Lipoprotein-free Serum Research Articles

Scavenger receptor-BI is a receptor for lipoprotein(a)

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.

An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes

Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A(2) (PLA(2))-like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA(2)-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA(2). Furthermore, specific inhibitors of PLA(2) such as p-bromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA(2)-like activity may be an intrinsic attribute of albumin.

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

Lipid Emulsion as Antisense Oligonucleotides Vector That Inhibits P-Glycoprotein Expression in a Sarcoma Cell Line through LDL Receptor

It is well known that antisense oligonucleotides (OAS) are able to inhibit gene expression in a sequence-specific way. The potencial use of an artificial lipid nanoemulsion (LDE) as a vector to carry OAS has been described. LDE was shown to bind 3'-cholesteryl- OAS (C-OAS) and to be internalized into cells through LDL receptors. Here we further explore these findings by examining the capacity of this complex to inhibit MDR-1 gene expression in a sarcoma cell line (MES-DX), which express P-glycoprotein. LDE was prepared as described (Bydlowski et al. ,1995). The capacity of LDE to bind C-OAS was examined by fluorescence spectra analysis using a Hitachi F4500 fluorimeter. Human plasma was obtained from healthy blood donors and LDL, HDL and lipoprotein free serum (LPDS) were separated by sequencial ultracentrifugation. C-OAS/LDE complex was incubated with HDL (apoE donor) before cell experiments were performed. Binding of [3H] LDE/C-OAS complex to LDL receptors from MES-DX cells was studied by competition assay. Two different C-OASs, both complementary to regions flanking the AUG initiation codon were used. Inhibition of MDR-1 gene expression was evaluated by RT-PCR. The binding constant for C-OAS/LDE was 4,2 × 10−3M−1 indicating a high specific capacitiy of conjugation.The C-OAS/LDE complex was shown, by the competition assays and confocal microscopy, to bind to LDL receptors and then to be internalized into cells. Both C-OAS/ LDE complexes strongly inhibited (70% inhibition) the MDR-1 gene expression after 48 hours of cell incubation. This inhibition was not observed when LDE was used alone or complexed with scrambled OAS sequences. The results show that this nanoemulsion binds to cholesteryl-OASs. Moreover, this nanoparticle is an efficient carrier for OAS to target cells expressing LDL receptors. This complex is able to internalize oligonucleotides into cells specifically through the LDL receptor-mediated pathway. The internalized ODN was able to act on nucleic acid sequences as determined by the inhibition of MDR-1 gene expression. Therefore, LDE/C-OAS is promissing nanoparticle complex to be used in gene therapy studies.

The Catalytic Histidine Dyad of High Density Lipoprotein-associated Serum Paraoxonase-1 (PON1) Is Essential for PON1-mediated Inhibition of Low Density Lipoprotein Oxidation and Stimulation of Macrophage Cholesterol Efflux

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by approximately 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.

Delayed removal of 125I-labeled very low density lipoprotein from plasma of rats with insulin-deficient diabetes mellitus.

The present studies demonstrate that the removal rate of exogenously labelled 125I-VLDL-protein is prolonged when total serum from insulin-deficient rats combined with isolated 125I-VLDL is injected into normal recipient rats (6.8 +/- 0.7 vs 4.2 +/- 0.4 min; p < 0.01), but not when 125I-VLDL-protein is isolated and injected alone (4.2 +/- 0.8 vs 4.3 +/- 0.8 min). Furthermore, the present studies demonstrate that when isolated 125I-VLDL-protein is recombined with either VLDL-free (d > 1.006 g/ml), or lipoprotein-free serum (d > 1.215 g/ml) from insulin-deficient rats, the defect in removal rate of VLDL-protein observed in total serum is reestablished (125I-VLDL + VLDL-free serum from insulin-deficient rat vs that from normal rat: 7.6 +/- 1.2 vs 4.6 +/- 0.7 min, p < 0.05; and 125I-VLDL + lipoprotein-free serum from insulin-deficient rat vs that from normal rat: 6.4 +/- 0.7 vs 4.1 +/- 0.4 min, p < 0.01). These data suggest that a factor or factors exist in lipoprotein-free serum of insulin-deficient rats which interfere with the normal removal of 125I-VLDL. Since we have previously demonstrated a prolongation in the removal rate of endogenously labeled VLDL-3H-TG, the defect in removal of VLDL from the plasma of insulin-deficient rats appears to include both the lipid and protein moieties of the VLDL particles.

