Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

Isolation and characterization of an apoA-II-containing lipoprotein (LP-A-II:B complex) from plasma very low density lipoproteins of patients with Tangier disease and type V hyperlipoproteinemia

Previous studies have shown that very low density lipoproteins (VLDL) from patients with Tangier disease are less effective as a substrate for human milk lipoprotein lipase (LPL) than VLDL from normal controls as assessed by measuring the first order rate constant (k1) of triglyceride hydrolysis. Tangier VLDL also has a higher content of apolipoprotein (apo) A-II than normal VLDL. To explore the possible relationship between the relatively high concentration of apoA-II in VLDL and low k1 values, Tangier VLDL were fractionated on an anti-apoA-II immunosorber. The retained fraction contained a newly identified triglyceride-rich lipoprotein characterized by the presence of apolipoproteins A-II, B, C-I, C-II, C-III, D, and E (LP-A-II:B:C:D:E or LP-A-II:B complex), whereas the unretained fraction consisted of previously identified triglyceride-rich apoB-containing lipoproteins free of apoA-II. In VLDL from patients with Tangier disease or type V hyperlipoproteinemia, the LP-A-II:B complex accounted for 70-90% and 25-70% of the total apoB content, respectively. The LP-A-II:B complexes had similar lipid and apolipoprotein composition; they were poor substrates for LPL as indicated by their low k1 values (0.014-0.016 min-1). In contrast, the apoA-II-free lipoproteins present in unretained fractions were effective substrates for LPL with k1 values equal to or greater than 0.0313 min-1. These results indicate that triglyceride-rich lipoproteins consist of several apoB-containing lipoproteins, including the LP-A-II:B complex, and that lipoprotein particles of similar size and density but distinct apolipoprotein composition also possess distinct metabolic properties.     

Isolation and partial characterization of high-density lipoprotein HDL1 from rat plasma by gradient centrifugation.

The lipoproteins isolated from rat plasma by flotation in the density range 1.019-1.063 g/ml were further characterized. Using rate zonal ultracentrifugation, we isolated two lipoproteins in almost equal proportions from this density range. Similar isolations may be accomplished with density gradients in a swinging-bucket rotor. On isopycnic-density-gradient ultracentrifugation one component banded at rho = 1.031 g/ml and the other at rho = 1.054 g/ml. More that 98% of the apoprotein of the lighter component was B protein, and hence this particle is LD (low-density) lipoprotein. Of the apoproteins of the rho = 1.054 g/ml particles, designated lipoprotein HDL1, over 60% was arginine-rich peptide, and the remainder was A-I, A-IV and C peptides. The molecular weight of these lipoproteins determined by agarose column chromatography was 2.36 x 10(6) for LD lipoprotein and 1.30 x 10(6) for lipoprotein HDL1. On electron microscopy the radius of LD lipoprotein was 14.0 nm and that of lipoprotein HDL1 was 10.0 nm, in contrast with molecular radii of 10.4 nm and 8.4 nm respectively determined from the gel-permeation-chromatography data. The lipid and phospholipid composition of both particles was determined. Lipoprotein HDL1 was notable for both the concentration of its esterified cholesterol, which was similar to that of LD lipoprotein, and the low triacylglycerol content, resembling that of HD lipoprotein. The possible origin of lipoprotein HDL1 is discussed.     

Heterogeneity of human plasma lipoprotein (a). Isolation and characterization of the lipoprotein subspecies and their apoproteins

Lipoprotein (a) (Lp(a] from the plasma of normolipidemic human donors was isolated by rate zonal and isopycnic density gradient ultracentrifugation. The final preparations usually contained varying amounts of isopycnic low-density lipoproteins (LDL), which were totally removed either by heparin-Sepharose column chromatography or by chromatofocusing. The Lp(a) preparations exhibited both inter- and intraindividual density heterogeneity which was accounted for by the differences in their protein and lipid composition. In addition, there was heterogeneity in the size of apoprotein (a) (apo(a] which was found to be linked to apoprotein B (apo-B) through disulfide bonds. Three different apo(a) species were obtained; they had a size either smaller, equal to, or larger than apo-B-100, the protein moiety of LDL. The apo(a) that was smaller than apo-B resided in a low-density Lp(a) particle whose peak was in the 1.019-1.063 g/ml density range. The larger apo(a) was a component of the dense Lp(a) particle and was responsible for the increased density in this Lp(a) species. The third apo(a) which was equivalent in size to apo-B resided in a density range intermediate between the other two Lp(a)s. It is concluded that Lp(a) may differ not only from one individual to another, but also within the same individual who may have more than one Lp(a) species. Part of this heterogeneity may be accounted for by differences in the (a) polypeptide.     

