Bovine Lipoprotein Research Articles

Isolation and characterization of bovine lipoproteins: incubation with hepatic lipase and isolated adrenal zona fasciculata cells

Isolation and characterization of bovine lipoproteins: incubation with hepatic lipase and isolated adrenal zona fasciculata cells

Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation.

Sedimentation equilibrium analysis demonstrated that preparations of bovine lipoprotein lipase contain a complex mixture of dimers and higher oligomers of enzyme protein. Enzyme activity profiles from sedimentation equilibrium as well as from gel filtration indicated that activity is associated almost exclusively with the dimer fraction. To explore if the enzyme could be dissociated into active monomers, 0.75 M guanidinium chloride was used. Sedimentation velocity measurements demonstrated that this treatment led to dissociation of the lipase protein into monomers. Concomitant with dissociation, there was an irreversible loss of catalytic activity and a moderate change in secondary structure as detected by circular dichroism. The rate of inactivation increased with decreasing concentrations of active lipase, but addition of inactive lipase protein did not slow down the inactivation. This indicates that reversible interactions between active species precede the irreversible loss of activity. The implication is that dissociation initially leads to a monomer form which is in reversible equilibrium with the active dimer, but which decays rapidly into an inactive form, and is therefore not detected as a stable component in the system.

Apolipoprotein AI and AII metabolism in patients with primary high-density lipoprotein deficiency associated with familial hypertriglyceridemia

Plasma high-density lipoproteins (HDL) and their major proteins—apolipoprotein (apo) AI and apo AII—are subnormal in most patients with familial hypertriglyceridemia. However, the pathophysiology of low-plasma apo AI and apo AII is unclear. The kinetic parameters (turnover) of HDL apo AI and apo AII were studied in six lean patients with primary HDL deficiency associated with familial hypertriglyceridemia and five normolipidemic controls. Autologous 125I labeled HDL were injected intravenously (IV; 25 μCi) and blood samples drawn ten minutes after the injection and periodically thereafter for 12 days. Urine samples were collected daily and their radioactivity measured. Kinetic parameters were calculated from the area under the decay curve using three exponentials. Mean plasma apo AI and apo AII were significantly lower ( P < 0.001) in patients than normals (70.4 ± 2.7 v 106.9 ± 7.0; 24.2 ± 1.6 v 39.2 ± 0.9 mg/dL, respectively). The mean fractional catabolic rates (FCR) obtained from plasma 125I-HDL, apo AI, apo AII radioactivity decay curves and by Berson and Yalow's method (urine/plasma radioactivity ratios) were significantly greater ( P < 0.05) in patients than in controls (0.387 v 0.299; 0.391 v 0.309; 0.361 v 0.275; 0.272 v 0.207/d; respectively). The mean synthetic rates (SR) of apo AI and apo AII were significantly lower in patients than in controls (11.12 v 14.17 mg/kg body weight/d, P < 0.05; 3.53 v 4.68 mg/kg body weight/d, P < 0.05, respectively). In vitro lipolysis of triglyceride (TG) rich lipoproteins by bovine lipoprotein lipase, and measurement of hepatic TG lipase and lipoprotein lipase in postheparin plasma were similar in patients and controls, indicating no abnormality in these factors that are linked to HDL and TG catabolism. However, a significant positive correlation between hepatic TG lipase and the FCR of apo AI and apo AII was found. The data suggest that in this series of patients with HDL deficiency the low plasma HDL-cholesterol, apo AI, and apo AII levels resulted from decreased synthesis and an increased fractional catabolic rate of apo AI and apo AII, the major proteins of HDL.

Purification and characterization of bovine lipoproteins: resolution of high density and low density lipoproteins using heparin-Sepharose chromatography.

The selective and reversible adsorption of bovine low density lipoproteins (LDL) by heparin-Sepharose has been exploited as the critical step in a procedure for the preparative isolation of very low density lipoproteins (VLDL)/chylomicrons, LDL, and high density lipoproteins (HDL) from bovine plasma. Molecular size exclusion chromatography and isopycnic density gradient separation steps are also involved in the method described. The resulting HDL and LDL fractions are free from contamination by one another as judged by electrophoretic mobility in agarose gels. The major lipid and apolipoprotein compositions of the three resolved lipoprotein classes have been determined.

