Metabolism of triglyceride-rich nascent rat hepatic high density lipoproteins

Nascent high density lipoprotein (HDL) and nascent very low density lipoprotein (VLDL) were isolated from rat livers that had been perfused with [3H]glycerol to label the triglyceride. When injected into intact rats, the labeled HDL-triglyceride disappeared as rapidly as the VLDL-triglyceride, with only 10% of the injected label remaining in the plasma after 30 min. The protein moiety of nascent HDL was labeled with [35S]methionine in a similar fashion and the labeled nascent HDL was separated into nonretained (NR) and retained (R) fractions by heparin-Sepharose affinity chromatography. When injected into rats, 55% of the injected label in nascent fraction NR and 72% of that in nascent fraction R was recovered from plasma at 30 min, compared to only 10% of the triglyceride label from unfractionated nascent HDL, indicating dissociation of triglyceride and apolipoprotein clearance. The plasma decay curves for both triglyceride and protein were biexponential. By 5 min, 15% of the 35S label remaining in plasma represented apoE and apoC that had been transferred from nascent HDL fractions NR and R to the d less than 1.063 g/ml fraction of plasma. Plasma HDL was labeled in vivo with [35S]methionine, separated into fractions NR and R, and the clearance of the two plasma HDL fractions was compared with that of the corresponding nascent HDL fractions. Except for a faster rate of removal of the nascent HDL fractions during the first 5 min, the serum decay curves were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

High density lipoprotein accumulation in perfusates of isolated livers of African green monkeys. Effects of saturated versus polyunsaturated dietary fat

To determine whether altered hepatic secretion of HDL is part of the mechanism by which polyunsaturated fat lowers plasma HDL concentration, we have studied HDL secretion in the isolated perfused livers of African green monkeys fed an atherogenic diet containing either safflower oil as the polyunsaturated fat or butter as the saturated fat. During recirculating perfusion with a lipoprotein-free medium, livers from safflower oil-fed animals produced 21% less HDL mass on the average than those from butter-fed animals. Newly secreted hepatic HDL were characterized after their isolation and subfractionation by a combination of agarose column chromatography and density gradient ultracentrifugation. In both diet groups the HDL were heterogeneous in size, morphology, and composition and consisted of discoidal particles ranging in diameter from greater than 200 A to as little as 50 A. Large, discoidal particles that were rich in apoE and apoA-I were separated from small particles that were poor in apoE but rich in apoA-I. All hepatic HDL subfractions contained only small amounts of cholesteryl ester and triglyceride. The hepatic particles resembled in composition and structure the large variety of HDL particles found in the plasma of patients with the familial deficiency of lecithin:cholesterol acyltransferase. Accordingly, perfusate LCAT activity was measured and found to be 2% or less than that in monkey plasma. We conclude that the perfused monkey liver produces a variety of nascent HDL that are relatively unmodified by the post-secretory metabolic events which normally occur in blood plasma in vivo, and that livers of polyunsaturated fat-fed monkeys secrete fewer plasma HDL precursor particles than do those of saturated fat-fed monkeys.     

Accumulation of an apoE-poor subfraction of very low density lipoprotein in hypertriglyceridemic men

Studies were undertaken to investigate the mechanism of the marked accumulation of an apoE-poor very low density lipoprotein (VLDL) subfraction in untreated Type IV and IIb hypertriglyceridemic subjects. Heparin-Sepharose chromatography was used to separate large VLDL (Sf 60-400) from fasted subjects, into an apoE-poor, unbound fraction and an apoE-rich, bound fraction. As a percent of total VLDL protein, the apoE-poor fraction comprised 40 +/- 4% of total VLDL in hypertriglyceridemic subjects versus 25% in normal subjects. Compared to the apoE-rich, bound fraction, this apoE-poor material was found to have a 5-fold lower ratio of apoE to apoC (0.20 +/- 0.06 vs 0.91 +/- 0.18, P less than 0.005), but a 1.5-fold higher ratio of triglyceride to protein (11.41 +/- 0.85 vs 7.97 +/- 0.77, P less than 0.01). In addition, the apoE-poor fraction was found to be enriched 2-fold in apoB-48 (10.30 +/- 2.41% vs 5.73 +/- 1.59% of total apoB, P less than 0.005) compared to the apoE-rich fraction, suggesting that the apoE-poor fraction contains more chylomicron remnants. The amount of this apoE-poor VLDL was markedly reduced following a reduction in VLDL triglyceride levels (a decrease from 40 +/- 4% to 21 +/- 2% of VLDL protein following a 50% reduction in VLDL triglyceride levels). The large VLDL from Type I, III, and V hyperlipoproteinemic subjects subfractionated using heparin-Sepharose showed an equal distribution of apoE between the two fractions in contrast with the Type IV and IIb subjects. The separation of VLDL from Type I, III, and V subjects using heparin-Sepharose involves a mechanism other than apoE binding. Separation in the latter likely results from apoB-100 binding to heparin, as opposed to apoE binding of VLDL from Type IV and IIb subjects.     

