Interaction of apolipoprotein(a) with apolipoprotein B-containing lipoproteins

Recombinant DNA-derived apolipoprotein(a) was used to demonstrate that the apo(a) moiety of lipoprotein(a) (Lp(a)) is responsible for the binding of Lp(a) to other apolipoprotein B-containing lipoproteins (apoB-Lp) including LDL2, a subclass of low density lipoproteins (d = 1.030-1.063 g/ml). The r-apo(a).LDL2 complexes exhibited the same binding constant as Lp(a).LDL2 (10(-8) M). Treatment of either recombinant apo(a) or Lp(a) with a reducing agent destroyed binding activity. A synthetic polypeptide corresponding to a portion of apo(a)'s kringle-4 inhibited the binding (K1 = 1.9 x 10(-4) M) of LDL2 to Lp(a). Therefore, we concluded that binding to apoB-Lp was mediated by the kringle-4-like domains on apo(a). Using ligand chromatography which can detect complexes having a KD as low as 10(-2) M, we demonstrated the binding of plasminogen to apoB-Lp. Like Lp(a), binding of plasminogen to apoB-Lp was mediated by the kringle domain(s). The differences in binding affinity may be due to amino acid substitutions in the kringle-4-like domain. In most of the kringle-4-like domains of apo(a), the aspartic residue critical for binding to lysine was substituted by valine. Consistent with this substitution, we found that L-proline and hydroxyproline, but not L-lysine, inhibited the binding of LDL2 to apo(a). Inhibition by L-proline could be reversed in the binding studies by increasing the amount of apo(a); and L-proline-Sepharose bound plasma Lp(a), suggesting that L-proline acted as a ligand for the kringle-4-like domain(s) of apo(a) involved in the binding of apoB-Lp. The binding of apo(a) to proline and hydroxyproline could be responsible for the binding of apo(a) to the subendothelial extracellular matrix, i.e. domains of proteins rich in proline or hydroxyproline (e.g. collagen and elastin).     

Bacterial expression and in vitro refolding of a single-chain fv antibody specific for human plasma apolipoprotein B-100

From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli. The expression vector was constructed so that the scFv gene (V(L)-linker-V(H)) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15-20 mg per 1-liter flask cultivation.     

Self-association of human apolipoproteins A-I and A-II and interactions of apolipoprotein A-I with bile salts: quasi-elastic light scattering studies

We employed quasi-elastic light scattering (QLS) to systematically study the aqueous self-association of human apolipoproteins A-I and A-II (apo A-I and apo A-II) and the interactions of apo A-I with common taurine-conjugated bile salts. Self-association of apo A-I was promoted by increases in apolipoprotein concentration (0.09-2.2 mg/mL) and ionic strength (0.15-2.0 M NaCl), inhibited by increases in temperature (5-50 degrees C) and guanidine hydrochloride concentration (0-2.0 M), and unaffected by hydrostatic pressures up to 500 atm. The mean hydrodynamic radius (Rh) of apo A-I micelles ranged from 38 A to a maximum asymptotic value of 68 A. We examined several possible models of apo A-I self-association; the model that best fitted the Rh values assumed that apo A-I monomers first interacted at low concentrations to form dimers, which then further associated to form ring-shaped limiting octamers. Comparison of the temperature-dependent and ionic strength dependent free energy changes for the formation of octamers from apo A-I dimers suggested that hydrophobic forces strongly favored self-association and that electrostatic repulsive forces were only weakly counteractive. Apo A-II self-association was also promoted by increases in apolipoprotein concentration (0.2-1.8 mg/mL) and inhibited by increases in guanidine hydrochloride concentration (0-1.0 M) but was unaffected by variations in temperature (10-37 degrees C): the largest Rh values observed were consistent with limiting tetramers. As demonstrated by equilibrium dialysis, bile salts in concentrations below their critical micellar concentrations (cmc) bound to apo A-I micelles but had no effect upon apo A-I self-association, as inferred from constant Rh values. When bile salt concentrations exceeded their aqueous cmc values, a dissociation of apo A-I micelles resulted with the formation of mixed bile salt/apo A-I micelles. These studies support the concepts that apo A-I and apo A-II form small dimeric micelles at low concentrations that grow sharply to reach limiting sizes over a narrow concentration range. The influences of bile salt concentration and species upon these micelles have relevance to the plasma transport of bile salts in high-density lipoproteins and to the physical-chemical state of apo A-I and apo A-II molecules in native biles.     

