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Biomedical subjects

A M Scanu

Publications and source records attributed to A M Scanu.

At least 181 records · Page 10Linked to original sources
PubMed1978

Sedimentation behavior of native and reduced apolipoprotein A-II from human high density lipoproteins.

The solution properties of human serum apolipoprotein A-II, both in the native and in the reduced forms, were investigated by the technique of sedimentation equilibrium in the analytical ultracentrifuge. For both proteins, the apparent weight average molecular weights determined in neutral buffer systems were found to be dependent on protein concentration and invariant with the rotor speeds used (16,000 to 44,000 rpm) indicating a reversible self-association. These results were also found to be independent of temperature between 5 and 30 degrees C. The pattern of self-association of native apolipoprotein A-II could best be described by a monomer-dimer-trimer equilibrium, in agreement with previously reported data (Vitello, L B., and Scanu, A. M. (1975), Biochemistry 15, 1161). The self-association pattern of apolipoprotein A-II reduced in the presence of 50 mM dithiothreitol conformed with a monomer-dimer-tetramer equilibrium similar to that reported for the native single chain apolipoprotein A-II of the rhesus monkey (Barbeau, D. L., et al. (1977), J. Biol. Chem. 252, 6745), but differing significantly from that reported for the reduced and carboxymethylated human product (Osborne, J. C. , et al. (1975), Biochemistry 14, 3741).

Apolipoproteins↗
PubMed1978

Ultrastructure of serum high density lipoproteins: facts and models.

The complexity of the structure of plasma high density lipoproteins (HDL) has invited numerous approaches which have been directed at the study of the intact particles, their apolipoproteins and reassembled complexes. Parameters such as flotation and sedimentation coefficients, size and molecular weight have been determined and in addition, through scattering techniques, an understanding has been obtained on the long range organization between core (cholesteryl esters and triglycerides) and surface components (unesterified cholesterol, phospholipids and apoproteins). In the case of the apolipoproteins, the knowledge of their primary structure has facilitated the study of their physicochemical properties in solution and at the air-water interface and has also permitted realistic predictions of the two dimensional organization, not only of their alpha-helical segments but also of the beta-pleated sheets, random coil and beta-turns, all of which have amphipathic properties. When all of the information from the physical and chemical studies is put together, the various HDL can be described as spherical structures having a liquid core of radius, r - 20.2 A, surrounded by a monolayer of cholesterol and phospholipids with closely packed hydrophobic ends on the surface of the core. The organization of the apoproteins at the lipoprotein interface is comparatively less understood. However, reasonable predictions can be made on secondary structure considerations and on their behavior at the air-water interface. The emerging overall structural information can be translated into a space-filling model that not only provides a useful representation of HDL, but, more importantly, a basis for planning future studies on the elucidation of the structure of these particles on a molecular level.

Chemical Phenomena↗
PubMed1978

Measurment of rhesus monkey (Macaca mulatta) apolipoprotein B in serum by radioimmunoassay: comparison of immunoreactivities of rhesus and human low density lipoproteins.

A sensitive and specific double antibody radio-immunoassay for the major apolipoprotein (apoB) of rhesus (Macaca mulatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The anti-serum was raised to LDL (d 1.030-1.040 g/ml) and the LDL(2) (d 1.020-1.050 g/ml) was labeled with (125)I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02-0.5 micro g of LDL(2), had an inter-assay coefficient of variation of 4.5%. This assay was successfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an "average American diet," a 25% peanut oil and 2% cholesterol-supplemented PMC diet, and a 25% coconut oil and 2% cholesterol-supplemented PMC diet. The control monkeys (n = 13) had a serum cholesterol of 146 +/- 28 mg/dl and an apoB of 50 +/- 18 mg/dl. In the monkeys maintained on the atherogenic diets the serum apoB was elevated: 103 +/- 28 mg/dl (American), 102 +/- 35 mg/dl (peanut oil), and 312 +/- 88 mg/dl (coconut oil). The values for serum total cholesterol were 333 +/- 65 mg/dl (American), 606 +/- 212 mg/dl (peanut oil), and 864 +/- 233 mg/dl (coconut oil) and were elevated relative to controls (P < 0.001). For each of the diets, total serum cholesterol correlated with serum apoB (P < 0.001). The slopes of the regression lines of serum apoB vs. cholesterol for the monkeys on the PMC, American, and coconut oil diets were similar (m = 0.531, 0.401, and 0.359, respectively), but differed from that of monkeys on the peanut oil diet (m = 0.121). The immunoreactivities of rhesus and human LDL were compared using specific antisera raised against these antigens. In homologous assay systems, monkey and human LDL exhibited unique immunological determinants. The same results were obtained with the delipidated preparations of the two LDLs using antisera raised against either monkey or human apoB. Crossover studies using a heterologous tracer with each anti-serum resulted in the selection of a specific population of antibodies directed against antigenic sites shared by these two LDL species.

