Fractionation of human serum high density lipoprotein in urea solutions. Evidence for polypeptide heterogeneity

Scanu, Angelo M.; Toth, John P.; Edelstein, Celina; Koga, Shunichi; Stiller, E. T.

doi:10.1021/bi00836a027

Cited by 318 publications

(89 citation statements)

References 21 publications

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“…The results obtained here demonstrate that the decreased HDL cholesterol values were accompanied by a marked reduction in the serum concentrations of the major HDL apoproteins, apo A-l, and apo A-ll, over 90% of which form the bulk of apo HDL. 20 Several studies 121 have also noted that heterozygotes with FH have modestly reduced HDL cholesterol levels. Similar findings 22 have been made in the cord blood of infants with FH.…”

Section: Discussionmentioning

confidence: 99%

Abnormalities of high density lipoproteins in homozygous familial hypercholesterolemia.

Goldberg¹,

Fless²,

Baker³

et al. 1984

Arteriosclerosis

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The possibility that low high density lipoprotein (HDL) levels may add to the risk of occlusive atherosclerosis in familial hyperchoiesteroiemia (FH) is supported by the frequency and severity of this finding in these patients, particularly homozygotes. To investigate this abnormality in greater detail, we measured HDL lipid and apoprotein values in nine homozygotes with FH. Compared to their unaffected relatives, they had markedly reduced HDL cholesterol and apo A-l and A-ll levels. The values found in heterozygote relatives were between those of homozygotes and those of controls. Density gradient ultracentrifugation of serum or isolated HDL from homozygotes demonstrated little or no HDL 2 ; there was also a shift of homozygotes' HDL to an above normal peak density (d = 1.15 g/ml). Heterozygotes had milder abnormalities. The reduced HDL in FH seems to be related to elevated low density lipoprotein levels although the pathophysiology is unknown. These findings add weight to the concept that HDL abnormalities contribute to the relentless course of atherosclerosis in familial hyperchoiesteroiemia. (Arteriosclerosis 4:472-478, September/October 1984)T he homozygous form of familial hyperchoiesteroiemia (FH) is characterized by extreme hyperchoiesteroiemia, the development of cutaneous xanthomata in childhood, and the occurrence of occlusive atherosclerotic vascular disease that is commonly fatal before age 20. 1 The disease is due to mutations of the gene that controls the cellular receptor for low density lipoprotein (LDL), which results in defective binding or internalization of this lipoprotein. Poorly regulated, receptor-independent pathways then form the predominant route for the uptake of LDL, which results in an excessive accumulation of cholesterol in the tissues. 2 The exact mechanism for the development of atherosclerosis in these patients is not well under-

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Section: Discussionmentioning

confidence: 99%

Abnormalities of high density lipoproteins in homozygous familial hypercholesterolemia.

Goldberg¹,

Fless²,

Baker³

et al. 1984

Arteriosclerosis

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“…Further, the conformations of the apoproteins of HDL appear to be stabilized by interaction with lipids (3,4 (6). HDL was delipidated by chloroform-methanol extraction (7), and apoA-I and apoA-2 were prepared by Sephadex G-200 column chromatography (8 [1] where R is the gas constant, ACd in cal/°C per g is the excess heat capacity at Td (see denaturation of several globular proteins has been shown to conform to a two-state model (9,11 The interpretation that the thermal transition of apoA-I is due to a cooperative unfolding process is supported by the circular dichroism and ultra-violet spectroscopic studies of solutions of apoA-I (12) and apo-HDL (3) which indicate a major conformational change associated with a helix-coil transition between 45-70°. Given that the denaturation of apoA-I is a two-state process, it is possible to estimate a AH value from the published spectroscopic data (12), using the relationship: aH = -Rd(ln K)/d(l/T) [2] where K is a thermodynamic equilibrium constant for the native-denatured transition (13 Fig.…”

