecithin-cholesterol vesicles: influence of apolipoprotein A-I concentration and vesicle composition

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small ( approximately 5-15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400-800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0-60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 microg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Forster resonance energy transfer; structures are confirmed by cryo TEM.     

A new method for the fractionation of human plasma high density lipoprotein.

We have devised a new method for the fractionation of human plasma high density lipoprotein (HDL). The HDL was chromatographed on DEAE-agarose columns using a continuous gradient of 0.06--0.15 M NaCl. The elution pattern obtained showed three phases, each with differing peptide composition. Examination of the three subfraction showed that each contained both apoA-I and apo A-II, but in different proportions. Subfraction 1 contained no apo C-II or C-III-1 and only a trace of apo C-III-2, subfraction 2 contained apo C-II and C-III-1 but no C-III-2, while subfraction 3 contained considerable apo C-III-2 with only traces of apo C-II or C-III-1.     

Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.     

Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor

A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 °C) when compared to PAMO in solution (no activity detected after 1 h at 60 °C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier.     

Activation of phosphatidylcholine-sterol acyltransferase by human apolipoprotein E isoforms

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.