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C Edelstein

Publications and source records attributed to C Edelstein.

At least 73 records · Page 4Linked to original sources
PubMed1983

In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.

Previous studies have established that human hepatocellular carcinoma cells (Hep G2) secrete into serum-free medium the pro form of apolipoprotein A-I (proapo-A-I) suggesting that its conversion to mature apo-A-I occurs after secretion. In order to assess the mode and site of proapo-A-I to apo-A-I conversion, we incubated the medium from [3H]proline-labeled Hep G2 cells with either human plasma, serum, lymph, or fractions thereof obtained by density gradient ultracentrifugation. The conversion was monitored by two-dimensional gel electrophoresis and by Edman degradation. Human plasma, serum, or mesenteric lymph all induced proapo-A-I to apo-A-I conversion; this was time dependent, unaffected by the serine protease inhibitor phenylmethylsulfonyl fluoride and inhibited by EDTA. Purified radiolabeled proapo-A-I bound to lymph chylomicrons and plasma high density lipoproteins. The converting enzyme was associated with both of these particles. Activity was also found in the d greater than 1.21-g/ml fraction and may have been derived from high density lipoprotein after displacement by high salts and/or ultracentrifugal force. We conclude that the conversion of proapo-A-I to apo-A-I occurs extracellularly and is probably effected by a metallo-enzyme which may act at the amphiphilic surface of either chylomicrons or high density lipoproteins.

Amino Acid Sequence↗
PubMed1983

Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease.

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.

Amino Acid Sequence↗
PubMed1983

Rapid isolation of apolipoprotein E from human plasma very low density lipoproteins by molecular sieve high performance liquid chromatography.

A rapid (less than 1 hour) and sensitive technique was developed for the isolation of apolipoprotein E from the other main components of human plasma very low density lipoproteins using molecular sieve high performance liquid chromatography with an approximate 80% recovery. The properties of the pure apoprotein were those reported in the literature for products isolated by conventional chromatographic and/or electrophoretic procedures.

Apolipoproteins↗
PubMed1981

Rapid fractionation of human high density apolipoproteins by high performance liquid chromatography.

A simple and rapid fractionation procedure (30 min) has been developed for the isolation of the major apoproteins from human serum high density lipoproteins by molecular sieving in a high performance liquid chromatographic column. Apo A-I, apoA-II and the C peptides are quantitatively resolved up to a protein load of 3 mg. The technique has also been successfully applied to the final purification of A apoproteins which had been isolated by conventional chromatographic procedures and as a sensitive analytical tool for assessing apoprotein purity.

Apolipoprotein A-I↗
PubMed1980

Characterization and measurement of human apolipoprotein A-II by radioimmunoassay.

The development of a radioimmunoassay for apolipoprotein A-II (apo A-II) is described. Initial studies revealed a lack of immunological identity between purified apo A-II used as the standard and serum or HDL. Extensive testing of different buffers, standards, antisera, tracers, utilization of a detergent, and heating of sera failed to resolve the problem. Gel filtration of iodinated and non-iodinated apo A-II on Sephadex G-100 columns showed that apo A-II, in dilute solution, elutes in a higher molecular zone than expected with a broad, assymetrical profile. The use of a subfraction of the tracer in the assay resulted in parallelism in the serum and standard dilution curves. The apo A-II assay was sensitive, specific, and reproducible. Apo A-II added to sera was fully recovered and delipidation did not affect the immunoreactivity of either serum or HDL. Apo A-II contributed approximately 20% to the protein mass of HDL. Comparison of these results with those obtained by radial immunodiffusion, and with previously reported data, indicates that the reactivity of apo A-II in its native and delipidated forms may be markedly influenced by different immunologic methodologies and their specific reagents. Caution should thus be shown at present in assigning absolute concentrations to apo A-II in serum or HDL.

Adult↗
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

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

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

Covalent structure of apolipoprotein A-II from Macaca mulatta serum high-density lipoproteins.

The covalent structure of apolipoprotein A-II, isolated from the serum high-density lipoprotein of a single male Rhesus monkey (Macaca mulatta), was determined. The amino acid sequence of this 77-residue polypeptide is: less than Glu-Ala-Glu-Glu-Pro5-Ser-Val-Glu-Ser-Leu10-Val-Ser-Gln-Tyr-Phe15-Gln-Thr-Val-Thr-Asp20-Tyr-Gly-Lys-Asp-Leu25-Met-Glu-Lys-Val-Lys30-Ser-Pro-Glu-Leu-Gln35-Ala-Gln-Ala-Lys-Ala40-Tyr-Phe-Glu-Lys-Ser45-Lys-Glu-Gln-Leu-Thr50-Pro-Leu-Val-Lys-Lys55-Ala-Gly-Thr-Asp-Leu60-Val-Asn-Phe-Leu-Ser65-Tyr-Phe-Val-Glu-Leu70-Arg-Thr-Gln-Pro-Ala75-Thr-Gln-COOH. A comparison of this structure to that of the monomeric form of human apolipoprotein A-II reveals a high degree of homology except for six conservative amino acid replacements (positions 3, 6, 40, 53, 59, and 71). Of particular structural significance is the replacement of cysteine by serine in position 6. This explaines why Rhesus A-II exists in monomeric form, contrary to the established dimeric nature of the human protein.

Amino Acid Sequence↗