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confidence: 99%

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

Tall¹,

Small²,

Shipley³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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Temperature-dependent conformational changes of the principal apoprotein of human plasma high density lipoprotein (HDL), apoA-I, have been studied in the isolated apoprotein, in complexes of apoprotein with phospholipid, and in intact HDL. Differential scanning calorimetry shows that in solution apoA-I undergoes a reversible, twostate thermal denaturation (midpoint temperature 540). The enthalpy (2.4 cal/g) (10.0 J/g) and specific heat change (0.08 cal/°C per g) (0.33 J/°C per g) associated with the denaturation were used to calculate the free energy difference (AG) between native and unfolded apoA-I at 37°. AG (2.4 kcal/ mol) (10.0 kJ/mol) is less than that of other globular proteins (typically 8-14 kcal/mol) (33-59 kJ/mol), indicating that at 370 native apoA-I has a loosely folded conformation. Turbidity studies show that apoA-I is able to solubilize phospholipidin its native but not in its denatured form. Mixtures of apo-HDL (the total apoprotein of HDL) or apoA-I with dimyristoyl lecithin show a thermal transition at about 850 n-ot present in the lecithin or the apoprotein alone, which indicates that the native conformation of the apoprotein is stabilized by phospholipid. Scanning calorimetry of intact HDL shows a high-temperature endotherm associated with disruption of the HDL particle, su esting that in HDL the conformation of apoA-I is also stabilized by interaction with lipid. The loosely folded conformation of native, uncomplexed apoA-I may be specially adapted to the binding of lipid, since this process may involve both hydrophobic sites on the surface of the protein and concealed apolar amino acid residues that are exposed by a cooperative, low energy unfolding process.A knowledge of the structure and thermodynamic stability of serum lipoproteins is important to an understanding of their metabolism and role in diseases such as atherosclerosis. The human plasma high density lipoproteins (HDL) are spherical particles (1, 2) consisting of about 50% protein, 22% phospholipids, 3% free cholesterol, 14% cholesterol esters, and 8% triglycerides (3). The proteins (apo-HDL) include approximately 60-65% apoA-I [molecular weight (Mr) 28,331], 30% apoA-2 (Mr 17,380) and 5-10% low Mr C-peptides (4, 5). The proteins and polar head groups of phospholipids occupy the surface of the lipoprotein particle while the apolar lipid moieties form a hydrophobic core (1, 2). Further, the conformations of the apoproteins of HDL appear to be stabilized by interaction with lipids (3, 4). In the present study, we show that the principal apoprotein of human HDL, apoA-I, undergoes a reversible temperaturedependent conformational change with unusual thermodynamic properties. A comparison with other water-soluble proteins suggests a loosely folded structure of the native apo- Fig. la and b). From the DSC thermogram, it is possible to measure both the calorimetric enthalpy of the transition in cal/g (AHcal) (1 cal = 4.184 J) from the area under the curve, and the effective enthalpy in cal/mol (AHvH) using an expression der...

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“…Human plasma high density lipoproteins (HDL) have been shown to contain two major apoprotein components that account for 90% of the total protein of the family (1,2). These two proteins, designated apolipoprotein-glutamine-I (apoLPGln-I) and apolipoprotein-glutamine-II (apoLP-Gln-II), have been extensively characterized in several laboratories, and their physiochemical and lipid-binding properties have been recently reviewed (3)(4)(5)(6).…”

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confidence: 99%

The Primary Structure of High Density Apolipoprotein-Glutamine-I

Baker

Delahunty

Gotto

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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The major protein constituent of human plasma high density lipoproteins has been isolated and its complete amino-acid sequence determined. The protein, designated apolipoprotein-glutamine-I by the presence of carboxyl-terminal glutamine, is a single polypeptide chain of 245 amino-acid residues, including three residues of methionine. The protein is devoid of cysteine, cystine, and isoleucine. Cleavage of apolipoprotein-glutamine-I with cyanogen bromide yields four fragments with 94, 90, 36, and 25 amino acids. The amino-acid sequence of each fragment was determined by conventional methods, with proteolytic digestion with trypsin, chymotrypsin, and thermolysin. The alignment of the cyanogen bromide fragments was determined by the isolation of the methionine-containing tryptic peptides from apolipoprotein-glutamine-I.Inspection of the sequence of apolipoprotein-glutamine-I suggests an interesting distribution of amino acids that may account for its helical structure and its ability to bind and transport lipid.Human plasma high density lipoproteins (HDL) have been shown to contain two major apoprotein components that account for 90% of the total protein of the family (1, 2). These two proteins, designated apolipoprotein-glutamine-I (apoLPGln-I) and apolipoprotein-glutamine-II (apoLP-Gln-II), have been extensively characterized in several laboratories, and their physiochemical and lipid-binding properties have been recently reviewed (3-6). The amino-acid sequence of apoLPGln-II is known (7-9). It contains two identical monomeric units of 77 amino acids each; the units are linked by a single disulfide bond.In the present communication, we describe the complete amino-acid sequence of the other major HDL apoprotein, apoLP-Gln-I. The protein contains 245 amino acids with a calculated molecular weight of 28,331. This new sequence information, coupled with that of apoLP-Gln-II and of apoLPAla (10) and apoLP-Ser (11, 12) from the very low density lipoproteins (VLDL), permits speculation as to the structural features of the plasma apoproteins that might account for their ability to bind and transport lipid. A theory to explain this ability has been recently presented (13). MATERIALS AND METHODSIsolation of ApoLP-Gln-l. Human plasma HDL was isolated by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml, as described (14). Lipid-free HDL (apoHDL) Abbreviations: VLDL, very low density lipoproteins; HDL, high density lipoproteins; apoLP-Gln-I (A-I) and apoLP-Gln-II (All), the two major apoproteins of human HDL, each with carboxyl-terminal glutamine; and apoLP-Ala (C-III) and apoLPSer (C-I), two apoproteins from human VLDL, with carboxylterminal alanine and serine, respectively. 3631 was prepared by delipidation of the lipoprotein with diethyl ether-ethanol (3: 1). ApoLP-Gln-I was isolated from apoHDL by a combination of gel filtration and DEAE-cellulose chromatography, as described (15).Sequence Strategy. The complete amino-acid sequence of apoLP-Gln-I was established from the four cyanogen bromide fragme...

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Fractionation of human serum high density lipoprotein in urea solutions. Evidence for polypeptide heterogeneity

Cited by 318 publications

References 21 publications

Abnormalities of high density lipoproteins in homozygous familial hypercholesterolemia.

Abnormalities of high density lipoproteins in homozygous familial hypercholesterolemia.

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

The Primary Structure of High Density Apolipoprotein-Glutamine-I

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