Large scale purification and structural characterization of squalene and sterol carrier protein.

Dempsey, Mary E.; McCoy, Kim E.; Baker, H N; Dimitriadou-Vafiadou, A; Lorsbach, Thomas; Howard, James B.

doi:10.1016/s0021-9258(19)69887-x

Cited by 119 publications

(15 citation statements)

References 40 publications

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“…If scp73 and sp71 do have roles in fatty acid metabolism in normal and stressed cells, respectively, they are more likely to specifically involve the metabolism of palmitate and stearate. However, it is interesting to note that sterol carrier protein (i.e., fatty acid binding protein) highly purified from rat liver contains primarily palmitic and stearic acids in a 1:1 ratio (Dempsey et al, 1981). One interesting possibility is that sp71 and scp73 may be involved in membrane remodeling.…”

Section: Resultsmentioning

confidence: 99%

Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins

Guidon¹,

Hightower²

1986

Biochemistry

103

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The major rat heat-shock (stress) protein and its cognate were purified to electrophoretic homogeneity from livers of heat-shocked rats. Both proteins exhibited similar behavior on a variety of column chromatography matrices but were separable by preparative isoelectric focusing under nondenaturing conditions by virtue of a 0.2 pH unit difference in isoelectric point. Both purified proteins had similar physical properties, suggesting the possibility that they may have similar biological functions as well. Both proteins were homodimers under nondissociative conditions (Mr 150 000) with isoelectric points of 5.0 (cognate) and 5.2 (major stress protein). After denaturation, both proteins had an increase in isoelectric point of 0.6 pH unit, and the resulting polypeptide chains had apparent molecular weights of 73 000 (cognate) and 71 000 (major stress protein). Similarities in the electrophoretic properties of these two proteins and serum albumin, which also undergoes a large basic shift in isoelectric point due to loss of fatty acids and conformational changes accompanying denaturation, prompted us to search for lipids associated with the purified 71-kilodalton stress protein and its cognate. Thin-layer chromatography of chloroform/methanol extracts of these two proteins revealed nonesterified fatty acids bound to both proteins. Palmitic acid, stearic acid, and a small amount of myristic acid were identified by gas chromatography/mass spectroscopy. Both proteins contained approximately four molecules of fatty acid per dimer with palmitate and stearate present in a one to one molar ratio. Possible roles of the major stress protein and its cognate as fatty acid associated proteins in cellular responses to stress are discussed.

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Section: Resultsmentioning

confidence: 99%

Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins

Guidon¹,

Hightower²

1986

Biochemistry

103

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“…Direct protein sequencing of H-ALBP yielded no detectable PTHamino acid, indicating that the amino terminus was blocked to sequencing. It has been demonstrated that the amino terminus of murine-ALBP is posttranslationally modified, with the terminal methionyl residue removed and the following residue, cysteine, acetylated.3 Blocked amino termini are commonly found in the lipid-binding protein family, the blocking group often being an acetyl moiety (Sacchettini et al, 1986;Lowe et al, 1987;Dempsey et al, 1981).…”

Section: Discussionmentioning

confidence: 99%

Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA

Baxa¹,

et al. 1989

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Human adipocyte lipid-binding protein (H-ALBP) was purified from normal subcutaneous adipose tissue to greater than 98% homogeneity, utilizing a combination of acid fractionation, gel filtration, covalent chromatography on activated thiol-Sepharose 4B, and anion-exchange chromatography. Human ALBP comprised about 1% of total cytosolic protein in human adipose tissue, had a relative molecular mass of about 15 kDa, and existed as a monomer in solution. The amino terminus of H-ALBP was blocked to sequencing. When a liposome ligand delivery assay was used, H-ALBP saturably bound oleic acid with about 1 mol of ligand bound per mole of protein. Additionally, H-ALBP saturably bound retinoic acid as determined by the quenching of intrinsic tryptophan fluorescence. A full-length H-ALBP cDNA has been cloned; the sequence predicts a 649-base mRNA comprised of a 62-base 5'-noncoding region containing an 18S ribosome-binding site, a single 396-base open-reading frame, and a 191-base 3'-noncoding region. Comparative sequence analysis indicated that the 132 amino acid H-ALBP is a member of a multigene family of intracellular lipid-binding proteins and contains the consensus substrate phosphorylation sequence for tyrosyl kinases.

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“…Rat liver sterol carrier protein (SCP)2 was isolated as described previously (Dempsey et al, 1981). Ergosterol was purchased from Sigma Chemical Co. (St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

“…Instead, the present investigation seeks to extend our knowledge of the properties of sterols themselves to sterol-protein interactions and to the behavior of sterols in membranes. In this report we address the characterization of the fluorescence properties of highly purified dehydroergosterol existing as (1) free, soluble molecules, (2) aqueous micelles, (3) components of LM fibroblast plasma membranes, and (4) bound complexes with purified rat liver sterol carrier protein.1 Furthermore, dehydroergosterol was introduced into LM fibroblast cell cultures to demonstrate in vivo binding of dehydroergosterol to soluble cytosolic sterol carrier proteins which may be responsible for intracellular transport of sterols to microsomal and mitochondrial membranes, a function proposed,for sterol carrier protein (Dempsey et al, 1981;Grinstead et al, 1983;Dempsey, 1984).…”

mentioning

confidence: 99%

Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

Fischer¹,

Cowlen²,

Dempsey³

et al. 1985

Biochemistry

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The fluorescent sterol analogue delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was synthesized and purified by reverse-phase high-performance liquid chromatography. Dehydroergosterol in aqueous solution had a critical micelle concentration of 25 nM and a maximum solubility of 1.3 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of dehydroergosterol with purified rat liver squalene and sterol carrier protein (SCP). SCP increased the maximal solubility of dehydroergosterol in aqueous buffer. The fluorescence emission spectrum of dehydroergosterol was blue shifted upon addition of SCP. The fluorescence lifetime of dehydroergosterol in aqueous buffer was 2.3 ns; addition of SCP resulted in the appearance of a second lifetime component near 12.4 ns. The SCP increased the fluorescence polarization of monomeric dehydroergosterol in aqueous buffer from 0.033 to 0.086. Scatchard analysis of the binding data indicated that dehydroergosterol interacted with purified rat liver SCP with an apparent KD = 0.88 microM and Bmax = 4.8 microM. At maximal binding, 1.0 mol of dehydroergosterol was specifically bound per mole of SCP. The close molecular interaction of dehydroergosterol with SCP was also demonstrated by energy-transfer experiments. The intermolecular distance between SCP and bound dehydroergosterol was evaluated by fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene series of double bonds in dehydroergosterol. The transfer efficiency was 36%, and R, the apparent distance between the tyrosine energy donor and the dehydroergosterol energy acceptor, was 19 A. The significance of these data obtained in vitro for dehydroergosterol interaction with SCP was also tested in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

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Large scale purification and structural characterization of squalene and sterol carrier protein.

Cited by 119 publications

References 40 publications

Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins

Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins

Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA

Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

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