Animals↗
PubMed1977

An ultracentrifugal study of the self-association of canine apolipoprotein A-I in solution.

The sedimentation behavior of canine apolipoprotein (apo) A-I in 0.02 M EDTA, pH 8.6, was studied as a function of protein concentration by the techniques of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge. At concentrations of less than 1 g/liter, apo-A-I exhibited a monomodal sedimentation pattern, with apparent sedimentation coefficients which varied from 2.3 to 3.5 S with increasing protein concentrations. Above 1.5 g/liter, apo-A-I had two well resolved peaks with s20,w values of 4.15 S and 5.75 S. The proportion of the 5.75 S component increased with increasing apo-A-I concentrations, with a concomitant decrease of the 4.15 S component. By sedimentation equilibrium ultracentrifugation with both the conventional and meniscus-depletion methods, the apparent weight-average molecular weight of apo-A-I was found to be concentration-dependent. At a protein concentration of 5.25 g/liter, an apparent weight average molecular weight of 138,000 was determined, indicating that molecular species larger than a tetramer (monomer molecular weight = 28,000) were present in solution. When analyzed in terms of a reversible self-associating system, the experimental data could best be described according to a monomer-dimer-tetramer-octamer model, as previously reported from human apo-A-I (Vitello, L. B., and Scanu, A. M. (1975) J. Biol. Chem. 251, 1131-1136). The equilibrium constants were: K2 = 4.5 liters/g, K4 = 470 liters3/g3, and K8 = 41,600 liters7/g7, respectively.

Animals↗
PubMed1977

Electron microscopy of negatively stained and freeze-etched high density lipoprotein-3 from human serum.

High density lipoproteins of d = 1.12 to 1.21 g/ml from human serum (HDL3) were studied by electron microscopy with both negative staining and freeze-etching techniques. For the negatively stained specimens, a modified conventional transmission electron microscope as well as a scanning transmission electron microscope were used. The freeze-etched specimens were examined by a conventional transmission electron microscope. The diameter of HDL3 was found to be 105 +/- 4 A by freeze-etching and 94 +/- 6 A by negative staining. The surface of the HDL3 particles exhibited about 12 discrete domains, 28 +/- 3 A (freeze-etched) and 28 +/- 4 A (negatively stained) in diameter, of undefined chemical composition. Moreover, the freeze-etched specimens revealed an inner core 40 +/- 2 A in diameter, corresponding to estimated values reported previously. All information is consistent with the HDL3 model proposed by B. W. Shen, F. J. Kézdy, and A. M. Scanu [(1977) Proc. Natl. Acad. Sci. USA 74, 837-841], with additional evidence for well-defined surface substructure. The consistency of the images obtained with the various electron microscopy techniques and the marked change in the appearance of the surface in the HDL3 preparations that were digested by phospholipase A2 (EC 3.1.1.4) support the validity of the interpretation.

Freeze Etching↗
PubMed1977

Structure of human serum lipoproteins inferred from compositional analysis.

Analysis of the correlations between size and chemical composition of lipoproteins of normolipidemic human plasma shows that the structure of all circulating lipoproteins is consistent with a spherical model of radius r in which a spherical liquid core of cholesterol esters and triglycerides of radius = r --20.2 A is surrounded by a monolayer of cholesterol and phospholipids with closely hydrophobic ends on the surface of the core. The average molecular areas at this inner surface are Spl = 68.5 A2/molecule for phospholipids and Sc= 39.1 A2/molecule for cholesterol. The proteins are closely packed with the hydrophilic head groups of phospholipids at the outer surface of the particle, with S' pl = 62.7 A2/molecule for phospholipids and Saa = 15.6 A2/amino acid for proteins. The polar head group of free cholesterol does not participate in the packing of the outer layer and thus must be masked by proteins. Free cholesterol is distributed among the circulating lipoproteins--with the exception of very high density lipoprotein and perhaps chylomicrons--according to a thermodynamic equilibrium governed by the curvature of the surface of the particle.

Blood Proteins↗
PubMed1977

Fractionation of human serum lipoproteins by single-spin gradient ultracentrifugation: quantification of apolipoproteins B and A-1 and lipid components.