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

Distribution of apolipoprotein E among lipoprotein fractions in the lactating cow

Apolipoprotein (apo) E plays a key role in regulating plasma levels of lipoproteins. We investigated the serum apoE concentrations in cows during different lactating stages by ELISA. To confirm the distribution of apoE in lipoprotein fractions, cow plasma was separated by gel filtration, ultracentrifugation and agarose gel electrophoresis. The apoE concentrations during early, mid- and late lactating stages in cows were significantly higher than that during the non-lactating stage. In lactating plasma, apoE eluted in high-density lipoprotein (HDL) fractions separated by gel filtration increased. The portion of this apoE in plasma was 49%. However, when lactating plasma was separated by ultracentrifugation, less then 5% apoE was recovered in the HDL fraction, and more apoE was recovered in the non-lipoprotein fraction (d>1.21 g/ml, 46%). In agarose gel electrophoresis, plasma apoE was found in β-migrating lipoprotein, but it was not present in α-migrating lipoprotein. To purify apoE-containing particles, the HDL fraction separated by gel filtration was pooled and the fraction retained on Heparin–Sepharose chromatography collected. Cholesterol was absent from this fraction. These results suggest that apoE-containing particles, which increased during the lactating stage, were not associated with HDL particles, and that lipid-free forms were included in cow plasma.     

Alterations of high density lipoproteins in experimental intrahepatic cholestasis in the rat induced by administration of alpha-naphthylisothiocyanate

It has previously been reported that abnormally enlarged high density lipoproteins (HDL) appear in rats with extrahepatic cholestasis induced by ligation of the common bile duct. To see whether similar changes in HDL occur in intrahepatic cholestasis in rats, we studied HDL alterations in rats treated with alpha-naphthylisothiocyanate (ANIT), which is known to produce a cholestatic response in rats similar to intrahepatic cholestasis in man. Findings were obtained which indicated changes in HDL similar to those in bile duct-ligated rat serum: HDL from ANIT-treated rats were separated into two subfractions, enlarged particles and smaller ones, on Bio-Gel A5m column chromatography. In electron micrographs, the two subfractions appeared spherical and the diameters of the enlarged particles and the other ones were 15.0 +/- 2.6 nm and 11.5 +/- 2.2 nm, respectively. Both subfractions showed slow alpha-mobility in agarose gel electrophoresis. The enlarged HDL had apoE as their major apoprotein, while apoA-I was the major apoprotein in the other HDL subfraction. The enlarged HDL contained less protein and more cholesterol than the other HDL subfraction. The two HDL subfractions were also separated by heparin-Sepharose affinity chromatography.     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.     

Isolation and partial characterization of two glycoproteins from human liver metastases of sigmoid colon carcinoma

1. Perchloric acid-soluble glycoprotein fraction (PASF) extracted from human liver metastases (LM) of sigmoid colon carcinoma was chromatographed on a DEAE-cellulose column. The main fraction (DEAE-nonadsorbed fraction) passed through the column was then subjected to Sephacryl S-200 superfine gel filtration and separated into 12 fractions. 2. Among 12 fractions, only both Fractions 3 and 4 were demonstrated to be chemically and immunologically homogeneous glycoproteins, respectively, by a combination of chemical composition analysis, SDS-PAGE and EITB assay using antisera against the DEAE-nonadsorbed fractions of PASFs from human LMs, normal liver (NL) and normal sigmoid colon (NSC). Each of Fractions 3 and 4 reacted with anti-LM serum to give one immuno complex on a nitrocellulose sheet in EITB assay, but did not react with anti-NL and -NSC sera. 3. Apparent molecular weights of 80,900 and 62,100, respectively, were found for Fractions 3 and 4. Both the fractions, respectively, had abnormal sugar compositions. Fraction 3 contained sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine, but lacked glucose and mannose, and Fraction 4 contained sialic acid, fucose, galactose and N-acetylglucosamine, but lacked glucose, mannose and N-acetylgalactosamine, as sugar components.     