Isolation and partial characterization of the plasma membrane from human spermatozoa

Ejaculated human spermatozoa were subjected to nitrogen cavitation (600 psi for ten min) to remove the plasma membrane (PM). Electron microscopic examination of the cavitated cells revealed that 33% of the PM was removed from the sperm which includes both the head and tail regions. The released membrane was separated from the cavitated cells by centrifugation followed by a discontinuous sucrose density gradient centrifugation. A single membrane population was resolved at the 1.0 M sucrose interface. Examination of the isolated membranes by electron microscopy revealed vesicles of various sizes displaying unit membrane structures. Biochemical analysis of the isolated membranes showed a threefold enrichment in the surface membrane marker 5' nucleotidase and also suggested little contamination by enzymes from the cytosol (lactate dehydrogenase) or mitochondria (cytochrome oxidase). Analytical lipid analysis of the isolated membranes revealed a 26-fold enrichment in the distribution of cholesterol, an 11-fold enrichment of phospholipids, and a cholesterol:phospholipid molar ratio of 0.83. Also found was a twofold increase in glycosphingolipids which are ubiquitous components of PM in eukaryotic cells. These data indicate that the membrane vesicles isolated after nitrogen cavitation are primarily PM.     

Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II

Two populations of A-I-containing lipoprotein particles: A-I-containing lipoprotein with A-II (Lp (A-I with A-II], and A-I-containing lipoprotein without A-II (Lp (A-I without A-II] have been isolated from plasma of 10 normolipidemic subjects by immunoaffinity chromatography and characterized. Both types of particles possess alpha-electrophoretic mobility and hydrated density in the range of plasma high-density lipoproteins (HDL). Lp (A-I without A-II) and Lp (A-I with A-II) are heterogeneous in size. Lp (A-I without A-II) comprised two distinct particle sizes with mean apparent molecular weight and Stokes diameter of 3.01 X 10(5), and 10.8 nm for Lp (A-I without A-II)1, and 1.64 X 10(5), and 8.5 nm for Lp (A-I without A-II)2. Lp (A-I with A-II) usually contained particles of at least three distinct molecular sizes with mean apparent molecular weight and Stokes diameter of 2.28 X 10(5) and 9.6 nm for Lp (A-I with A-II)1, 1.80 X 10(5) and 8.9 nm for Lp (A-I with A-II)2, and 1.25 X 10(5) and 8.0 nm for Lp (A-I with A-II)3. Apoproteins C, D, and E, and lecithin:cholesterol acyltransferase (LCAT) were detected in both Lp (A-I without A-II) and Lp (A-I with A-II) with most of the apoprotein D, and E, and LCAT (EC 2.3.1.43) in Lp (A-I with A-II) particles. Lp (A-I without A-II) had a slightly higher lipid/protein ratio than Lp (A-I with A-II). Lp (A-I with A-II) had an A-I/A-II molar ratio of approximately 2:1. The percentage of plasma A-I associated with Lp (A-I without A-II) was highly correlated with the A-I/A-II ratio of plasma (r = 0.96, n = 10). The variation in A-I/A-II ratio of HDL density subfractions therefore reflects different proportions of two discrete types of particles: particles containing A-I and A-II in a nearly constant ratio and particles containing A-II but no A-II. Each type of particle is heterogeneous in size and in apoprotein composition.     

Isolation and partial characterization of human kidney gangliosides.

1. Eight gangliosides were purified from chloroform/methanol extracts of human kidneys by using modified Folch partition, dialysis, ethanol precipitation, silicic acid column chromatography and preparative thin-layer chromatography. 2. By thin-layer chromatographic behaviour and gas-liquid chromatographic determinations the main gangliosides in human kidney are N-acetylneuraminyllactosylceramide (74% of total) and di-N-acetylneuraminyllactosylceramide (19% of total). 3. Five hexosamine-containing fractions were isolated. Four of them were homogeneous on thin-layer chromatography, and one contained two gangliosides. By gas-liquid chromatography-mass spectrometry it was shown that two gangliosides (together 5% of total) contain glucosamine, and one (1% of total) contains galactosamine. The other of the glucosamine gangliosides contains fucose in addition to the usual sugars found in gangliosides. Of the two remaining hexosamine positive fractions (together 1% of total) one was homogeneous on thin-layer chromatography, the other contained two gangliosides. These two fractions contained both glucosamine and galactosamine. 4. The main long-chain base in all fractions was sphingosine.     

Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. Two forms of PLTP exist in human plasma, one catalytically active (high activity form, HA-PLTP) and the other inactive (low activity form, LA-PLTP) (Oka, T., Kujiraoka, T., Ito, M., Egashira, T., Takahashi, S., Nanjee, N. M., Miller, N. E., Metso, J., Olkkonen, V. M., Ehnholm, C., Jauhiainen, M., and Hattori, H. (2000) J. Lipid Res. 41, 1651-1657). The two forms are associated with macromolecular complexes of different size. The apparent size of LA-PLTP is 520 kDa and that of HA-PLTP is 160 kDa. Of the circulating PLTP mass only a minor portion is in the HA-PLTP form in normolipidemic subjects. In the present study we have isolated and partially characterized the LA and HA forms of PLTP. Both LA- and HA-PLTP bind to heparin-Sepharose and can be separated by elution with 0-0.5 m NaCl gradient, with HA-PLTP displaying higher affinity for the matrix. LA-PLTP was further purified using hydrophobic butyl-Sepharose and anti-PLTP immunoaffinity chromatography steps. HA-PLTP was subjected to a second heparin-Sepharose step and hydroxylapatite chromatography. Analysis of the two forms of PLTP by SDS-PAGE, Western blotting, immunoprecipitation, and gel filtration demonstrates that LA-PLTP is complexed with apoA-I whereas HA-PLTP is not. Instead, HA-PLTP copurified with apoE. Based on these findings we suggest a model in which nascent PLTP enters the circulation as a high specific activity form not associated with apoA-I. During or after the transfer of lipolytic surface remnants to HDL, PLTP is transferred to apoA-I-containing HDL particles and thereby becomes part of the low activity complex.     