Identification of high-density lipoprotein in serum to determine anti-cancer efficacy of doxorubicin in HeLa cells.

The cytotoxic effects of doxorubicin (DOX) and daunorubicin (DAU) on HeLa cells cultured under different serum conditions were analyzed by the "nucleophosmin translocation" assay using immunofluorescence. Bright nucleolar fluorescence was observed in untreated cells. A shift from nucleolar to nuclear fluorescence was observed with increasing doses of DOX or DAU, with longer incubation times. A lesser degree of nucleophosmin translocation from nucleoli to nucleoplasm was observed in serum-deprived cells under the same DOX or DAU treatment. These results correlated well with those of cell-growth-reversibility and colony-formation studies, showing decreased inhibitory effects of growth on cells cultured in medium without serum. Furthermore, cells cultured in medium supplemented with the lipoprotein-free serum responded to DOX in a similar way to cells cultured without serum. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) were then added to the lipoprotein-free serum. Cells cultured in medium with the HDL-supplemented, serum showed increased sensitivity to DOX. Inhibition of cell growth and colony formation was observed in such HDL-supplemented cells upon DOX treatment (30 min). LDL, on the other hand, did not show an increase in the anti-cancer response. These results suggested that the variation in response of cells to DOX anti-cancer treatment under different growth conditions may be due to their varied concentrations of HDL. "Nucleophosmin translocation", which is useful for monitoring and ensuring the efficacy of the drug during anti-cancer treatment, provides an improved potential for successful chemotherapy.

Serum factors influencing cultured hepatocyte exogenous and endogenous triglyceride

Rat hepatocyte monolayer triglyceride content was increased modestly by incubations with apolipoprotein E-containing triglyceride emulsions or chylomicrons (exogenous) and was increased substantially by increasing media free fatty acid concentrations (endogenous). The secretion of both endogenous and exogenous triglyceride into media was enhanced by additions of serum and serum components. These additions not only enhanced hepatocyte triglyceride secretion but also, because of the absence of media lipolysis, more triglyceride was recovered in the system. Lipoprotein-free serum replicated the effect of whole serum. Lipoprotein produced a more modest secretory response. The apolipoprotein C components were the only ones that enhanced hepatic triglyceride secretion. Both lipoprotein-free plasma and lipoprotein enhanced the in vitro transfer of hepatocyte exogenous triglyceride to fibroblast.

Comparison of the clearances of serum chylomicron triglycerides enriched with eicosapentaenoic acid or oleic acid

Rat mesenteric lymph chylomicrons containing triglycerides enriched with either [14C]oleic acid (OA) or [14C]-eicosapentaenoic acid (EPA) were prepared by ultracentrifugation of lymph samples collected for 6 hr after a single duodenal infusion of an emulsion containing either fatty acid. These chylomicrons were injected into the jugular vein of recipient rats and, at various time intervals, blood was drawn and serum was assayed for radioactivity. In separate animals, serum lipoprotein fractions were separated by ultracentrifugation, and the redistribution of labeled fatty acid among circulating lipoproteins was determined by liquid scintillation spectrometry. When the early disappearance rates (10 min) of either total serum radioactivity or specifically the chylomicron fraction were compared, there were no differences between the groups receiving OA- or EPA-enriched chylomicrons. However, disappearance rates of EPA-enriched chylomicrons were slower than those of OA-enriched chylomicrons from 25 to 90 min. The small but significant differences in the disappearance rates for the longer time periods cannot be ascertained without further studies. At 5 min after injection of either type of chylomicron, the d less than 1.006 g/ml lipoprotein fraction of serum chylomicrons and very low density lipoproteins contained almost 90% of the original radioactivity. By 240 min, when less than 2% of the radioactivity remained, this radioactivity in the d less than 1.006 g/ml fraction was 43-46%, with concomitant increases in the low and high density lipoprotein fractions and in the lipoprotein-free serum.

