Bovine Lipoprotein Research Articles

Isolation and characterization of lipid-protein complexes present in commercial albumin preparations.

Most commercially available albumin preparations examined by us contain phospholipids, cholesterol and apolipoprotein impurities. As these albumin preparations are frequently used in large amounts in systems involving lipoprotein metabolism these impurities may reach remarkable levels to introduce exogenous effects in these studies. We have studied in detail tow bovine albumin preparations differing in their content of these contaminants. Using preparative ultracentrifugation, we have isolated from both albumins a lipid protein complex at a buoyant density of d = 1.063-1.21 g/ml with a chemical composition resembling plasma high density lipoproteins. This complex when further characterized proved also to have a similar apoprotein composition to bovine plasma high density lipoproteins. Electron microscopic study of this complex revealed discoidal particles closely resembling nascent high density lipoproteins recovered from rat liver or lymph. The similarity of these lipid-protein complexes to high density lipoproteins, accounts for some reported effects caused by commercially available albumin preparations on cholesterol excretion from cells in tissue culture and their ability to act as acceptors for surface remnants released upon VLDL catabolism in vitro.

Bovine apolipoproteins C: I. Isolation and spectroscopic investigations of the phospholipid binding properties

Seven low-molecular weight proteins of the C class of apolipoproteins have been isolated from bovine serum high density lipoprotein. Amino acid analysis has shown five of these to be equivalent to the apolipoproteins previously described (Lim C.T. and Scanu A.M. (1976) Artery 2, 483–496). A spectroscopic examination of these proteins in the presence of increasing amounts of l-α-dimyristoyl phosphatidylcholine single-bilayer vesicles indicates that all bovine apolipoproteins C exhibit changes in secondary and tertiary structure as shown by intrinsic fluorescence intensity, wavelength, and polarization changes, and increases in α-helical content as seen by circular dichroism. Evidence is presented to show that bovine apolipoproteins C, like all human apolipoproteins of the A and C classes, can cause phospholipid multilamellar liposomes to disrupt and/or rearrange into a smaller complex which scatters less light. This paper details the screening of the bovine apolipoproteins for their phospholipid binding properties, whereas the following paper will examine the nature of their complexes with phospholipid in more detail. Together, these papers represent the first investigation of protein-lipid interactions involving any nonhuman apolipoproteins C.

Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase

In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydro lized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.

Electrophoretic Characterization of Bovine Lipoprotein Subfractions Isolated by Agarose Gel Chromatography

Lipoproteins isolated from whole bovine serum were applied to agarose gel columns and fractionated by molecular size. Three distinct lipoprotein absorbance peaks (280nm) emerged from the columns (Peaks I, II, and III). A typical elution profile exhibited lipoprotein distribution of approximately 11% Peak I, 6% Peak II, and 83% Peak III. Characterization of Peak I lipoproteins on agarose gel electrophoresis and polyacrylamide gel electrophoresis suggested that Peak I consisted of very low density lipoproteins. Comparison of polyacrylamide gel electrophoresis patterns of successive tube fractions in Peak II demonstrated the progressive emergence of three bands. The slowest migrating band appeared in fractions from the ascending portion of Peak II. Two additional bands progressively emerged from fractions in the central portion of the peak. These bands may represert low density lipoprotein and its metabolic precursors (intermediate density lipoproteins). Successive tube fractions in the area between Peaks II and III displayed a progressive change from β to α migration on both polyacrylamide gel electrophoresis and paper electrophoresis. This heterogeneous group of lipoproteins is probably equivalent to high density lipoprotein-subfraction 1. Lipoproteins from the center of Peak III demonstrated a electrophoretic migration patterns.

Interaction of bovine serum high density lipoprotein with mixed vesicles of phosphatidylcholine and cholesterol.