Molecular size of bovine lipoprotein lipase as determined by radiation inactivation.

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.

Metabolism of cholesteryl ester in monolayers of bovine adrenal cortical cells. Effect of an inhibitor of acyl-CoA : cholesterol acyltransferase

The effect of Sandoz compound 58-035 on cholesterol metabolism in monolayers of bovine adrenal cortical cells was studied. 58-035 did not inhibit cholesterol ester hydrolase, cholesterol side-chain cleavage, cholesterol synthesis from acetate, or cortisol synthesis in cells stimulated with ACTH or in unstimulated cells. It was, however, an effective inhibitor of formation of cholesteryl ester. The rate of formation of cholesteryl ester in the cells was increased by additional cholesterol derived from mevalonic acid or from the hydrolysis of intracellular lipid droplets. 58-035 caused an increase in the secretion of cortisol from cells maintained on a limited supply of cholesterol from bovine lipoproteins added to the medium when the cells were not stimulated with ACTH. This effect was not observed in stimulated cells. The results suggest that the bovine adrenal cortical cell can direct the flux of exogenous cholesterol very precisely according to its metabolic state.

Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized.

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.

Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase

Rats were injected intravenously with 125I-labeled bovine lipoprotein lipase. The lipase disappeared within minutes from the blood due to uptake both in the liver (about 50% of the injected dose) and in extrahepatic tissues. Lipase enzyme activity disappeared in parallel to the 125I radioactivity. Thus, there was no inactivation of lipase in the circulating blood. Similar results were obtained when lipoprotein lipase purified from guinea pigs was injected into guinea pigs. Using supradiphragmatic rats we could show that the extrahepatic uptake was saturable and that the amounts of lipase that could be bound far exceeded the amounts of endogenous lipase expected to be present on the endothelium. When the lipase was denatured before injection, its removal in supradiaphragmatic rats became slower, and in intact rats the fraction of the uptake that occurred in extrahepatic tissues was much decreased. It is concluded that recognition by the extrahepatic receptors depends on the native conformation of the lipase. The extrahepatic uptake was strongly impeded by injection of heparin prior to injection of the lipase, and the uptake could to a large extent be reversed by injection of heparin after the lipase. Even after 1 h lipase that had been taken up by extrahepatic tissues reappeared immediately in the blood on injection of heparin. This was true both for enzyme activity and for enzyme radioactivity. Thus, internalization-inactivation-degradation occur only slowly in extrahepatic tissues. It is possible that the extrahepatic binding occurs to the enzyme's physiological receptors. The hepatic uptake was not dependent on the native conformation of the lipase, was less sensitive to heparin, could not be reversed by heparin and was not saturable. The enzyme was not rapidly inactivated after uptake; its activity could be detected in liver homogenates even after 1 h. Degradation to acid-soluble products in the liver was relatively slow; the t 1 2 for native lipase was about 1 h. In comparison, in parallel experiments asialofetuin was degraded with a t 1 2 of about 15 min

Correlation of 13C and 1H chemical shifts in bovine high-density lipoprotein from two-dimensional NMR

Two-dimensional heteronuclear 13C 1H chemical-shift correlation spectra were obtained for bovine high-density lipoprotein (HDL). The 13C and 1H shifts of the lipid components are assigned. No evidence for dynamic equilibration of lipid classes between different structure environments was found at this limited resolution. Heteronuclear correlation spectroscopy can be effectively applied to macromolecular complexes as large as HDLs.

