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J T Sparrow

Publications and source records attributed to J T Sparrow.

At least 73 records · Page 4Linked to original sources
PubMed1984

Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins.

The purpose of this report is to develop a correlation between the hydrophobicity of a phospholipid as measured by reversed-phase high-performance liquid chromatography and its rate of spontaneous transfer and to use this correlation to predict the rate of transfer of any homologous lipid from any lipoprotein. We have studied the mechanism of transfer of a series of fluorescent or radiolabeled phospholipids among natural and reassembled serum lipoproteins. Fluorescent phosphatidylcholines included those with 9-(1-pyrenyl)nonanoic acid in the sn-2 position and lauric, myristic, palmitic, stearic, oleic or linoleic acid at sn-1. The radioactive phosphatidylcholines contained [3H]oleic acid in the sn-2 position and lauric, myristic, or palmitic acid at sn-1. The kinetics of transfer of the pyrene-labeled lipid were followed by changes in the excimer fluorescence, and that of the radioactive lipids by separation of the donor (lipid-apolipoprotein recombinant) from the acceptor (single bilayer vesicles) on a column of Sephacryl S-200. The retention time of each lipid was measured by high-performance hydrophobic chromatography through a Waters radially compressed C18 column eluted with 75% isopropanol and 25% triethylammonium phosphate (0.15 M). A linear relationship was observed between the rate-constant of transfer and the retention time which suggest that the rate of desorption of phosphatidylcholines from lipoproteins and vesicles is controlled predominately by the hydrophobic effect. For a homologous series of lipids, the rate of transfer can be predicted from retention times obtained from hydrophobic chromatography. The kinetics of transfer of 1-lauroyl-2-[9-(1-pyrenyl)nonanoyl] phosphatidylcholine between isolated human serum lipoproteins exhibits a linear correlation between the transfer half-time and the size of the donor lipoproteins. As a consequence, transfer from very-low-density lipoprotein is 10-times slower than that observed from high-density lipoproteins. The observed correlations between phospholipid transfer rates and both the Stokes radius of the donor and the retention time of the phospholipid on a hydrophobic column permit one to calculate the rate of transfer of homologous molecules between lipid-protein complexes. The results predict that the spontaneous transfer of phospholipids between plasma lipoproteins would be too slow to be a physiologically important phenomena.

Chromatography, High Pressure Liquid↗
PubMed1984

Interactions of myristic acid with bovine serum albumin: a 13C NMR study.

Interactions of myristic acid with bovine serum albumin were studied by 13C NMR spectroscopy at 50.3 MHz using 90% isotopically substituted [1-13C]-, [3-13C]-, and [14-13C]myristic acids, either individually or in a combination of all three with albumin. At pH 7.4, two or more resonances of different intensities were observed for each 13C-enriched myristic acid. Carboxyl and methylene C-3 resonances corresponding to the major myristic acid environment(s) exhibited pH-dependent chemical shift changes indicative of protonation below pH 6.7; in contrast, carboxyl groups in minor environments were resistant to protonation. 13C NMR spectra obtained as a function of the molar ratio of [3-13C]- and [14-13C]myristic acid to bovine serum albumin (from 0.7 to 5.6) revealed at least two narrow resonances for each carbon at all molar ratios. Thus, bovine serum albumin binding sites for myristic acid are heterogeneous with respect to titration behavior and with respect to the local magnetic environment at both the polar and the nonpolar ends of the fatty acid. The narrow resonances observed for the methylene and methyl carbons are inconsistent with complete immobilization of the protein-bound acid molecules. Together with spin- lattice relaxation times and nuclear Overhauser enhancements, the linewidth results indicate that bound myristic acid has internal motions that are rapid compared with overall protein tumbling and that the C-3 methylene carbon is more restricted than the terminal methyl carbon.

Binding Sites↗
PubMed1983

The receptor-binding domain of human apolipoprotein E. Monoclonal antibody inhibition of binding.