Cholestyramine treatment in early life: Immediate and delayed effects on arterial cholesteryl ester metabolizing enzymes in the rabbit

Feeding of cholestyramine-enriched diet to weaned normocholesterolemic rabbits resulted in: (a) lowering of plasma cholesterol and (b) distinctly decreased activity of aortic acyl-CoA cholesterol acyl transferase with no changes in aortic acid and neutral cholesteryl esterase activity. At 9 weeks after cessation of cholestyramine treatment enhanced activity of both aortic esterases were noted despite normalization of plasma cholesterol. No evidence for the presence of plasma factor influencing esterases activity was found in lipoprotein-free serum from cholestyramine-treated animals. These studies show that cholestyramine treatment in early life causes immediate and delayed changes in rabbit arterial cholesteryl ester metabolizing enzymes.

The role of lipoproteins and receptor-mediated endocytosis in the transport of bacterial lipopolysaccharide.

The addition of bacterial lipopolysaccharide (LPS) from Escherichia coli 0111:B4 to human monocyte-macrophages cultured in serum results in suppression of scavenger receptor activity. The present studies were performed to examine if the effect on scavenger receptor activity was mediated by LPS alone or by LPS in association with lipoproteins. Radioiodinated LPS (125I-LPS) was added to human plasma in vitro and to normal and hyperlipidemic rabbit plasma in vitro and in vivo to determine the distribution of 125I-LPS among the lipoprotein classes. It was found that all lipoprotein classes bound LPS in direct proportion to their plasma cholesterol concentration. LPS alone was compared to LPS bound to low density lipoprotein (LDL), high density lipoprotein, or reductively-methylated LDL for their abilities to suppress scavenger receptor activity in monocyte-macrophages in lipoprotein-free serum. Only LPS bound to LDL (LPS-LDL) demonstrated an effect similar to that observed when LPS was added to cells in serum. Either unlabeled LDL or unlabeled LPS-LDL complexes competed with the uptake of 125I-LPS-LDL complexes, which appeared to proceed by receptor-mediated endocytosis. In contrast to the uptake of 125I-LDL, the uptake of 125I-LPS-LDL by cultured monocyte-macrophages was not followed by its hydrolysis and the release of its radioactive degradation products into the medium. The association of LPS with lipoproteins was very stable and appeared to be mediated by a lipid-lipid interaction. We hypothesize that LPS bound to lipoproteins may be transported into the artery wall and may initiate the atherosclerotic reaction.

Factors affecting the conversion of high-density lipoproteins: experiments with pig and human plasma

The conversion of pig high-density lipoproteins (HDL) (mainly HDL 3) to fractions of lower densities was studied by incubating pig plasma for 24 h at 37° C in the presence and absence of lipoprotein lipase from bovine milk, lecithin: cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles (very-low-density lipoproteins (VLDL) or Intralipid). The results can be summarized as follows. (1) In the presence of lipoprotein lipase and at a VLDL/HDL mass ratio of 2, the F-1.210 of pig HDL was shifted from 3.3 to 4.2, which is characteristic for human HDL 2. This shift was caused by the excessive increase in the free fatty acid content in HDL. If 50 g/l of bovine serum albumin were added prior to incubation, the flotation rate of HDL remained in the HDL 2a region. (2) If lecithin: cholesterol acyltransferase was active in fasting pig plasma during incubation, we observed only a negligible increase of F-1.210 in HDL. (3) If pig lipoproteins were incubated with human lipoprotein-free serum as a source of cholesteryl ester transfer activity, a slight increase in the flotation rate of HDL was observed, which was amplified in the presence of active lecithin: cholesterol acyltransferase. (4) Pig HDL was converted to a fraction with F-1.210 of 4.2, which is typical for human HDL 2, only if active lecithin: cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles were present in the incubation mixture. (5) From our results we also concluded that apolipoprotein A-II plays no role in the HDL 2 formation.