Gangliosides released by the perfused rat liver are associated to high density lipoprotein.

We examine here the delivery of gangliosides from the perfused rat liver into the perfusate. One hour after the administration of [3H]GM1 to recirculating perfused livers, almost 80% of the perfusate radioactive gangliosides were recovered associated to the HDL fraction. This fraction was relatively enriched in radioactive GD1a. The pattern of endogenous gangliosides from perfused livers, rat serum and perfusates were very different: GM3 was the main liver ganglioside, GM1 and GD1a were the most abundant in perfusates being GM3 almost absent; GM3, GM1 and GD1a were present in rat serum in similar proportions. Using a non-recirculating perfusion protocol, radioactive gangliosides were found in the HDL fraction since 15 minutes after the administration of [3H]GM1. These results suggest that rat liver supplies the perfusates with some gangliosides and that they are associated to HDL. These facts arise the possibility that the liver is one of the source of serum gangliosides.     

Isolation and characterization of Chromatium vinosum membranes

A fractionation of Chromatium vinosum into an outer layer (cell wall) and three intracellular membrane fractions by isopycnic sucrose density centrifugation of a total membrane fraction obtained by lysis of lysozyme-EDTA spheroplasts is decribed. The three intracellular fractions (I, II, and III) have apparent densities of 1.11, 1.14, and 1.16, respectively, and contain the bulk of the photosynthetic pigments. Fraction II is enriched in bacteriochlorophyll and contains about 49% of the total membrane protein and 60% of the membrane bacteriochlorophyll. The outer membrane fraction (IV, cell wall) has a density of 1.23 and contains 5% of the membrane protein and 0.8% of the bacteriochlorophyll. Fraction I is enriched in lipids and phosphorus and has only a trace of diaminopimelate (DAP). Fractions II and III both contain a significant content of DAP. Fraction IV has no DAP, but has a fatty acid composition similar to that of the envelope fraction. Electrophoresis of the fractions on sodium dodecylsulfate-containing gels yielded from 8–13 bands of protein. Fractions I, II, and III contained the same series of unique proteins, while fraction IV contained another group of unique proteins. In fraction IV the bulk of the proteins traveled in one band with a molecular weight of 41,500. Examination of the fractions and whole spheroplasts in the electron microscope showed that fractions I, II and III were composed of large membrane structures in the form of membrane reticulum with bud-like appendages, and elongated flattened tubes. Fraction IV was composed of large ovoid structures which were seen to lie on the outer surface of the whole spheroplasts. These results suggest that the normal in vivo state of the intracellular membranes is that of an interconnected series of tubules and vesicles extending throughout the cell cytoplasm.     

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.     

Lecithin:cholesterol acyltransferase-induced modifications of liver perfusate discoidal high density lipoproteins from African green monkeys

The role of lecithin:cholesterol acyltransferase (LCAT) in the formation of plasma high density lipoproteins (HDL) was studied in a series of in vitro incubations in which perfusates from isolated African green monkey livers were incubated at 37 degrees C with partially purified LCAT for between 1 and 13 hr. The HDL particles isolated from monkey liver perfusate stored at 4 degrees C and not exposed to added LCAT contained apoA-I and apoE, were deficient in neutral lipids, and were observed by electron microscopy as discoidal particles. Particle sizes, measured as Stokes' diameters by gradient gel electrophoresis (GGE), ranged between 7.8 nm and 15.0 nm. The properties of perfusate HDL were unchanged following incubation at 37 degrees C in the presence of an LCAT inhibitor. However, HDL subfractions derived from incubations at 37 degrees C with active LCAT contained apoA-I as the major apoprotein, appeared round by electron microscopy, and possessed chemical compositions similar to plasma HDL. The HDL isolated from perfusate incubations at 37 degrees C with low amounts of LCAT had a particle size and chemical composition similar to plasma HDL3a. In three of four perfusates incubated with higher levels of LCAT activity, the HDL products consisted of two distinct HDL subpopulations when examined by GGE. The major subpopulation was similar in size and composition to plasma HDL2a, while the minor subpopulation demonstrated the characteristics of plasma HDL2b. The data indicate that the discoidal HDL particles secreted by perfused monkey livers can serve as precursors to three of the major HDL subpopulations observed in plasma.     