Abetalipoproteinemia with an ApoB-100-lipoprotein(a) glycoprotein complex in plasma. Indication for an assembly defect

Patients with autosomal recessive abetalipoproteinemia (ABL) lack in their plasma all lipoproteins containing apolipoprotein (apo)B-100 or B-48. Previous studies have suggested that this is due to the complete absence of apoB. We have investigated whether such patients (n = 10) are able to secrete the lipoprotein(a) (Lp(a] glycoprotein (apo(a] which, in normal plasma, exists as a complex with low density lipoproteins containing apoB-100 (Lp(a) lipoprotein). All 10 patients had reduced but detectable apo(a) levels in plasma (mean, 0.49 mg/dl; range, 0.2-2.03 mg/dl) but no Lp(a) lipoprotein. However, we also detected small amounts (0.2-2.8 mg/dl) of apoB in all patients with ABL. The apoB in the ABL patients had the size of apoB-100 and occurred as a lipid-poor complex with the Lp(a) glycoprotein in a fraction of density 1.22 g/ml. This material may represent partially assembled Lp(a) lipoprotein. There was also uncomplexed apo(a) and apoB-100 in the ABL plasma. The distribution and relative concentration of both proteins in the density fraction greater than 1.06 g/ml varied among patients. The data suggest that in ABL, the assembly of apoB-containing lipoproteins is defective and that apoB-100 may be secreted without its full lipid complement when complexed with apo(a).     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.     

Alterations in plasma lipoproteins and apolipoproteins associated with estrogen-induced hyperlipidemia in the laying hen

The laying hen represents a physiological model in which the mechanisms of action of estrogens on lipid transport can be evaluated. The plasma lipoproteins in the laying hen were subfractionated into discrete particle species by isopycnic density gradient ultracentrifugation and the physicochemical properties and apolipoprotein contents of individual subfractions evaluated. The qualitative and quantitative aspects of this estrogen-specific profile were then compared to those of the immature chicken. As observed earlier, estrogens induced dramatic elevation in very-low-density lipoproteins (VLDL) (up to 900 mg/dl). Indeed, triglyceride-rich lipoproteins with densities up to 1.035 g/ml, i.e. VLDL and their remnants, behaved as a continuum which displayed little variation in size (20.5-21 nm), electrophoretic mobility (beta-like) and apolipoprotein content; apo B-100 (540 kDa) predominated while apo A-I (27 kDa), apo VLDL-II (19 kDa) and an apo-C-like protein (13 kDa) were present as minor components. The typical high-density lipoproteins (HDL) in the immature chicken were replaced by a lipoprotein population whose physicochemical properties were quite distinct. Thus these particles were distributed as a single, asymmetric peak over the density range 1.030-1.158 g/ml, a wide interval which overlapped that of apo-B-rich particles at its lower limit. The rho 1.030-1.158 g/ml lipoproteins were present at concentrations (approximately equal to 200 mg/dl) some twofold to threefold lower than those of HDL in immature birds. Furthermore, they displayed physical and chemical properties in common with both low-density lipoproteins (LDL) and HDL and were LDL-like in exhibiting beta mobility but HDL-like in size (9-15 nm diameter). Their protein moiety was also HDL-like in its predominant content of apo A-I; small amounts of apo VLDL-II and the apo-C-like protein were also detected. Substantial amounts of lipid were found at rho greater than 1.195 g/ml: such substances are absent in the immature chicken and may reflect the presence of vitellogenins. The hyperestrogenic state in the laying hen is therefore associated with major modifications in lipoprotein and apolipoprotein profile. Such modifications may be of relevance to clinical disorders involving estrogen-induced hyperlipidemia.     

Stimulation of lecithin:cholesterol acyltransferase activity by apolipoprotein A-II in the presence of apolipoprotein A-I

Various combinations of incorporation and addition of apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) individually or together to a defined lecithin-cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A-II on lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A-I (0.1-3.0 nmol/assay) alone was incorporated or added to the liposome, apo A-I effectively activated the enzyme. By contrast, when apo A-II (0.1-3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A-II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A-I. Addition of apo A-II (0.1-3.0 nmol/assay) together with apo A-I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A-I alone. On the other hand, addition of apo A-II (0.1-3.0 nmol/assay) or addition of lecithin-cholesterol liposome containing apo A-II (0.1-3.0 nmol/assay) to lecithin-cholesterol liposome containing apo A-I (0.8 nmol/assay) did not significantly alter apo A-I activation of LCAT activity. However, when the same amounts (0.1-3.0 nmol/assay) of apo A-II were incorporated together with apo A-I (0.8 nmol/assay) into the liposome, apo A-II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A-I alone. The maximal stimulation was obtained with 0.4 nmol apo A-II/assay for both purified and plasma enzyme. At this apo A-II concentration, approximately 4-fold and 1.8-fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A-II must be incorporated together with apo A-I into lecithin-cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A-II in high-density lipoprotein may play an important role in the regulation of LCAT activity.     

Hepatitis B virus surface antigen binds to apolipoprotein H.

