Abstract 547: Proteomic Correlation of Gel Filtration Lipoprotein Subfractions with Atheroprotective Functions of HDL

HDL has been shown to possess a variety of cardio-protective functions, including removal of excess cholesterol from the periphery, inhibition of oxidation, and stimulation of endothelial function. It has been proposed that various HDL sub-particles exist, each with distinct protein and lipid compositions. We hypothesized that fractionation of plasma would separate HDL particles with different protein compositions into distinct populations responsible for different HDL functions. Plasma from 10 healthy adults was fractionated by gel filtration and the protein composition of phospholipid containing fractions was analyzed by mass spectrometry. Each fraction was assessed for its ability to efflux cholesterol from macrophages, as well as its ability to inhibit LDL oxidation. Correlations were made between individual proteins in the HDL fractions and their ability to participate in both functional assays. One peak of activity was found in the cholesterol efflux assay when analyzing fractions in the HDL range. Cholesterol efflux activity did not correlate strongly with any protein in the HDL range of fractions, though there was a weak correlation with Protein S (r=0.337, p<0.01). However, the phospholipid (PL) and cholesterol (CH) concentration of the fractions correlated strongly with efflux (r=0.75. and r=0.61 respectively, both p<0.00001) across all fractions (both LDL and HDL). In contrast, the PL or CH content were not correlated with anti-oxidation activity. However, there were 2 strong peaks of anti-oxidation activity in the HDL range that did correlate with specific proteins (all values of r>0.3 and p<0.05). Activity in the first peak, fraction 25, in the small HDL range, correlated most strongly with ceruloplasmin and inter-α-trypsin inhibitor 4, while activity in the second peak, fraction 28, in the minimally lipidated/free protein range, correlated with gelsolin and albumin. In conclusion, we have shown that PL and CH correlate more strongly with cholesterol efflux than any protein in the HDL sub-fractions, while the protein composition of particular HDL sub-fractions is a better indicator of its anti-oxidative capacity than the PL or CH concentration.

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Zinc- and copper-binding proteins in the plasma of winter flounder (Pseudopleuronectes americanus)

Canadian Journal of Zoology ◽

10.1139/z80-086 ◽

1980 ◽

Vol 58 (4) ◽

pp. 609-613 ◽

Cited By ~ 11

Author(s):

P. E. Fletcher ◽

G. L. Fletcher

Keyword(s):

Molecular Weight ◽

Binding Proteins ◽

Binding Protein ◽

Gel Filtration ◽

Protein S ◽

Egg Yolk ◽

Winter Flounder ◽

Zinc Binding ◽

Copper Binding ◽

Copper Binding Proteins

Zinc- and copper-binding proteins were isolated from the plasma of winter flounder using gel filtration chromatography. A single copper-binding protein fraction of molecular weight 170 000 was isolated from the plasma of both sexes.In male and female flounder over 95% of the plasma zinc was associated with a zinc-binding protein(s) with a molecular weight of 76 000. In male flounder the remaining zinc appeared to be bound to a protein(s) of molecular weight 186 000. In female flounder the remaining 5% of the zinc was associated with two zinc-binding fractions with apparent molecular weights of 186 000 and 340 000 – 370 000.Extracts of plasma vitellogenin and egg yolk proteins revealed significant quantities of zinc and copper. It is hypothesized that the female specific zinc-binding protein (340 000 – 370 000) was vitellogenin.

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Structure of glycerol dehydrogenase fromSerratia

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x13034444 ◽

2014 ◽

Vol 70 (2) ◽

pp. 166-172 ◽

Cited By ~ 5

Author(s):

Paul Musille ◽

Eric Ortlund

Keyword(s):