The interaction of sonicated, small vesicles of egg phosphatidylcholine and cholesterol (2:1, mol/mol) with bovine high density serum lipoproteins was examined in terms of lipid transfer between both types of particles and the resulting changes in lipoprotein structure. Saturation of high density lipoprotein preparations with vesicle lipids gave final lipoprotein particles with essentially unchanged protein content and composition, unchanged cholesterylester and nonpolar lipid content, but with markedly increased phospholipid content (59% increas by weight) and moderately increased cholesterol content (20% increase by weight). The lipoproteins enriched in lipid were relatively uniform, spherical particles, 110 +/- 3.6 A in diameter (6 A larger than the original lipoproteins); they had a markedly decreased intrinsic protein fluorescence, a red-shifted fluorescence wavelength maximum, and more fluid lipid domains. These results indicate that the direct addition of excess lipids from membranes or other lipoproteins is a possible mechanism for lipid transfer to high density lipoproteins. Also they suggest a structural flexibility of high density lipoproteins that allows the addition of significant amounts of surface components.

Active Ca 2+ transport by vesicles reconstituted from triton X-100-solubilized pigeon erythrocyte membrane

Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidylethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of 45Ca 2+. ATP-dependent 45Ca 2+ uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca 2+ uptake rate depended on the square of the Ca 2+ concentration and had similar [Ca 2+] 1 2 values, 0.16 μM and 0.18 μM, respectively.

Dissociation of apolipoprotein A-I from porcine and bovine high density lipoproteins by guanidine hydrochloride

Dissociation of apolipoprotein A-I from pig and steer high density lipoproteins (HDL) deficient in apoA-II was determined by exposing native HDL fractions to 6 M guanidine hydrochloride (Gdn-HCl) at 37°C for periods from 5 min to 18 h. Bovine high density lipoprotein (HDL-B) was isolated at d 1.063–1.100 g/ ml while porcine high density lipoprotein (HDL-P) was isolated at d 1.125–1.21 g/ ml. Incubation for 5 min with Gdn-HCl resulted in a 45 and 3% loss of apoA-I from HDL-P and HDL-B, respectively. Exposure to the denaturant for 3 h resulted in a 75% loss of apoA-I from HDL-P and a 30% loss from HDL-B. Analytic ultracentrifugation patterns paralleled the degree of apoA-I dissociation from each HDL species. The initial flotation peak for HDLP shifted from F° 1.20 2.68 to F° 1.20 10.75 after 3 h exposure while HDL-B showed only a small shift from F° 1.20 8.30 to F° 1.20 8.96 after 3 h exposure. HDL-P particle diameter increased 25% after 5 min of Gdn-HCl treatment and large, flattened structures predominated after 3 h. There was no change in the size of HDL-B after 5 min exposure and only 16% increase in particle diameter after 3 h. The difference in behavior of HDL-B and HDL-P to Gdn-HCl exposure is discussed in terms of differences in apolipoprotein A-I amino acid composition, interaction of apolipoprotein A-I with phospholipids and the possible involvement of the cholesteryl ester core.

Kinetics and mechanism of phosphatidylcholine and cholesterol exchange between single bilayer vesicles and bovine serum high-density lipoprotein

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKinetics and mechanism of phosphatidylcholine and cholesterol exchange between single bilayer vesicles and bovine serum high-density lipoproteinAna Jonas and Gregory T. MaineCite this: Biochemistry 1979, 18, 9, 1722–1728Publication Date (Print):May 1, 1979Publication History Published online1 May 2002Published inissue 1 May 1979https://doi.org/10.1021/bi00576a014RIGHTS & PERMISSIONSArticle Views78Altmetric-Citations69LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (829 KB) Get e-Alerts Get e-Alerts