Domain map of the LDL receptor: Sequence homology with the epidermal growth factor precursor

The nucleotide sequence of a partial cDNA for the bovine low-density lipoprotein (LDL) receptor revealed an open reading frame of 264 amino acids that encodes the COOH-terminal 25% of the receptor protein. The sequence predicts a cytoplasmic domain of 50 amino acids at the COOH terminus, followed in order by a membrane-spanning region of 27 hydrophobic amino acids and an externally disposed stretch of 42 amino acids, that is rich in serine and threonine residues and appears to be the site of O-linked glycosylation. This orientation was confirmed by proteolysis experiments in which the relevant fragments were localized by blotting with antipeptide antibodies and a galactose-specific lectin. The extracytoplasmic domain of the LDL receptor contains a region that is 38% identical with a 96 amino acid sequence in the precursor to mouse epidermal growth factor (EGF), a peptide hormone. This unexpected homology raises the possibility that proteins involved in growth stimulation (e.g., EGF precursor) and nutrient delivery (e.g., LDL receptor) may have a common evolutionary origin.

Effect of particle size and lipid composition of bovine blood high density lipoprotein on its function as a carrier of β-carotene

As part of a study on the influence of dietary lipids on vitamin transport and metabolism in lactatinf cows, we have examined the β-carotene content and other properties of fractions of the high-density lipoprotein (HDL, density 1.05–1.16 g/ml) of bovine blood. Our purpose was primarily to explain previous results indicating that feeding cows polyunsaturated lipids alters the properties of the HDL and increases the concentration of β-carotene in the blood but not in the milk. Fractions of HDL of different particle size were prepared by gel-filtration chromatography and the particle diameters measured by electron microscopy. We found that large HDL particles contain more β-carotene per unit weight than small particles. Furthermore the HDL from cows fed lipid-rich diets with a high proportion of linoleic-acid residues, which had been protected against microbial degradation in the rumen, had a high percentage of HDL particles with large diameters. The blood from these cows had a higher concentration of β-carotene than before feeding polyunsaturated lipids, but their milk had a lower concentration. We suggest that HDL is the main store of β-carotene in bovine blood. Moreover the concentration of β-carotene in blood is increased by feeding polyunsaturated lipids largely because of the increase in the percentage of large HDL particles, which contain more β-carotene. The effect on the concentration of β-carotene in milk implies that the transfer mechanism is less efficient as a result of feeding polyunsaturated lipids. This lower efficiency may be due in part to the higher percentage of large HDL particles.

CDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA.

The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of approximately equal to 5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of approximately equal to 5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns.

Comparison of ultracentrifugation and gel filtration for the isolation of bovine lipoproteins.

Lipoproteins from the plasma of three nonlactating Holstein cows were isolated using either preparative ultracentrifugation or gel filtration chromatography. Lipoprotein classes obtained by ultracentrifugation were very low density plus chylomicra, less than 1.006 g/ml; low density, 1.007-1.039 g/ml; high density1, 1.040-1.063 g/ml; and high density, 1.064-1.22 g/ml. These lipoprotein classes were individually applied to an agarose gel column to determine at what volume they eluted in comparison to lipoproteins that were separated after applying total bovine lipoproteins to the column. Three major peaks corresponding to very low density lipoproteins plus chylomicra, low density, and high density lipoproteins resulted after gel filtration of total lipoproteins. Very low density lipoproteins plus chylomicra, obtained by ultracentrifugation, eluted as a single peak, as did low density and high density lipoproteins. However, high density1 lipoproteins eluted as two peaks. The first peak eluted at the same volume as low density lipoproteins, and the second peak eluted at a volume similar to that of the ascending slope of the high density lipoprotein peak. Results from disc polyacrylamide gel electrophoresis, immunoelectrophoresis and double immunodiffusion of lipoprotein fractions, and SDS polyacrylamide gel electrophoresis of their apoproteins, similarly indicated that the lipoproteins present in the 1.040-1.063 g/ml density interval are a mixture of low and high density lipoproteins rather than a unique class of lipoproteins.