Protein Expression ◽

Structure Determination ◽

Gel Filtration ◽

Protein Composition ◽

Glycerol Dehydrogenase ◽

Metabolic Enzyme ◽

X Ray ◽

Unknown Protein ◽

Crystallization Experiment ◽

Additional Protein

The 1.90 Å resolution X-ray crystal structure of glycerol dehydrogenase derived from contaminating bacteria present during routineEscherichia coliprotein expression is presented. This off-target enzyme showed intrinsic affinity for Ni2+-Sepharose, migrated at the expected molecular mass for the target protein during gel filtration and was crystallized before it was realised that contamination had occurred. In this study, it is shown that liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) can efficiently identify the protein composition of crystals in a crystallization experiment as part of a structure-determination pipeline for an unknown protein. The high-resolution X-ray data enabled sequencing directly from the electron-density maps, allowing the source of contamination to be placed within theSerratiagenus. Incorporating additional protein-identity checks, such as tandem LC-MS/MS, earlier in the protein expression, purification and crystallization workflow may have prevented the unintentional structure determination of this metabolic enzyme, which represents the first enterobacterial glycerol dehydrogenase reported to date.

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HUMAN AMYLOID PROTEIN: CHEMICAL VARIABILITY AND HOMOGENEITY

Journal of Histochemistry & Cytochemistry ◽

10.1177/19.1.1 ◽

1971 ◽

Vol 19 (1) ◽

pp. 1-15 ◽

Cited By ~ 62

Author(s):

M. HARADA ◽

C. ISERSKY ◽

P. CUATRECASAS ◽

D. PAGE ◽

H. A. BLADEN ◽

...

Keyword(s):

Amino Acid ◽

Amyloid Fibril ◽

Gel Filtration ◽

Protein S ◽

Amyloid Protein ◽

X Ray Diffraction ◽

Amyloid Proteins ◽

X Ray ◽

Chemical Variability ◽

Sepharose 4B

The morphology of the fibril of amyloid derived from different individuals is similar, but occasionally significant differences are noted. All human amyloid filaments have a "β-pleated sheet" conformation as revealed by x-ray diffraction, and those examined after orientation show a "cross-β" pattern. All amyloid fibril concentrates studied so far can be fractionated to obtain the major amyloid protein component(s) by sequential gel filtration with 5 M guanidine-HCl in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns with the removal of over 28% of proteins representing minor constituents. The major amyloid protein(s) obtained from the spleen and/or liver of six patients is found to contain tryptophan, to be deficient in hydroxylysine and hydroxyproline and usually at least one commonly occurring amino acid and to have a high content of dicarboxylic acid and short chain amino acids and unreactive (blocked) NH2-terminal groups or aspartic acid-asparagine (Asx). However, the amyloid protein(s) from each individual differs from that of the others in molecular weight, in amino acid composition and in the presence or absence of specific tryptic peptides. Amyloid protein(s) from the liver and spleen of the same individual is identical. No chemical characteristics distinguish amyloid proteins derived from cases classified clinically as "primary" from those classified as "secondary." There is a striking chemical similarity between amyloid proteins and the NH2-terminal variable fragment of the light and heavy chain of immumoglobulin proteins.

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Abstract 126: Effects of Rosuvastatin on the Protein Composition of Lipoprotein Subfractions

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.126 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Scott M Gordon ◽

Georgina Kemeh ◽

Michael B Fessler ◽

Alan T Remaley

Keyword(s):

Lipoprotein Metabolism ◽

Protein Composition ◽

Protein Detection ◽

Fold Increase ◽

Cholesterol Content ◽

Vascular Lesions ◽

Lipid Lowering ◽

Lipoprotein Subfractions ◽

The Impact

Introduction: Statins, by inhibiting HMG-CoA reductase and up regulating hepatic LDL receptors, effectively lower plasma LDL-C by as much as 50%, thus reducing future CVD events. However, the physiological effects of statins are diverse and not all are related to lowering of LDL-C. Goal: The goal of this study was to test our hypothesis that some of these pleiotropic alternative effects from statins may be driven by compositional changes to lipoproteins distinct from their cholesterol content. We, therefore, performed a small clinical pilot study to assess the impact of statins on lipoprotein associated proteins in healthy individuals. Methods: Ten subjects with normal LDL-C (<130 mg/dL) were given rosuvastatin (20 mg/day) for 28 days. Plasma samples collected at baseline and after treatment were used for lipid measurement, nuclear magnetic resonance (NMR) lipoprotein profiling and lipoprotein proteomics. Results: The effects of rosuvastatin treatment on clinical lipid measures and NMR profile were consistent with established findings. Proteomic analysis of FPLC fractions representing LDL, HDL-1 (large) and HDL-2 (small) identified a total of 124 different proteins. Spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool: LDL = 9, HDL-1 = 9 and HDL-2 = 4. These changes included both increases and decreases in proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the treatment was a profound increase in alpha-1-antirypsin (A1AT) spectral counts association with HDL-1 particles. Quantitative measurement by ELISA revealed an average 5.7 fold increase in HDL-1 associated A1AT. Preliminary in vitro studies indicate a potential functional role for A1AT enriched HDL in the formation of neutrophil extracellular traps (NETs), a pro-inflammatory component of vascular lesions. Summary: Based on these results, statins can significantly change the protein composition of both LDL and HDL. Some of these changes, such as the up regulation of A1AT on HDL, may convey anti-inflammatory functionality on lipoproteins and might contribute to some of the non-lipid lowering effects of statins.