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

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)。     

Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100

We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.     

Binding of human apolipoprotein A-IV to human hepatocellular plasma membranes

We have investigated the binding of human apolipoprotein A-IV (apo A-IV) to human hepatocellular plasma membranes. Addition of increasing concentrations of radiolabeled apo A-IV to hepatic plasma membranes, in the presence and absence of a 25-fold excess of unlabeled apo A-IV, revealed saturation binding to the membranes with a KD of 154 nM and a binding maximum of 1.6 ng/microgram of membrane protein. The binding was temperature-insensitive, partially calcium-dependent, abolished when apo A-IV was denatured by guanidine hydrochloride or when the membranes were treated with Pronase and decreased when apo A-IV was incorporated into phospholipid/cholesterol proteoliposomes. In displacement studies using purified apolipoproteins and isolated lipoproteins, only unlabeled apo A-IV, apo A-I and high-density lipoproteins effectively competed with radiolabeled apo A-IV for membrane binding sites. We conclude that human apo A-IV exhibits high-affinity binding to isolated human hepatocellular plasma membranes which is saturable, reversible and specific.     

Sandwich enzyme-linked immunosorbent assay of rat apolipoprotein E

A sandwich ELISA for rat apo E was developed. Blood was drawn from rats that had been administered with triamcinolone diacetate to increase apo E-rich HDL in serum. Apo E was purified from the d less than 1.225 g/ml lipoproteins, and antiserum was raised in rabbits. Diluted samples and standards were added into the wells of polystyrene microtiter plates precoated with immunoaffinity-purified IgG and incubated for 90 min. After washing, immunoaffinity-purified Fab'-horseradish peroxidase conjugate was added to each well and incubated for 90 min. The bound enzyme was assayed by a colorimetric method. Samples and standards were pretreated with 6 M guanidine. HCl to maximize the antigenic response of apo E. The sensitivity lies around 1 pg apo E, and the working range was 0.1 to 1.0 ng. All assay procedures were completed within 4-5 hr. The mean intra- and interassay coefficients of variation were 1.8 and 4.1%, respectively. Serum apo E concentrations were 21.2 +/- 2.1 and 61.3 +/- 17.0 mg/dl (mean +/- SD) for young (8-12 weeks old, n = 9) and old (36-40 weeks old, n = 16) rats, respectively. As determined by gel filtrations, most of the apo E in fasted rat serum was associated with larger HDL particles (or HDL1) and a small portion of apo E was present in a free form.     

Purification and properties of phospholipase A2 from human seminal plasma

The soluble Ca2+-dependent phospholipase A2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 10(-4) M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2.     

Effects of alcohol on the major steps of reverse cholesterol transport

The effects of moderate alcohol consumption on the capacity of blood sera to promote acceptance of cholesterol (C) from Fu5AH hepatoma cells, esterification of delivered free C, and transfer of produced cholesteryl esters to apolipoprotein (apo) B-containing lipoproteins have been studied. Twenty male subjects with relatively high (>50 mg/dl, n = 10) and low (<50 mg/dl, n = 10) high density lipoprotein (HDL) C levels consumed for eight weeks red grape wine (0.3 g ethanol/kg body mass per day). Alcohol consumption reduced total C and low density lipoprotein C levels in both groups of subjects. Low HDL C subjects showed an increase in HDL C, apo AI, apo AII, and lipoprotein (Lp) AI particle levels after alcohol consumption. Alcohol did not affect free C efflux from the cells. However, after the following period of substitution of alcohol with an isocaloric amount of red grape juice, cellular C efflux markedly reduced. While lecithin:cholesterol acyltransferase (LCAT) activity increased during alcohol consumption only in subjects with low HDL C, high HDL C subjects showed a significant decrease in cholesteryl ester transfer protein (CETP) activity. At the same time, alcohol consumption reduced the endogenous C esterification rate and increased the transfer of endogenous cholesteryl esters to apo B-containing lipoproteins in both groups. Thus, alcohol consumption in moderate doses enhanced the anti-atherogenicity of the serum lipoprotein spectrum, supporting more effective C efflux from peripheral cells and transport of accepted C to apo B-containing lipoproteins. The effects of alcohol on the reverse cholesterol transport depend on the initial HDL C level.     

Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I

An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (gamma 1, kappa), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography. The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). The affinity constant was determined by a biosensor method using the BIAcore system. The results showed that the yield of correctly refolded scFv was more than 20 mg l-1 of E. coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74 x 10(-8) M (Kd). A notable thing is that guanidine-HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was. This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies.     