Immunochemical properties of lipoprotein lipase: Development of an immunoassay applicable to several mammalian species

The reaction of bovine lipoprotein lipase with its antibodies was found to be conformation-dependent. One aspect of this was that most antisera were more reactive with denatured than with native 125I-labeled lipoprotein lipase. Another aspect was that denatured lipase did not compete effectively with native lipase for those antibodies which caused inhibition of the enzyme's activity. This latter observation leads to the conclusion that the inhibiting antibodies recognize conformation-dependent determinants on the native enzyme. F ab fragments prepared from an inhibiting antiserum blocked the binding of the lipase to triacylglycerol/phospholipid droplets. This suggests that the inhibition results from reaction of the antibodies with the enzyme as it exists in solution, either covering the lipid-binding site on the enzyme or making it impossible for the enzyme to go through the conformational transitions necessary for binding to lipid. Most rabbit antisera did not react with rat or mouse lipoprotein lipase, but some sera showed a weak cross-reaction. Antisera raised in hens showed a much stronger cross-reaction, enough to be useful for heterologous immunoassays. An immunoassay for the bovine lipase was developed. For reproducible results it was necessary to have tracer, standard and samples in denatured form. This was accomplished by heating them in SDS, and running the immunoreaction in a Triton X-100-containing medium

Isolation and Characterization of Lipoprotein Lipase Activators from Bovine Blood Serum

Bovine serum lipoproteins were fractionated into high density and low and very low density lipoproteins by precipitation with sodium phosphotungstate and magnesium chloride. From each lipoprotein fraction seven apo C peptides were isolated by gel filtration and ion exchange chromatography. The two lipoprotein fractions probably contain identical apo C peptides but in different proportions. Two of the apo C peptides activated lipoprotein lipase from milk in vitro. Their specific activities were about 2000 times as high as that of the original serum. The apo C fraction from low-very low density lipoproteins had a specific activity three times that from high density lipoproteins. Also, the activator peptides from low and very low density lipoproteins gave one band on alkaline urea gel electrophoresis whereas corresponding peptides from high density lipoproteins were slightly heterogeneous. The low and very low density lipoproteins, therefore, seem to be the fraction of choice for isolation of activators for lipoprotein lipase.

Fractionation of Plasma Triglyceride-Rich Lipoproteins of the Dairy Cow: Evidence of Chylomicron-Size Particles

Electron micrographs of bovine triglyceride-rich lipoproteins demonstrated spherical particles with diameters up to 3000Å. Chylomicron-size particles (larger than 750Å) were isolated specifically by rate zonal centrifugation. Subfractionation by rate zonal centrifugation, isopycnic centrifugation, and gel filtration chromatography all indicated a wide range of bovine triglyceride-rich lipoproteins from chylomicrons to very low density lipoproteins. Chemical analysis of subfractions from gel filtration showed changes in the ratio of triglyceride/protein characteristic of chylomicrons versus very low density lipoproteins. Electrophoretic analysis on agarose gels of bovine chylomicrons obtained by centrifugation and gel filtration showed migrating bands similar to very low density lipoproteins, unlike human chylomicrons. Evidence of bovine chylomicrons provides support for the concept of a significant contribution to milk fat from intestinally absorbed lipid in the dairy cow.

Molecular Properties of Lipoprotein Lipase

The monomer molecular size of bovine lipoprotein lipase was evaluated by sedimentation equilibrium measurements and by gel permeation chromatography in 6 M guanidinium chloride. To establish molecular weight unequivocally we determined the partial specific volume (v) experimentally. This was done by analyzing equilibrium concentration profiles from analytical ultracentrifugation in 6 M guanidinium chloride using buffers made up in H2O and 2H2O. The combined results gave a v of 0.71 +/- 0.007 ml/g and a molecular weight of 41,700 +/- 1000 for monomeric bovine lipoprotein lipase. This value did not change upon mild tryptic digestion; the elution volume upon gel permeation chromatography in 6 M guanidinium chloride was also unaffected by treatment with trypsin. Sedimentation equilibrium measurements of the trypsin-treated material in the presence of reducing agents gave limiting molecular weights of 19,000 and 23,000, demonstrating that mild trypsin digestion cleaved lipoprotein lipase into two polypeptide chains of similar size held together by disulfide bonds. Mild trypsin digestion also resulted in a loss of secondary structure as determined by circular dichroic measurements. Discussion centers around the correlation between these effects of trypsin on the molecular properties of lipoprotein lipase and the previously reported effects on the kinetic properties of the enzyme.