動脈壁脂肪酸代謝の研究

Acyl-CoA synthetase was estimated as an amount of acyl-CoA synthesis from radioactive palmitic acid in the presence of ATP, CoASH, and MgCl2.Characteristics of acyl-CoA synthetase in arterial wall extract were examined. Km values for palmitate or ATP or CoASH or Mg2+ were 0.29, 3.45, 0.03, 4.0mM, respectively. The optimum pH was around 8.0 and the highest specific activity was found in microsomal fraction. These characteristics were almost similar to those of liver acyl-CoA synthetase.Acyl-CoA synthetase was inhibited by divalent cations such as Cu2+, Fe2+, Zn2+ and Ca2+ and was activated by serum, bovine serum albumin and lipoproteins. Changes of these enzyme activities were found in some pathological conditions. Increase in aged or hypercholesterolemic rat was found and in experimental hypertensive or diabetic or tocopherol deficient condition, the activities were found to be decreased. The relationship between regulatory factors and changes of this enzyme activities in pathological conditions was discussed.

Effects of triton X-100 treatments on the composition and activities of membrane vesicles from pigeon erythrocytes

Membrane vesicles were prepared from pigeon erythrocytes. The effect of various treatments on the ability of these vesicles to trap and transport glycine was measured. Most of the work concerned the effects of the nonionic detergent, Triton X-100 (Triton). Triton inhibits the capacity of membrane vesicles to trap and transport glycine. The effects of Triton depend on temperaature and pH. At neutral pH and 41°C, the half-inactivating dose of Triton is 6 μl/g wet weight of membrane, while at neutral pH and O°C it is approx. 60 μl/g. At pH 8.5–8.8 and 0°C the half-inactivating dose is 15–20 μl/g. The Triton effect depends on the ratio of Triton to membrane, not the Triton ‘concentration’ in the aqueous phase. Protein is released from the membrane by Triton treatment at 0°C. At pH 8.5–8.8, the dose-response curve for protein release is very similar to the dose-response curve for inhibition of the capacities to trap and take up glycine. All three curves have steep and shallow regions, respectively below and above a Triton dose of 15 μl/g. The protein released by Triton treatment at 0°C is largely the lower molecular weight classes and the maximum protein release is 50–60% of the total membrane protein. The percent of this protein class released by a given Triton dose equals the percent inhibition of the capacity to trap glycine. Triton is bound by the membranes. In the dose range of 0–10 μl Triton/g wet membrane, about half of the added Triton is bound. This bound Triton is not readily removed by washing. Like protein release and inhibition of trapping and transport capacities, the binding process has two phases, one above and one below a dose of 15 μl/g. The membrane is saturated with Triton at a dose of 15 μl/g with 4.5 μl Triton bound/g wet weight, corresponding to 85–90 μl bound/g remaining membrane protein. Bovine serum high density lipoprotein efficiently removes bound Triton from the membrane. By removing Triton bound at 0°C with lipoprotein, the effects of Triton at 0 and 41°C could be distinguished and the time course of Triton action at 0°C could be measured. Triton action was nearly complete by 10 min at 0°C. Possible modes of action of Triton on these membranes are discussed. At least part of the action of Triton can be described as a process in which membrane proteins are partitioned between the membrane phase and an extramembranal Triton micelle phase.

Incorporation of excess cholesterol by high density serum lipoproteins

Excess cholesterol was added to human HDL 3 and to bovine mammalian high density serum lipoprotein (HDL) by incubating aqueous lipoprotein solutions with solid dispersions of [4- 14C]cholesterol on Celite. Lipoprotein · cholesterol complexes were isolated by centrifugation and filtration through a Sepharose 4B column. The pure complexes were analyzed for protein and lipid content and composition and were subsequently investigated by physical methods (analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy), in order to detect any structural changes induced by added cholesterol. The rates of cholesterol uptake varied as an inverse function of the intrinsic cholesterol present in the native lipoproteins. The maximum cholesterol taken up by human HDL 3 increased the free cholesterol content from 3–4% (initial) up to 22% of the total lipoprotein weight. Bovine HDL was observed to increase its free cholesterol content from 2–4% (initial) up to 11–17% of the total lipoprotein weight, before denaturation. At maximum levels of added cholesterol, both lipoproteins had increased molecular weights and sedimentation velocity coefficients corresponding to the increased mass of the particles. No major changes in the hydrodynamic properties were observed. At the molecular level, the protein components only showed a 15–20% decrease in fluorescence intensity, possibly a consequence of a modified environment of the aromatic amino acid residues. In the human HDL 3, added cholesterol increased the microviscosity of the lipid domains by 1.2 P at 25°C (from 3.4 to 4.6 P), but did not affect the fluidity of bovine HDL lipids (5.9 P).