Effect of low density lipoprotein on glycosaminoglycan secretion by cultured human smooth muscle cells and fibroblasts: Influence of serum concentration and cell proliferation rate

Glycosaminoglycan (GAG) secretion was studied in cultures of human fibroblasts and arterial smooth muscle cells. Supplementation of culture medium with whole human serum increased the secretion of GAG but this effect disappeared as cell density increased. Lipoprotein-free serum (LFS) supported cell growth but led to a decrease in GAG secretion and in cell cholesterol. Addition of human low density lipoprotein (LDL) to the medium containing 10% LFS produced increases in GAG secretion (200%) and cell cholesterol (300%) and a decrease (60%) in cell population. The effects of LDL were considerably smaller in medium containing 5% LFS; this was related to the lower rate of proliferation in this medium, since there was a close relationship between rate of proliferation and stimulation of GAG secretion by LDL independent of serum concentration. In addition, fetal smooth muscle cells showed a qualitatively different response to LDL in 5% LFS, with a biphasic dose-response of GAG secretion and cell number. It is concluded that: (1) whole human serum stimulates GAG secretion by sparse cell cultures, (2) LFS can support cell growth but not GAG secretion, (3) LDL stimulates GAG secretion but has a cytotoxic effect, (4) the degree of GAG stimulation by LDL is dependent on the proliferative state of cells, (5) at low serum concentrations fibroblasts and fetal smooth muscle cells show differences in response to LDL which are not evident at higher serum concentration.

Standardization of immunoassays for the quantitation of plasma apo B protein

The apo B quantitation by electroimmunoassay and immunonephelometry are compared for normal and pathological sera. The parameters affecting the accuracy and sensitivity of the assays, such as the selection of a suitable standard and the addition of a detergent, are discussed. Both assays were optimized by the addition of a lipoprotein-free serum to the low-density lipoprotein standard and dilution of the samples with a detergent. Under these conditions normal values for plasma lie in the range 80–90 mg/dl and the assay can be carried out on either plasma or serum from nonfasting or triglyceride-rich subjects with a variation coefficient of 6–7%.

Monitoring of detergent binding to amphiphilic proteins by means fo micelles containing the hydrophobic dye Sudan Black B

A simple method for detecting micellar binding of Triton X-100 to amphiphilic proteins is described. The hydrophobic dye Sudan Black B is incorporated into Triton micelles. Binding of the coloured micelles to serum apoliproteins, as well as to amphiphilic proteins, of erythrocyte and fat globule membranes renders these visible as dark bands after sucrose density gradient centrifugation. In contrast, the hydrophilic proteins present in lipoprotein-free serum do not show detergent binding. The method does not permit accurate quantification of detergent binding, but may serve as a pilot procedure for initial detection of amphiphilic proteins and for monitoring their isolation from crude solubilized membrane material. The sensitivity of the assay corresponds to that obtained with [ 3H]Triton X-100.

Influence of lipoprotein concentration on the exchanges of triacylglycerol between rabbit plasma low density and high density lipoproteins

Molecular exchanges of triacylglycerol between rabbit serum low density lipoproteins (LDL) and high density lipoproteins (HDL) have been studied in 37°C incubations performed in the presence of rabbit lipoprotein-free serum as a source of the triacylglycerol transfer protein. The molar rate of exchange of triacylglycerol between the two fractions increased with increasing incubation concentrations of LDL but was decreased as the HDL concentration was increased. When the concentration of both LDL and HDL was increased in parallel there was an increase in the molar rate of triacylglycerol exchange between the two fractions which flattened at higher concentrations, suggesting that the process was saturable. Fractionation of rabbit lipoprotein-free serum on a column of Sephadex G-200 resulted in the elution of the triacylglycerol transfer activity in a single peak. Addition of LDL to the lipoprotein-free serum had no effect on the position of elution of the triacylglycerol transfer activity. Addition of HDL, however, resulted in an elution of the transfer activity as two peaks, one in the original position and the other in the same position as HDL. The results of the kinetic studies have been interpreted in terms of a binding of the triacylglycerol transfer protein to HDL, but not to LDL.

Characterization of an auto-rosette inhibitory fraction in mouse serum.