Physicochemical characterization of ten fractions of bovine alpha lipoproteins.

With the onset of milk production, serum concentrations of alpha lipoproteins in the dairy cow steadily increase, frequently attaining values greater than 1.5 g/dl. Since these lipoproteins comprise a highly polydisperse system, we have carried out studies to explore differences among bovine alpha lipoproteins in the density interval between 1.05 to 1.21 g/ml. Separation into ten fractions was achieved ultracentrifugally in an isopycnic gradient. Agarose gel electrophoresis showed that all but the bottom fraction contained alpha lipoproteins as either the major or sole lipoprotein class. Compositional analyses revealed an increasing percentage of both protein and phospholipid and a decreasing percentage of cholesterol with increasing fraction density. The esterified to unesterified cholesterol ratio ranged from 3 to 8 from the top to the bottom of the gradient. The densities of the particles obtained from the various fractions were calculated both from sedimentation velocity measurements and from compositional analyses. The resulting density values agreed well with the solution densities of these isopycnic gradient fractions. The major apoprotein of each fraction was apoA-I. Combining diffusion coefficient data obtained by intensity fluctuation spectroscopy with sedimentation velocity data, we were able to calculate molecular weights, frictional ratios, and Einstein-Stokes radii for three of the fractions. Results are discussed in terms of previously published data on bovine lipoproteins as well as other mammalian data.

Characterization of unusual intermediate density lipoproteins.

We report on the physicochemical properties of unusual lipoproteins isolated from both lymph and blood of ruminating cattle. The densities of most of these particles fall within the range between 1.006 and 1.020 g/ml, although densities of 0.97-0.99 g/ml are calculated from chemical composition, assuming a liquid core. The triglycerides of these particles have a high content of saturated fatty acids. The major apoprotein has a mobility on polyacrylamide-SDS gels consistent with a molecular weight of 40,000. The negatively-stained particles appear flattened and asymmetric in electron micrographs. The particles are very large, with molecular weights in the 20 to 250 million dalton range, and they scatter light strongly. The hydrodynamic frictional ratio is about 1.4, consistent with oblate ellipsoids with axial ratios of about 8 to 1. The flat appearance, asymmetric shape, and anomalous densities of the particles would be explained if these lipoproteins consisted of a core of crystallized triglycerides encapsulated within a phospholipid monolayer. Crystallization of the saturated triglycerides could occur during routine lipoprotein isolation, in which temperatures much lower than the melting points of their core lipids are employed. when protocols are done entirely at 37 degrees C, the unusual structures are not observed in the intermediate density class. Although the saturated fats in these bovine lipoproteins are derived from ruminal fermentation, we feel that any triglyceride-rich lipoprotein highly enriched in saturated fats will behave similarly if isolation temperatures are well below the melting points of the core lipids.

Lipoprotein lipase secretion by human monocyte-derived macrophages.

Human monocyte-derived macrophages in culture produced lipoprotein lipase. Although freshly isolated blood monocytes did not secrete much lipase activity, 1 d in culture was sufficient to trigger measureable enzyme production. During 3 wk in culture, maximal activity was attained after 7 d. At all times, the culture medium contained more enzyme activity than did a serum-heparin eluate or a detergent extract of the cell layer. The lipase activity was stimulated by serum and was inhibited by preincubation with antiserum to bovine lipoprotein lipase or when assayed at a high salt concentration. Furthermore, the enzyme bound to a heparin-Sepharose affinity column at physiological ionic strength. Cells cultured from a subject with primary lipoprotein lipase deficiency secreted no detectable enzyme. Since macrophages are prominent components of atherosclerotic lesions in man, their ability to synthesize and secrete lipoprotein lipase may be important to atherogenesis.