Interaction of human and bovine A-1 apolipoproteins with L-alpha-dimyristoyl phosphadicylcholine and L-alpha-myristoyl lysophosphatidylcholine.

The major protein components from human and bovine high density serum lipoproteins (apo-A-I proteins) were investigated in their interactions with L-alpha-myristoyl lysophosphatidylcholine and L-alpha-dimyristoyl phosphatidylcholine. Complex formation was followed by 25 degrees by observing changes in fluorescence polarization, rotational relaxation times (ph), and CD spectra of the proteins, covalently labeled with fluorescent dimethylaminonaphthalene sulfonyl groups. Monomeric human apo-A-I and initially oligomeric bovine apo-A-I interact with similar efficiency with the same amphiphiles. During binding of L-alpha-myristoyl lysophosphatidylcholine and L-alpha-dimyristoyl phosphatidylcholine, the structure of both proteins changes drastically, exhibiting about 35% increases in secondary structure with the phospholipid and about 20% increases with the lysophospholipid. Simultaneously, regions of the proteins adjacent to the fluorescent probes become more mobile. With the bovine protein, binding of both amphiphiles results in changes in the oligomeric structure: dissociation with L-alpha-myristoyl lysophosphatidylcholine and dissociation or rearrangement with L-alpha-dimyristoyl phosphatidylcholine. The complexes formed in the presence of excess L-alpha-myristoyl lysophosphatidylcholine are flexible structures with considerable rotational freedom. The largest rotational unit in these complexes has ph = 60 ns for both proteins; by comparison, the monometic human apo-A-I has a ph = 73.5 ns. Interactions of both proteins with the lysophospholipid take place well above its critical micelle concentration, but probably involve binding of monomeric lipid. With L-alpha-dimyristoyl phosphatidylcholine the complexes are different from those formed with the lysophospholipid. They have limiting ph values of 250 +/- 50 and 280 +/- 60 ns with the human and bovine proteins, respectively. These ph values are consistent with particles of the general size of human high density lipoprotein rather than of liposomes and indicate the formation of distinct, relatively small structures upon interaction of the self-associated lipid with the apo-A-I-proteins.

Reassembly [formula omitted] of lung surfactant lipoprotein

This report describes a successful attempt to reassemble, in vitro , two fractions obtained from bovine lung surfactant lipoprotein. An apoprotein isolated by gel filtration in the presence of sodium deoxycholate was recombined with lipid extracts of the surfactant, in a highly alkaline buffer (pH 10) containing 10 mM sodium deoxycholate. Sonication, dilution 1 to 10, dialysis, and washing by means of centrifugation were used to produce a lipid-protein complex. Centrifugation in a continuous sucrose density gradient revealed that this material had a density of 1.081 gm/ml and a phospholipid/protein ratio respectively almost the same as those of the original lipoprotein.

Fat emulsions with added free cholesterol or fatty acid cholesteryl esters. Studies on removal mechanisms in vivo and hydrolysis by lipoprotein lipase in vitro.