Partial separation of the pituitary proteins of Labeo umbratus, Smith (mudfish)

L. umbratus pituitary glands were partially separated by means of preparative polyacry-lamide electrophoresis and chromatography on DEAE-cellulose and Sephadex G-50. Eight fractions were obtaìned and it was found that fraction 1 contained potent adrenocorticotropic stimulating activity, with a molecular weight in the region of 6000. Fractions 3 and 4 contained potent gonadotropic and a lesser degree of exopthalmic producing activity, with molecular weights in the region of 14 000 to 20 000. Fraction 6 displayed pigeon-crop stimulating activity, with a molecular weight above 30 000. Fractions 7 and 8 displayed M.S.H. and vasopressinoxytocic activity respectively, with a molecular weight in the region of 5500–6500. Fractions 2 and 5 could not be identified. The fractions seemed to be glycoproteins and the results are discussed in relation to previous findings.     

Mechanism of the HDL2 stimulation of progesterone secretion in cultured placental trophoblast

Lipoprotein cholesterol (C) supports the high rate of progesterone production by the human placenta as endogenous cholesterol synthesis is low. To study underlying mechanisms whereby lipoproteins, including high density lipoprotein-2 (HDL2), stimulate progesterone secretion, trophoblast cells were isolated from human term placentas and maintained in primary tissue culture. Lipoproteins were added at several concentrations and medium progesterone secretion was determined. HDL2 (d 1.063-1.125 g/ml) as well as low density lipoproteins (LDL) (d 1.019-1.063 g/ml) but not HDL3 (d 1.125-1.21 g/ml) stimulated progesterone secretion in a dose-dependent manner, with HDL2 cholesterol entering the cell and serving as substrate for progesterone synthesis. Conversely, LDL and HDL2 produced a significant decrease in [2-14C]acetate incorporation into cell cholesterol. Cholesterol-depleted lipoproteins did not stimulate progesterone secretion. The stimulating effect of LDL was abolished by apolipoprotein modification by cyclohexanedione or reductive methylation and by the addition of anti-LDL receptor antibody or 10 microM chloroquine to the medium. [14C]acetate conversion into cholesterol was accelerated by these procedures. However, HDL2 stimulation of progesterone secretion and reduction of [14C]acetate incorporation into cholesterol was not blocked by chemical modification of apolipoproteins, anti-LDL receptor antibody, or chloroquine. Treatment of HDL2 with tetranitromethane or dimethylsuberimidate also did not block the stimulation of progesterone. To determine whether the capacity of HDL2 to deliver cholesterol to the trophoblast cells was restricted to subfractions differing in apoE content, HDL2 was chromatographed on heparin-Sepharose and three fractions (A, B, and C) were obtained. Fraction A was poorest in apoE and free cholesterol, fraction B contained the majority of cholesterol, and fraction C was the richest in apoE and free cholesterol. When added to trophoblast cells, fraction A stimulated little progesterone secretion, fraction B stimulated moderately, and fraction C did so greatly. Modification of these subfractions with cyclohexanedione or reductive methylation did not inhibit these effects. In conclusion, HDL2 stimulated progesterone secretion in human trophoblast cell culture. Contrary to LDL, the HDL effect was not mediated by apolipoproteins or the LDL receptor pathway. The ability of HDL2 to stimulate progesterone secretion is consistent with the passive transfer of free cholesterol to the cell membrane from a physicochemically specific subfraction of HDL. This mechanism may be an auxiliary source of cholesterol for human steroidogenic cells.     