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Na+-K+-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1991.261.4.l87 ◽

1991 ◽

Vol 261 (4) ◽

pp. L87-L91

Author(s):

Mikhail P. Danilenko ◽

Vera C. Turmukhambetova ◽

Oleg V. Yesirev ◽

Vsevolod A. Tkachuk ◽

Mikhail P. Panchenko

Keyword(s):

Atpase Activity ◽

Pertussis Toxin ◽

Inhibitory Concentration ◽

Gel Filtration ◽

Protein S ◽

Half Maximum ◽

Inhibitory Effects ◽

Cardiac Sarcolemma ◽

Dependent Inhibition ◽

Zwitterionic Detergent

The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 μM) decrease in the Na+-K+-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[ggr-thio]triphosphate (0.1 μM GTPggrS) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 μM) and is abolished in sarcolemma treated with pertussis toxin (20 μg/ml) in the presence of 100 μM NAD. GTPggrS alone reduces Na+-K+-ATPase activity by 45% (half-maximum inhibitory = 1 μM). The apparent affinity of the enzyme for GTPgγS is increased ≈10-fold in the presence of 1 μM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTPgγS-dependent inhibition of the Na+-K+-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL-6B column resulted in a separation of Na+-K+-ATPase and pertussis toxin-sensitive Gi activities. Na+-K+-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20–30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTPggrS, or Gpp(CH2)p] at micromolar concentrations were restored when the Na+-K+-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive Gi protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5' -[beta-thio]diphosphate, or App(NH)p were unable to induce the Gi protein-mediated attenuation of Na+-K+-ATPase activity in the reconstitution system. These results suggest that a pertussis toxin-sensitive Gi protein may act as a transducer of the inhibitory hormonal signals on Na+-K+-ATPase in the sarcolemma. cardiac sarcolemma

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Radioimmunoassay Studies of Intestinal Calcium-binding Protein in the Pig. II. The Distribution of Intestinal CaBP in Pig Tissues

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y75-158 ◽

1975 ◽

Vol 53 (6) ◽

pp. 1135-1140 ◽

Cited By ~ 27

Author(s):

B. M. Arnold ◽

M. Kuttner ◽

D. M. Willis ◽

A. J. W. Hitchman ◽

J. E. Harrison ◽

...

Keyword(s):

Small Intestine ◽

Calcium Binding ◽

Binding Protein ◽

Gel Filtration ◽

Protein S ◽

Binding Activity ◽

Calcium Binding Protein ◽

Distal Small Intestine ◽

Specific Radioimmunoassay ◽

Kidney Liver

Using a specific radioimmunoassay for porcine intestinal calcium-binding protein (CaBP), we have measured the concentration of CaBP in the various tissues and organs of normal pigs. Intestinal CaBP was present in highest concentration in the upper small intestine, with lower concentrations in the distal small intestine. Intestinal CaBP was also found, in lower concentrations, in kidney, liver, thyroid, pancreas, and blood. In all other tissues, including parathyroid, bone, skeletal muscle, and brain, CaBP immunoreactivity was undetectable or less than in blood. The elution profile of calcium-binding activity and immunoreactivity from gel filtration analysis of kidney and parathyroid extracts suggest that the calcium-binding protein in the parathyroid gland, and the major calcium-binding protein(s) in the kidney, are chemically and immunochemically different from intestinal CaBP.