The fractional elimination rate of fat emulsions of soybean oil, emulsified with egg yolk phosphatides with 1% addition of cholesterol or various cholesteryl fatty acid esters, was studied in rabbits. The fractional removal rate k2%/min was the same after addition of free cholesterol or esters with fatty acids up to eight carbon esters. The k2 values were twice as high for emulsions with cholesteryl-stearate, three times higher with added cholesteryl-palmitate and four times higher when cholesteryl-linoleate was added. The triglyceride (TG) lipase activity was determined with human or rabbit post-heparin plasma and with purified bovine lipoprotein lipase. All these enzyme sources gave similar results. Addition of saturated cholesteryl esters did not affect the lipase activity, but addition of 1% cholesterol markedly decreased the lipase activity. Furthermore, addition of cholesteryl-linoleate and linolenate reduced post-heparin TG lipase activity.

Use of a cholesterol-rich bovine lipoprotein to enhance cholesterol concentrations in the preparation of serum control materials.

We describe a process for preparing a cholesterol-rich lipoprotein extract from bovine serum. The material is used to enhance cholestrol concentration up to 3.5 g/liter in a clear control material from human serum. The optical clarity of the enriched serum is only slightly affected by lyophilization and reconstitution. The lipoprotein additive neither produces any evidnet interference in the measurement of the commonly analyzed consitutents nor affects the stability of components after lyophilization and reconstitution.

Interaction of phosphatidylcholine with bovine serum albumin. Specificity and properties of the complexes

Phosphatidylcholine dispersed on Celite was rapidly solubilized by neutral bovine serum albumin solutions. Stable protein-lipid complexes were isolated by Agrose gel filtration or by ultracentrifugal flotation in high density solvents, and the physicochemical properties of the complexes were investigated in terms of the stoichiometry of binding, effect of fatty acid ligands on phosphatidylcholine binding, effect of high ionic strength on the stability of the complexes, intrinsic fluorescence and circular dichroism spectra, and sedimentation velocity coefficients. Complexes containing from 2 to 30 phosphatidylcholine molecules per protein molecule were observed; however, no saturation of binding sites could be detected in this range of molar ratios. Oleic acid binding by serum albumin prevents interaction of the protein with phosphatidylcholine, indicating possible competition of these ligands at low contents of the phospholipid. For molar ratios of up to 10 phosphatidylcholine molecules per serum albumin, binding is primarily due to hydrophobic interactions that have no effect on the overall shape and secondary structure of the native protein except for local modifications at tryptophan residues, whose fluorescence becomes quenched and blue shifted on phosphatidylcholine binding. Similar phosphatidylcholine uptake experiments performed with a series of globular proteins indicated that the lipid extraction from Celite surfaces is a non-specific process, accelerated by several other proteins (e.g. aldolase, egg albumin, chymotrypsinogen, soybean trypsin inhibitor, and the major apolipoprotein from bovine serum high density lipoprotein). Formation of stable protein-lipid complexes, however, was only observed with bovine serum albumin, which in contrast to the other proteins is known to have high affinity binding sites for anions with hydrophobic side chains.

Immunological Properties of Bovine Serum Lipoproteins and Chemical Analysis of Their Protein Moieties

Four classes of bovine serum lipoproteins were isolated by precipitation with dextran sulfate, ultracentrifugation, and preparative electrophoresis on polyacrylamide gel. Very low density lipoprotein (d<1.019 g/ml) was related immunologically to low density lipoprotein-two (d 1.039 to 1.060 g/ml) and high density lipoprotein (d 1.060 to 1.210 g/ml) was related immunologically to low density lipoprotein-one (d 1.019 to 1.039 g/ml), but the two pairs were immunologically distinct. The major N-terminal amino acid of both high density lipoprotein and low density lipoprotein-one was aspartic acid, and that of low density lipoprotein-two was glutamic acid. Very low density lipoprotein had both aspartic acid and glutamic acid as the major N-terminal amino acids. None of the lipoproteins was identical with any other with respect to amino acid composition, but high density lipoprotein and low density lipoprotein-one were similar to each other and different from low density lipoprotein-two. Very low density lipoprotein was similar to both low density lipoprotein-one and low density lipoprotein-two. It is concluded that the proteins of high density lipoprotein and of low density lipoprotein-one are related and are different from that of low density lipoprotein-two. The protein of very low density lipoprotein is related to that of low density lipoprotein-two but may contain polypeptides of high density lipoprotein or low density lipoprotein-one.