Plasmodium berghei: I. Immunochemical properties of fractions of a soluble extract

Soluble extracts of Plasmodium berghei were separated into 12 fractions following preparative disc electrophoresis in polyacrylamide gel. One or two protein bands were detected in each fraction by analytical disc electrophoresis. Similarly, one or two precipitinogens were generally detected in each of Fractions 1 through 11 by immunoelectrophoresis and by double immunodiffusion in agar gel, while the unfractionated extract contained 10 precipitinogens. Antisera produced in rabbits against each fraction each contained two or three (sometimes five) antiplasmodial precipitins demonstrable by immunoelectrophoresis. Serial fractions obtained in separate runs were closely similar to each other, although some degree of overlapping sometimes occurred between neighboring fractions. Glycoproteins were detected in all the fractions, but chiefly in Fractions 4 and 12. The bulk of the RNA in the extract was located in Fraction 4, while hemoglobin was usually confined to Fraction 6. The molecular weights of the soluble components of P. berghei range between 8000 and 130,000.     

A multicomponent protein of a fission yeast that promotes joint molecule formation from homologous DNAs.

We developed a quantitative assay ("homologous pairing gel assay") adequate for the purification of the activity promoting the formation of joint molecules, an intermediate of homologous recombination ("homologous pairing"). With this assay, one can measure the extent of homologous pairing between a single-stranded DNA and a strand of 3H-labeled double-stranded DNA by crude enzyme preparations. Since the total activity did not significantly change during the sporulation process, we tried to purify the activity from a whole cell extract of mitotic cells of a fission yeast, (Schizosaccharomyces pombe). Through quantitative assaying of a single fraction or of mixed fractions, we obtained three fractions, all of which were required for the maximum level of the ATP-independent homologous pairing: Fractions 65, 100, and 30. In Fractions 100 and 30, polypeptides of approximately 100 and approximately 30 kDa (the 100- and 30-kDa polypeptides), respectively, were the sole detectable components. Fraction 65 contained a polypeptide of approximately 65 kDa (the 65-kDa polypeptide) as the major component and also small amounts of the 30- and 100-kDa polypeptides. Fraction 65 by itself promoted homologous pairing, but the reaction was saturated at a level of approximately 20% of the maximum level achieved with the recA protein. Even when added in excess, Fraction 30 or 100 alone did not promote detectable homologous pairing. A mixture of Fractions 65 and 100 at a rather strict optimum ratio only promoted homologous pairing, the level being 50-70% that with the recA protein, suggesting a stoichiometric complex of these polypeptides as the active form. Fraction 30 alone did not enhance the reaction with Fraction 65, but stimulated homologous pairing promoted by the optimum mixture of Fractions 65 and 100 to the maximum level achieved with the recA protein. Therefore, the homologous pairing-promoting protein from the fission yeast is likely to be a multicomponent protein.     

Subfractionation of 1.006-1.063 g/ml components of badger plasma lipoproteins by using heparin-Sepharose affinity chromatography: relevance to the endocrine regulation of lipoprotein metabolism

Badger plasma lipoproteins with density 1.006-1.063 g/ml have been subfractionated by means of affinity chromatography on a heparin-Sepharose column, using a modification of the method reported by Weisgraber and Mahley (1980. J. Lipid Res. 21: 316-325). These experiments have provided evidence for the presence of three lipoprotein subfractions hereinafter termed fractions I, II, and III. Fraction I was cholesteryl ester- and phospholipid-rich (ca. 35% and 30% of lipoprotein mass, respectively), and contained apoA-I as its prominent apolipoprotein constituent. In contrast, triglyceride-rich fractions II and III both exhibited a complex apolipoprotein pattern, including apoB-100, apoA-I, and apoE whose amino acid composition and NH2-terminal sequence in the badger are reported. However, fraction III appeared markedly enriched in apoE when compared to fraction II. On polyacrylamide gel electrophoresis, fraction I presented as a spectrum of particles with diameters in the 140-190 A range. In contrast, fraction II migrated as a single band with a diameter of approximately 200 A, and fraction III presented as a single band or a doublet with a diameter of 195-200 A. The respective plasma concentrations and chemical compositions of the three chromatographic fractions were determined at four different dates of the year (i.e., April, August, November, and January), each of which corresponded to a different endocrine status in the badger. Thus hypothyroidism appeared to be associated with an increase in the concentration of fraction I, while the lowering in summer of the plasma level of testosterone correlated well with an increase in the concentration of fraction II. At the same time, the respective proportions of hydrophobic lipids in this latter material modified with an increase of triglycerides. Finally, both the apolipoprotein pattern of fraction III, and the chronologic profile of the successive variations of its concentration, suggest that it could represent a metabolic precursor of fraction II. These results suggest that the respective metabolism of the lipoproteins constituting the three chromatographic fractions could be under control by thyroid and testis secretions, operating via a complex combined regulation of the activities of the enzymes and receptors involved in these metabolic processes.     