Normal mouse sera inhibited auto-erythrocyte rosette formation by mouse thymus cells. Serum inhibitory factor was associated with high density lipoprotein (HDL) isolated from mouse sera with sequential flotation ultra-centrifugation techniques. Other portions of normal mouse sera, i.e., very low density lipoprotein (VLDL), low density lipoprotein (LDL), lipoprotein-free serum, and delipidated HDL had little or no inhibitory activity. A 20-fold purification with 68% recovery of total serum activity was obtained by this isolation procedure. HDL from the serum of BALB/c, C57BL/6, C3H, and NZB/BIN mice inhibited BALB/c auto-rosettes. Inhibition was observed when lymphocytes were preincubated with HDL and then washed before assaying, but there was no inhibition when erythrocytes were pretreated.

Transfers of esterified cholesterol and triglyceride between high density and very low density lipoproteins: In vitro studies of rabbits and humans

Mixtures of rabbit very low density lipoproteins (VLDL) and high density lipoproteins (HDL), one of which was endogenously labeled with 3H in the free and esterified cholesterol moieties and with 14C in the triglyceride (TG) moiety, were incubated at 37°C in vitro in the presence of rabbit lipoprotein-free serum. There was a net mass transfer of esterified cholesterol from HDL to VLDL and of TG from VLDL to HDL. Both esterified 3H-cholesterol and 14C-TG, however, transferred in each direction between the two fractions. In the presence of parachlormercuryphenyl sulfonate (PCMPS), a thiol group blocker that inhibits lecithin:cholesterol acyltransferase (LCAT), the net mass transfers of esterified cholesterol and TG were reduced, as were the bidirectional transfers of 14C-TG, but the bidirectional transfers of esterified 3H-cholesterol were much less affected. Qualitatively similar results were obtained with incubations of human VLDL and HDL containing tracer amounts of either VLDL or HDL labeled with 3H in the esterified cholesterol moiety. In incubations of unlabeled human VLDL, HDL, and lipoprotein-free plasma, the molar ratio of the net mass transfers of TG:esterified cholesterol was 0.55 ± 0.10 (mean ± SE, n = 8) in the absence of PCMPS, and 0.25 ± 0.04 in the presence of PCMPS. The difference between these ratios was statistically significant ( p < 0.02). Therefore, it has been concluded that the net mass transfers of esterified cholesterol and TG between VLDL and HDL may be independent processes.

Partial purification and characterization of a triacylglycerol-transfer protein from rabbit serum

Rabbit lipoprotein-free serum was found to contain a protein fraction that mediates the transfer of [3H]triacylglycerol from low-density lipoprotein to high-density lipoprotein.Fractionation of rabbit lipoprotein-free serum by DEAE-Sephadex chromatography, ammonium sulphate precipitation, concanavalin-A-Sepharose chromatography, Sephadex G-200 gel filtration, phenyl-Sepharose chromatography and Sephadex G-100 gel filtration, yielded a preparation that had a 500-fold increase in transfer activity compared to that of the starting sample. The transfer activity appeared to reside in a glycoprotein of molecular weight in the range 100000–115000 and an isoelectric point at pH 9.

Activation of milk lipase by serum proteins: possible role in the occurrence of lipolysis in raw bovine milk.

The increase in unesterified fatty acid content of unpasteurized bulk milk in storage at 4 degrees C in the presence of known effectors of bovine milk lipoprotein lipase originating from bovine serum was studied. Bovine serum and high density lipoprotein (HDL) caused an increase in developed unesterified fatty acid levels whilst lipoprotein-free serum, apo-HDL, all individual apo-HDL tested, and the unfractionated C-peptide fraction were without lipolytic effect. In the presence of HDL-lipids, 2 C-peptides stimulated considerable lipolysis, as did the combination of HDL-lipid with unfractionated C-peptides. These characteristics of unesterified fatty acid development could be duplicated in milk whose endogenous lipolytic activity had been destroyed by heat treatment (75 degrees C for 5 min) and then restored by addition of purified bovine milk lipoprotein lipase. Radioactively labelled glycerol trioleate in milk was not hydrolysed in the same way as milk fat on the addition of serum liproproteins.