Antonio Gotto

Evidence for the identity of the major apoprotein in low density and very low density lipoproteins in normal subjects and patients with familial hyperlipoproteinemia

The major apoprotein(s) from human plasma low density lipoproteins was isolated and compared with a major protein fraction (fraction I) from very low density lipoproteins (VLDL). Fraction I had been previously found to comprise approximately 40% of the total protein of VLDL. Fraction I from VLDL and apoLDL from normal subjects were indistinguishable in amino acid compositions and circular dichroic spectra. They yielded indistinguishable displacement curves of LDL-(125)I by radioimmunoassay and formed immunoprecipitin lines of complete identity. Fraction I from VLDL of normal subjects was compared with the fraction isolated from patients with familial types II, III, IV, and V hyperlipoproteinemia. There were no detectable differences between any of these fractions in amino acid compositions, circular dichroic spectra, and immunochemical properties. It was, therefore, concluded that short of peptide mapping or determination of amino acid sequence, fraction I from VLDL of each subject with familial hyperlipoproteinemia appears to be identical with fraction I and apoLDL from normal individuals.A new convenient method of preparation of soluble apoLDL, modified from a procedure previously described from this laboratory, is presented.

Human serum beta-lipoprotein: Preparation and properties of a delipidated, soluble derivative

Abstract An investigation of the protein moiety of human serum β-lipoprotein has been hampered by the insolubility of the delipidated protein in aqueous media ( Scanu et al ., 1958; Banasak and McDonald, 1962; Margolis and Langdon, 1966 ). The purpose of this communication is to describe a method which can conveniently be used for the preparation in high yield of a delipidated form of β-lipoprotein. This is accomplished by succinylation, delipidation and solubilization of the protein moiety in the presence of relatively low concentrations of decyl sulfate, an ionic detergent which may be removed readily by dialysis. Pertinent properties of this delipidated soluble protein are presented.

Application of electron spin resonance to the study of the structure of human serum lipoproteins

Abstract Human serum high density (HDL) and low density (LDL) lipoproteins were spin labeled with isothiocyanate and maleimide derivatives of the nitroxide radical N-(1-oxyl-2, 2, 6, 6-tetramethyl-4-piperidine). The ESR spectra of the HDL and LDL derivatives demonstrated at least two kinds of protein binding sites involving amino groups, one with slight and another with strong constraint on the tumbling of the nitroxide radical. There was relatively more of the strongly constrained component in labeled HDL than in LDL. The strongly immobilized signal was due to the binding of lipid as was shown by the effect of delipidation.

Solid phase radioimmunoassay of apolipoprotein B (apo B) in normal human plasma.

A solid phase radioimmunoassay (RIA) has been developed for apolipoprotein B (apoB), a major constituent of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in man. Antisera were prepared against apoB in goats and rabbits, coupled to bromoacetyle cellulose, and the complex was incubated with [125I] LDL. The RIA was based on the displacement of [ 1252]-LDL by unknown samples, as compared with unlabeled LDL standards, using a logit transformation to calculate results. The RIA was found to be satisfactory in terms of precision, sensitivity, reproducibility and specificity. Control subjects had mean apoB levels of 94 mg/100 ml in whole fasting plasma, of which 3.6 mg/100 ml plasma were in the VLDL, while 86 mg/100 ml plasma were in the LDL. Both the triglyceride and apoB content of VLDL, and the cholesterol and apoB content of LDL were positively correlated. It is concluded that the solid phase radioimmunoassay described in the present report provides a rapid and relatively simple means of quantitating apoB in normal human plasma.

Preparation and properties of an apoprotein derivative of human serum β-lipoprotein

The aim of this study was to develop a convenient method for the preparation and study of a soluble delipidated form of human serum β-lipoprotein. This was achieved by succinylation and delipidation with ether-ethanol (3∶1). The succinylated apoprotein was soluble in either 0.13 M Tris-HCl buffer, pH 8.2 (for β-lipoprotein prepared by ultracentrifugation) or in the same Tris buffer to which 5 mM sodium decyl sulfate was added (for heparin-Mn precipitated β-lipoprotein). The immunological activity of β-lipoprotein or its apoprotein were markedly altered by succinylation. Whereas the succinylated β-lipoprotein appeared as one peak in the analytical ultracentrifuge, the succinylated apoprotein appeared as two. Under the electron microscope β-lipoprotein and succinylated β-lipoprotein were indistinguishable, appearing as uniform preparations of spherical particles 215 to 220 A in diamater.