Intestinal lymph and plasma lipoproteins in the preruminant calf: partial resolution of particle heterogeneity in the 1.040-1.090 g/ml interval.

Our previous studies in the preruminant calf have provided evidence for the heterogeneity of lipoprotein particles in the 1.040-1.090 g/ml density interval in both plasma and postprandial intestinal lymph (Bauchart, D. et al., 1989. J. Lipid Res. 30: 1499-1514; and Laplaud, P. M. et al., 1990. J. Lipid Res. 31: 1781-1792). We therefore attempted to resolve this heterogeneity by use of heparin-Sepharose affinity chromatography. Experiments were performed on three calves; portal vein plasma and intestinal lymph were obtained simultaneously 10 h after a meal, i.e., at peak lipid absorption. In both fluids, the chromatographic profile presented three fractions, I, II, and III. Fraction I was characterized by the presence of cholesteryl ester-rich particles (approximately 35-37% of lipoprotein mass), which migrated electrophoretically as typical high density lipoproteins and exhibited Stokes diameters in the 130-160 A range; apoA-I was the predominant protein. In addition to this polypeptide, fraction II contained small amounts of a supplementary protein (Mr approximately 51,000), exhibiting heparin-binding properties. In the light of results reported in the literature, we suggest that this latter protein could correspond to beta 2 glycoprotein I. The chemical composition of each fraction II closely resembled that of the corresponding fraction I, while their electrophoretic migrations appeared slightly slower and their Stokes diameters slightly larger (155-165 A). Apart from the presence of small amounts of apoA-I, two high Mr proteins (Mr approx. 560,000 and 300,000) were typical of the apolipoprotein moiety of fractions III. The lower Mr form was present as a trace component only in fraction III originating from plasma; its proportion increased in lymph fraction III so as to approximately match that of the higher Mr (i.e., 560,000) protein. In both plasma and lymph, fraction III was electrophoretically heterogeneous, exhibiting a doublet of bands with migration and Stokes diameters (250 A) typical of low density lipoprotein particles. However, no evidence for the presence of a particle resembling lipoprotein[a] in fraction III could be obtained. In lymph only, fraction III contained a supplementary population of lipoproteins with migration intermediary between those of conventional low and high density lipoproteins and with Stokes diameters in the 190-200 A range. Other specific features of lymph fraction III included a sevenfold increase in its triglyceride content (8.5 +/- 3.4% vs. 1.2 +/- 1.1% in the corresponding fraction from plasma), to the detriment of cholesteryl esters, and a higher proportion of protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apolipoprotein C-III displacement of apolipoprotein E from VLDL: effect of particle size.

ApoC-III and apoE are important determinants of intravascular lipolysis and clearance of triglyceride-rich chylomicrons and VLDL from the blood plasma. Interactions of these two apolipoproteins were studied by adding purified human apoC-III to human plasma at levels observed in hypertriglyceridemic subjects and incubating under specific conditions (2 h, 37 degrees C). As plasma concentrations of apoC-III protein were increased, the contents in both VLDL and HDL were also increased. Addition of apoC-III at concentrations up to four times the intrinsic concentration resulted in the decreasing incremental binding of apoC-III to VLDL while HDL bound increasing amounts without evidence of saturation. No changes were found in lipid content or in particle size of any lipoprotein in these experiments. However, distribution of the intrinsic apoE in different lipoprotein particles changed markedly with displacement of apoE from VLDL to HDL. The fraction of VLDL apoE that was displaced from VLDL to HDL at these high apoC-III concentrations varied among individuals from 20% to 100% its intrinsic level. The proportion of VLDL apoE that was tightly bound (0% to 80%) was found to be reproducible and to correlate with several indices of VLDL particle size. In the group of subjects studied, strongly adherent apoE was essentially absent from VLDL particles having an average content of less than 50,000 molecules of triglyceride.Addition of apoC-III to plasma almost completely displaces apoE from small VLDL particles. Larger VLDL contain tightly bound apoE which are not displaced by increasing concentration of apoC-III.     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)