Retention of lipolytic products in chylomicrons incubated with lipoprotein lipase: electron microscope study

Early effects of lipolysis on the structure of chylomicrons in vitro were studied in rat chylomicrons incubated with purified bovine mild lipoprotein lipase at pH 8.1. The amount of the albumin added to the incubation medium was limited so that free fatty acids (FFA) and partial glycerides formed during lipolysis would accumulate in the chylomicrons. The structures visualized in lipolyzed chylomicrons was found to be affected by pH during preparation of specimens for microscopy, whether fixed with OsO4 and sectioned, or stained with sodium phosphotungstate and examined as whole mounts. Circular aqueous spaces were present in the triglyceride core of lipolyzed chylomicrons processed at pH 8.1 and 7.4. Sometimes the spaces contained aggregates of osmiophilic material and whorls of bilayered lamellae. The spaces were replaced by lamellar structures having a periodicity of 40 A, in chylomicrons processed at pH 5.5, and the spaces and lamellae were both absent at pH 3.0. The findings indicate that these spaces were lined by a lipid monolayer which formed bilayered lamellae under certain conditions. It is concluded that the monolayer lining the aqueous spaces is an inward extension of the chylomicron surface film produced by the accumulation and movement of lipolytic products, FFA and partial glycerides, in the interfacial plane between core triglyceride and water.

Determination of Physical Properties of Bovine Serum Lipoproteins by Analytical Ultracentrifugation

Pure samples of low density lipoprotein-one (d 1.019 to 1.039 g/ml), low density lipoprotein-two (d 1.039 to 1.060 g/ml), and high density lipoprotein (d 1.060 to 1.210 g/ml) were isolated from bovine serum and their properties studied in the analytical ultracentrifuge. Approach-to-equilibrium experiments indicated that the lipoprotein classes were homogeneous. Molecular weights of the lipoproteins given by this method (low density lipoprotein-one, 1.14×106; low density lipoprotein-two, 2.37×106; high density lipoprotein, .576×106) agreed well with those obtained by a high-speed-equilibrium method (1.03×106, 2.15×106, and .567×106). Linear plots of flotation rate (corrected for viscosity) against the density of the medium were obtained in sedimentation velocity experiments and on extrapolation gave values for the hydrated density of 1.032 g/ml for low density lipoprotein-one, 1.044 g/ml for low density lipoprotein-two, and 1.071 g/ml for high density lipoprotein. The density of the material which would have to be added to high density lipoprotein to give it the physical properties of low density lipoprotein-one was less than 1 g/ml, which suggested that it was predominantly lipid.

Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements

The microviscosity of the lipid phase of bovine serum high density lipoprotein was determined by fluorescence polarization measurements on a lipophilic probe (1,6-diphenyl-1,3,5-hexatriene) dissolved in the lipoprotein. At 25°C the average microviscosity was 6.1 ± 0.5 poise, and the activation energy calculated from a plot of log η versus 1 T was 13±3Kcal/mole. A constant slope for the Arrhenius plot from 0 to 46°C indicated no apparent phase transitions in this temperature range. Comparison of the present results with reported microviscosity values for rat lymphocyte membranes and liposomes [Shinitzky and Inbar (1974) J. Mol. Biol. 85 , 603] indicates a more rigid environment of the probe in the high density lipoprotein system fluidity of the lipid appears to be considerably decreased in the lipoprotein relative to organic solvent or oil solutions of lipids, probably as a result of the anisotropic environment of the probe, high total cholesterol, and presence of protein in these particles.