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Age and SPARC Change the Extracellular Matrix Composition of the Left Ventricle

BioMed Research International ◽

10.1155/2014/810562 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 23

Author(s):

Lisandra E. de Castro Brás ◽

Hiroe Toba ◽

Catalin F. Baicu ◽

Michael R. Zile ◽

Susan T. Weintraub ◽

...

Keyword(s):

Extracellular Matrix ◽

Left Ventricle ◽

Protein Composition ◽

Extracellular Proteins ◽

Matrix Composition ◽

Matricellular Protein ◽

Middle Aged ◽

Lv Dysfunction ◽

Collagen Iii ◽

Specific Proteins

Secreted protein acidic and rich in cysteine (SPARC), a collagen-binding matricellular protein, has been implicated in procollagen processing and deposition. The aim of this study was to investigate age- and SPARC-dependent changes in protein composition of the cardiac extracellular matrix (ECM). We studied 6 groups of mice (n=4/group): young (4-5 months old), middle-aged (11-12 m.o.), and old (18–29 m.o.) C57BL/6J wild type (WT) and SPARC null. The left ventricle (LV) was decellularized to enrich for ECM proteins. Protein extracts were separated by SDS-PAGE, digested in-gel, and analyzed by HPLC-ESI-MS/MS. Relative quantification was performed by spectral counting, and changes in specific proteins were validated by immunoblotting. We identified 321 proteins, of which 44 proteins were extracellular proteins. Of these proteins, collagen III levels were lower in the old null mice compared to WT, suggestive of a role for SPARC in collagen deposition. Additionally, fibrillin showed a significant increase in the null middle-aged group, suggestive of increased microfibril deposition in the absence of SPARC. Collagen VI increased with age in both genotypes (>3-fold), while collagen IV showed increased age-associated levels only in the WT animals (4-fold,P<0.05). These changes may explain the previously reported age-associated increases in LV stiffness. In summary, our data suggest SPARC is a possible therapeutic target for aging induced LV dysfunction.

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Structure-Function Studies of Factor VIII/Von Willebrand Protein(s)

10.1055/s-0039-1680464 ◽

1977 ◽

Author(s):

Patrick A. McKee

Keyword(s):

Factor Viii ◽

Single Molecule ◽

Gel Filtration ◽

Protein S ◽

Fold Increase ◽

Procoagulant Activity ◽

Void Volume ◽

Pas Staining ◽

Von Willebrand ◽

Willebrand Factor

Factor VIII (FVIII) procoagulant activity was initially thought to be a glycoprotein with a molecular weight (MW) >1 million and composed of disulfide-1inked ~200,000 MW subunits. A protein with similar properties, except lacking procoagulant activity, is in hemophilic plasma; it was identical to normal FVIII by SDS-gel analyses, isoelectric focusing, and PAS staining. Subsequently it was shown that the FVIII glycoprotein also has von Willebrand factor (vWF) activity, suggesting that both FVIII and vWF activities might be properties of the same molecule. When the FVIII/vWF protein(s) is rechromatographed on 4% agarose and 0.25 M CaCl2, virtually all the protein and vWF activity elute in the void volume, but most of the FVIII procoagulant activity elutes much later. The extent of separation of the two activities depends on the amount of protein applied to the column. Also, exposure of the FVIII/vWF to thrombin before gel filtration strikingly accentuates separation of the two activities. The reduced SDS-gel pattern of the void volume protein peak showed the 200,000 MW subunit while that of the procoagulant peak contained several subunit bands which ranged from ~30,000–100,000 MW. Removal of sialic acid from FVIII/vWF is associated with reduced ristocetin induced platelet aggregation and causes a 50-fold increase in the rate of clearance of protein from the circulation by the hepatocyte. Currently, our data suggest that FVIII procoagulant and vWF activities are properties of a single molecule composed of disulfide-bound identical subunits. Cleavage by thrombin then results in FVIII procoagulant activity. Additional cleavages, to which the molecule appears very sensitive, results in FVIII inactivation. The vWF activity is very stable—even to proteolysis—and it appears to be a function of the carbohydrate side chains of the molecule.

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