High affinity binding between lipoprotein lipase and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity, and is modulated by glycosaminoglycans

Lipoprotein lipase (LPL) physically associates with lipoproteins and hydrolyzes triglycerides. To characterize the binding of LPL to lipoproteins, we studied the binding of low density lipoproteins (LDL), apolipoprotein (apo) B17, and various apoB-FLAG (DYKDDDDK octapeptide) chimeras to purified LPL. LDL bound to LPL with high affinity (K(d) values of 10(-12) m) similar to that observed for the binding of LDL to its receptors and 1D1, a monoclonal antibody to LDL, and was greater than its affinity for microsomal triglyceride transfer protein. LDL-LPL binding was sensitive to both salt and detergents, indicating the involvement of both hydrophobic and hydrophilic interactions. In contrast, the N-terminal 17% of apoB interacted with LPL mainly via ionic interactions. Binding of various apoB fusion peptides suggested that LPL bound to apoB at multiple sites within apoB17. Tetrahydrolipstatin, a potent enzyme activity inhibitor, had no effect on apoB-LPL binding, indicating that the enzyme activity was not required for apoB binding. LDL-LPL binding was inhibited by monoclonal antibodies that recognize amino acids 380-410 in the C-terminal region of LPL, a region also shown to interact with heparin and LDL receptor-related protein. The LDL-LPL binding was also inhibited by glycosaminoglycans (GAGs); heparin inhibited the interactions by approximately 50% and removal of trace amounts of heparin from LPL preparations increased LDL binding. Thus, we conclude that the high affinity binding between LPL and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity and is modulated by GAGs. It is proposed that LPL contains a surface exposed positively charged amino acid cluster that may be important for various physiological interactions of LPL with different biologically important molecules. Moreover, we postulate that by binding to this cluster, GAGs modulate the association between LDL and LPL and the in vivo metabolism of LPL.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Purification and study of the physicochemical properties of angiotensin converting enzyme from the human liver

Angiotensin-converting enzyme (ACE) from human liver was first purified 9000-fold by chromatofocusing with 22% yield. The enzyme had a specific activity of 10 U/mg. The enzyme molecular weight was 150000, as determined by electrophoresis in a 7.5% polyacrylamide gel. The enzyme pI determined by chromatofocusing was 4.2-4.3. KM of human liver ACE, measured using hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine as substrates, was 5 mM and 0.1 mM, respectively. Human liver ACE was inhibited by SQ 20881 with IC50 equal to 1.8 X 10(-8) M.     

Antipeptide antibodies reveal interrelationships of MBP 200 and MBP 235: unique apoB-specific receptors for triglyceride-rich lipoproteins on human monocyte-macrophages

Two human monocyte-macrophage (HMM) membrane binding proteins, (MBP) 200 and 235, are receptor candidates that bind to the apolipoprotein (apo)B-48 domain in triglyceride-rich lipoproteins for uptake independent of apoE. Microsequence analysis of the purified reduced MBP 200R characterized tryptic peptides of MBP 200R. A synthetic peptide mimicking a unique, unambiguous 10-residue sequence (AEGLMVTGGR) induced antipeptide antibodies that specifically recognized MBP 200, 235 and 200R, in 1- and 2-dimensional analyses, indicating 1) the ligand binding protein was sequenced and 2) MBP 200 and 235 yielded MBP 200R upon reduction. These antibodies identified the MBPs in human blood-borne, THP-1, U937 MMs, and endothelial cells (EC) but not in human fibroblasts or Chinese hamster ovary (CHO) cells. Fluorescence activated cell sorting (FACS) analysis located the MBPs on the MM surface as necessary for receptor function. The 10-residue, unambiguous MBP 200-derived sequence is unique, with no matches in extant protein databases. Antipeptide antibodies bind to the MBPs in reticuloendothelial cells that have this receptor activity, but not to proteins in cells that lack this receptor activity. These studies provide the first direct protein sequence and immunochemical data that a new, unique apoB receptor for triglyceride-rich lipoproteins exists in human monocytes, macrophages, and endothelial cells.     

Purification and Properties of UDP-galactose 4-Epimerase from Bifidobacterium bifidum

UDP-galactose 4-epimerase (EC 5.1.3.2) was purified to a homogeneous state from Bifidobacterium bifidutn grown on a glucose medium. The molecular weight was estimated to be about 90,000. The purified enzyme was very stable and 60 % of its initial activity survived three months of storage at 4°C even at a low protein concentration (0.2 mg/ml). The optimum pH was 9.0, and the Km values for UDP-galactose and UDP-glucose were 5.4 × 10^-4 M and 1.4×10 ^-3 M. UDP was a competitive inhibitor. The enzyme activity was stimulated by various sugar phosphates, but was slightly inhibited by fructose 1,6-diphosphate (FDP). A high concentration of galactose or glucose, which had no effect by itself, inhibited the activity in combination with UMP. The inhibition by FDP was also enhanced by combination with UMP.