A sensitive and reproducible method has been developed for separation of the major serum lipoproteins from 1 ml or less of human serum by isopycnic density gradient ultracentrifugation. The serum, applied to a step gradient (total volume 12.8 ml), was spun for 48 hr at 38,000 rpm at 10 degrees C and, in each of the fractions, apolipoproteins B and A-I were quantified by the respective radioimmunoassays. The markers for lipid distribution used were [4-(14)C]cholesterol and [U-(14)C]lecithin, each incubated with an aliquot of serum at 20 degrees C for 75 min prior to ultracentrifugation. In control sera, three main fractions, very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins were clearly separated from a bottom fraction. Their flotational, electrophoretic, and chemical properties were in good agreement with those reported for the corresponding lipoproteins separated by conventional ultracentrifugation. Both apo B and apo A-I were fully recovered. Essentially all of the apo B was found in VLDL (9.3 +/- 3.5%) and LDL (87 +/- 4.6%); of the apo A-I, 81.0 +/- 5.7% was in HDL and the remainder (17.0 +/- 5.8%) was in the bottom fraction. The peak activities of [(14)C]cholesterol coincided with the peak of apo B in both LDL and VLDL, and with the peak of apo A-I in HDL. The results with the radiolabeled cholesterol were in good agreement with those obtained by chemical analyses. Carbon 14-labeled lecithin, although fully recovered, was not an accurate marker of phospholipid distribution because, under our experimental conditions, a significant amount of the lecithin was converted into its lyso derivative. The mechanism of the conversion was not established; it appeared to be unrelated to the activities of either lecithin-cholesterol acyl transferase or a Ca(2+)-dependent phospholipase. Besides its validity in the study of control sera, our method also proved successful in the separation of the serum lipoproteins of the few patients with dyslipoproteinemia (abetalipoproteinemia and familial hypercholesterolemia) who were examined. However, the applicability of the method to all dyslipoproteinemias was not assessed. Taken together, the results indicate that the single-spin method could be useful in clinical studies as a complement to other established techniques.

Abetalipoproteinemia↗
PubMed1976

Study of abnormal plasma low-density lipoprotein in rhesus monkeys with diet-induced hyperlipidemia.

Male rhesus monkeys were divided into three groups: five were fed a regular primate chow diet and were used as controls; four received an "average" American diet; and five a special low-fat primate chow diet supplemented with 25% coconut oil and 2% cholesterol. In all of these animals, the plasma low-density lipoproteins (LDL) were isolated by ultracentrifugal flotation between densities of 1.019 and 1.050 g/ml. The LDL of the five control monkeys had variable molecular weights, with a mean value of 3.12 +/- 0.21 X 10(6) (range: 2.92 X 10(6) to 3.45 X 10(6)), and an average partial specific volume of 0.969 +/- 0.003 ml/g; both were assessed by flotation equilibrium analysis in the analytical ultracentrifuge. In the individual animals, however, the physical properties of LDL were invariant with time. The administration of either an "average" American diet or a coconut oil-cholesterol diet was accompanied by hypercholesterolemia associated with changes in LDL which were characterized by increases in molecular weight to 3.52 +/- 0.21 X 10(6) (average of nine monkeys) and in partial specific volume to 0.973 +/- 0.002 ml/g. These changes were particularly evident when the molecular weight of LDL from monkeys in the normolipidemic state was compared with that obtained from the same monkeys during the hyperlipidemic state. Chemical analyses revealed that the particles from the hyperlipidemic animals had a relatively higher cholesteryl ester content, a slight increase in phospholipids, and a marked decrease to nearly complete absence of triglycerides. The other lipoprotein components, protein, carbohydrate, free cholesterol, and fatty acids, did not vary significantly from those of control LDL. It is concluded that the administration of atherogenic diets causes structural changes in LDL which appear to be accounted for, at least in part, by changes in the composition of the lipid moiety. The changes in physical and chemical properties noted in the LDL of rhesus monkeys with experimentally induced hypercholesterolemia contrast with the apparent structurally normal LDL from rhesus monkeys with spontaneous hypercholesterolemia reported previously.

Animals↗
PubMed1976

Ultracentrifugal behavior of apolipoprotein A-I of rhesus monkey (Macaca mulatta).