Isolation and partial characterization of chick brain synaptic plasma membranes.

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones, D. H. and Matus, A. I. (1974) Biochim. Biophys. Acta 356, 276-287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organelles. Fraction 2 was recovered from the 28.5-34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na+ +K+)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5'-nucleotidase (EC 3.1.3.5) were, respectively, 4.5, 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Isolation and partial characterization of rabbit plasma alpha1-antitrypsin.

Alpha1-Antitrypsin was isolated from rabbit plasma by salting out with (NH4)2SO4 followed by ion-exchange chromatography either on DEAE-Sephadex or DEAE-cellulose (each at pH8.8 and 6.5), and affinity chromatography on Sepharose-Cibacron Blue and Sepharose-concanavalin A. The protein thus obtained was homogeneous during crossed immunoelectrophoresis by using an antiserum to whole rabbit plasma, but it migrated as two broad bands when electrophoresed in alkaline polyacrylamide gels. Under optimal loading conditions, two or three subcomponents could be distinguished in each band. The two major forms of rabbit alpha1-antitrypsin, designated components F and S, were separated by preparative polyacrylamide-gel electrophoresis, and some of their physico-chemical properties were established. Both forms reacted with trypsin at a molar ratio of 1:1. Their elution volumes from a Sephadex G-200 column were identical, corresponding to a mol.wt. of 58000; however, some heterogeneity was observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing in polyacrylamide gel in a pH 4-6 gradient revealed a multiple-band pattern for each form in the range of pH4.4-4.9. The two forms of rabbit alpha1-antitrypsin possessed the same N-terminal amino acid (glutamic acid) and had very similar amino acid and carbohydrate compositions.     

Identification and characterization of apolipoprotein(AII-E2-AII) complex in human plasma lipoprotein.

A new apolipoprotein complex designated as the apo(AII-E2-AII) complex was identified in the lipoprotein fractions of human plasma with apoE phenotypes containing apoE2 (E4/E2, E3/E2, and E2/E2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by an immunoblotting assay using anti-apoE or anti-apoAII antibodies, established that the apo(AII-E2-AII) complex, with a molecular weight of 58,000, was identical to the complex consisting of apoE and apoAII, and that it also dissociated following reduction with beta-mercaptoethanol. This new complex was also demonstrated to be distinct from the apo(E-AII) complex and apoE monomer by isoelectric focusing, in the samples that were not treated with beta-mercaptoethanol. In apoE phenotype E3/E2, the apo(AII-E2-AII) complex was primarily included in the high-density lipoprotein (HDL, 1.063 < d < 1.21 g/ml) fraction, but was also observed in a small quantity in the very-low-density lipoprotein (VLDL, d < 1.006 g/ml) fraction. For further characterization, the apo(AII-E2-AII) complex was isolated by preparative SDS-PAGE, and no contamination of apo(E-AII) complex and apoE monomer was detected by immunoblotting assay using an anti-apoE antibody. It was confirmed by an enzyme-linked immunosorbent assay (ELISA) system that a molecular ratio between apoAII monomer and apoE in the isolated apo(AII-E2-AII) complex was approx. 2, when the apo(E-AII) complex was used as a standard with the ratio of 1:1. It indicates that the apo(AII-E2-AII) complex is formed from two molecules of apoAII monomer and one molecule of apoE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of human platelet vinculin

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.     

Isolation and partial characterization of an arginine-rich apolipoprotein from human plasma very-low-density lipoproteins: apolipoprotein E.

A water-insoluble apoprotein was isolated from apo-VLDL by column chromatography on Sephadex G-200 in sodium dodecylsulfate followed by preparative polyacrylamide gel electrophoresis in a discontinous sodium dodecylsulfate system, or by preparative electrophoresis alone. The protein was similar in amino acid composition to the "arginine-rich protein" reported by Shore and Shore. It represented about 10% of the total protein mass of VLDL. The apoprotein showed one single band with an apparent Mr of 39000 in sodium dodecylsulfate gel electrophoresis, and was homogeneous in gel electrophoresis at pH 8.9 In 8M urea. Immunochemical studies also showed homogeneity of this protein, and antisera prepared against it did not react with any other of the well known apolipoproteins, but did react with VLDL and apo-VLDL preparations. Analytical isoelectric focusing in 8M urea resulted in a heterogeneous banding pattern showing three major polypeptides with pI values of 5.5, 5.6 and 5.75. Thus this apolipoprotein clearly differs from the apo-B and apo-C polypeptides of VLDL as well as from apoproteins A and D in its molecular weight, amino acid composition, focusing behavior and immunochemical properties.