The sedimentation equilibrium and sedimentation velocity of apolipoprotein AI (apo-A-I) from high density lipoproteins of rhesus monkey plasma were studied in aqueous solutions of 0.02 M EDTA, pH 8.6, as a function of protein concentration. The sedimentation equilibrium results showed that, in the concentration range between 9 and 36 muM (0.25 to 1.0 g/liter), apo-A-I behaved as a single species of molecular weight 28,200. This molecular weight corresponds to that of the monomeric form as previously estimated from electrophoretic and chemical data. As the apo-A-I concentration was increased above 36 muM, the log c versus r2 plots from sedimentation equilibrium experiments became curvilinear, suggesting self-association. An analysis of these data generated nonlinear, nonoverlapping molecular weight versus concentration plots pointing at the existence of a heterogeneous population of apo-A-I. By sedimentation velocity studies apo-A-I at concentrations between 68 and 330 muM exhibited two distinct components, with S0/20,w = 1.9 and S0/20,w = 3.9. When separated by gel permeation chromatography, these two components had an identical amino acid composition and retained similar S20,w values as before column fractionation. The slow component had a diffusion coefficient of 8.27 X 10(-7) cm2/s and, assessed by the criteria of sedimentation equilibrium ultracentrifugation, remained monomeric even at concentrations of 125 muM. The fast component, when analyzed by low speed sedimentation equilibrium, behaved as a self-associating system which could best be fitted into a monomer-hexamer model in a rapid equilibrium. The equilibrium constant for this association was found to be 9.5 X 10(23) M(-5). Thus, rhesus apo-A-I, dissolved in 0.02 M EDTA, pH 8.6, consisted of two distinct species, one monomeric over a relatively wide range of concentrations, the other readily self-associating. Their structural relationship remains to be established. This ultracentrifugal behavior of rhesus apo-A-I differs markedly from that reported for human apo-AI (Vitello, L. B., and Scanu, A. M. (1976) J. Biol. Chem. 251, 1131-1136).

Animals↗
PubMed1976

Isolation and characterization of a dog serum lipoprotein having apolipoprotein A-I as its predominant protein constituent.

The serum high density lipoproteins (HDL) of normolipemic dogs (beagles) were isolated in the density range of p 1.063 to 1.21 g/ml, and characterized in terms of composition and physical properties (flotation and diffusion coefficients, partial specific volume, molecular weight, electrophoretic mobility, ultraviolet absorption, and circular dichroism). The results indicated that canine HDL is a relatively homogeneous class with a molecular weight of about 230 000 and general properties similar to those reported for human HDL. After delipidation, the resulting apolipoprotein, apo-HDL, was fractionated by Sephadex G-200 column chromatography in urea or guanidine hydrochloride solutions. About 90% of the apo-HDL consisted of a protein with a molecular weight of about 28 000, similar in amino acid composition to human apolipoprotein A-I and having the same NH2 terminus (aspartic acid) and COOH terminus (glutamine) and no carbohydrates. Two other proteins were isolated, one having an apparent mol wt of 55 000 and representing, at least in part, an aggregate of apolipoprotein A-I and the other component with a mol wt of 8000, not yet characterized. The results indicate that canine HDL, as an intact complex, has general physical properties that lie between those reported for human HDL2 and HDL3, and that it differs compositionally from the human products mainly in its predominant content of apo-A-I. These findings together with evidence for the relatively homogeneous nature of the canine HDL provide new prospects for unraveling the relationship between polypeptide composition and HDL structure.

Amino Acids↗
PubMed1976

Kinetic study of the action of snake venom phospholipase A2 on human serum high density lipoprotein 3.

The hydrolysis of the phospholipids of intact human serum high density lipoprotein 3 (HDL3) by pure alpha-phospholipase A2 from Crotalus adamanteus was studied by pH-stat titration. The enzyme quantitatively hydrolyzed phosphatidylcholine and phosphatidylethanolamine and left sphinogomyelin intact, yielding a stable and water-soluble modified HDL. Lysophospholipids and free fatty acids, the products of hydrolysis, remained in the lipoprotein. When 1 mol of defatted bovine serum albumin/mol of substrate phospholipids was added to the reaction mixture, up to 60% of the fatty acids and 85% of the lysophospholipids were removed from the modified lipoprotein. The immunological reactivity of the hydrolyzed HDL remained unaltered in both the presence and absence of albumin. The changes in the physical properties of the lipoprotein during hydrolysis were rather small, the most notable being an increase in the hydrated density and in the electrophoretic mobility in alkaline buffers. The hydrolysis followed an apparent first order time course with product inhibition (KI) and yielded values of kcat/Km = 7 X 10(5 M(-1)s(-1) and KI congruent to 1 X 10(-4) M. Addition of albumin to the reaction mixture relieved the product inhibition without any alteration of the kinetic parameters. High concentrations of albumin protected some of the substrate phospholipids from hydrolysis, presumably through complexation to the lipoprotein. The Arrhenius plot for the experimental first order rate constant in the absence of albumin (kexp = kcat (KI/Km)) was linear between 15 degrees and 47 degrees, indicating the absence of any phospholipid phase transitions and yielding an activation energy of 15.2 kcal/mol. From the accessibility of the HDL phospholipids to phospholipase A2 one concludes that the phosphatidylcholine and phosphatidylethanolamine are located at, or are in rapid equilibrium with, the surface of this lipoprotein. It also appears that these phospholipids are not essential for maintaining the supramolecular properties of the lipoprotein in vitro. Thsu the study of the modified Hdl should provide valuable information concenring the structure and function of this lipoprotein particularly with regard to the role played by shiingomyelin.

Binding Sites↗