To investigate the potential of monoclonal antibodies as probes to determine the receptor-binding domain of apolipoprotein E (apo-E), five apo-E antibodies were tested to see if any of them inhibited 125I-apo-E3 . dimyristoylphosphatidylcholine binding to apo-B,E receptors on cultured fibroblasts. Only one of the five antibodies, referred to as 1D7, was found to inhibit binding, blocking greater than 90% of the receptor-binding activity of apo-E3 dimyristoylphosphatidyl-choline. The 1D7 Fab fragments were also effective inhibitors. The 1D7 bound to a Mr = 22,000 NH2-terminal thrombolytic fragment of apo-E (residues 1-191) and to a 93-residue cyanogen bromide fragment of apo-E (residues 126-218). The four noninhibitory antibodies bound only to the NH2-terminal thrombolytic fragment. These results suggested that the 1D7 epitope is contained between residues 126 and 191, and that the epitopes of the other antibodies are not contained in this region. The use of synthetic apo-E fragments, which cover various lengths of the sequence from residues 129-169, and human apo-E variants with substitutions at residues 145, 146, or 158, narrowed the location of the 1D7 epitope to residues 139-169 and, most likely, to the immediate vicinity of residues 140-150. It is of interest that 1D7 was found to bind to the same region of apo-E that has been implicated as the receptor-binding domain in receptor-binding studies using human apo-E variants and apo-E3 fragments.

Amino Acid Sequence↗
PubMed1983

Interaction of synthetic N-5-dimethylaminonaphthalene-1-sulfonyl-apolipoprotein C-II peptides with lipoprotein lipase.

Synthetic fragments of apo-C-II, specifically labeled on their NH2-terminals with the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl or DNS) fluorophore, have been prepared by solid phase peptide synthesis. When a complex is formed between bovine milk lipoprotein lipase and N-dansyl-apo-C-II peptides, resonance energy transfer occurs from the tryptophan residues of the enzyme to the dansyl-labeled peptides upon excitation at 280 nm. In the absence of lipid, the association constant increases 10-fold when the length of the DNS peptide is increased from apo-C-II-DNS(64-78) (0.04 X 10(6) M-1) to apo-C-II-DNS(60-78) (0.3 X 10(6) M-1). In the presence of lipid, the association constants are dependent on peptide chain length, and increase from 0.4 X 10(6) M-1 for apo-C-II-DNS(64-78) to 2.2 X 10(7) M-1 for apo-C-II-DNS(43-78). The interactions are specific for lipoprotein lipase, are disrupted by guanidinium chloride, are not affected by 1.0 M NaCl, and are competitive with the corresponding nondansylated peptide. Apolipoproteins C-III and A-I, at 5 to 1 molar ratios, had no effect on the interaction. These findings demonstrate the importance of the COOH-terminal region in the lipoprotein lipase-apo-C-II interaction and show that activation of the enzyme involves a specific protein-protein interaction.

Amino Acids↗
PubMed1983

Esterase-type of activity possessed by human plasma apolipoprotein C-II and its synthetic fragments.

Human plasma apolipoproteins apo A-I, A-II, C-I, C-II and C-III (with the exception of apoE), porcine pancreatic colipase and procolipase hydrolyze 4-methylumbelliferyloleate. In all cases, liberation of 4-methylumbelliferone could be inhibited by phenylmethylsulfonyl-fluoride, thus suggesting the involvement of serine residues. To the best of our knowledge this is the first report on the esterase activities of these peptides. Synthetic fragments of the lipoprotein lipase activator, apoC-II, prepared according to the known sequence, also possessed this esterase-type of activity. Furthermore, the esterase-type of activities of the synthetic apoC-II fragments with different chain lengths bore a relatively good correlation to the reported abilities of these peptides to produce activation of lipoprotein lipase. We propose a model for the mechanism of activation of lipoprotein lipase by apolipoprotein C-II. ApoC-II would enhance the apparent catalytic rate constant of lipoprotein lipase by functioning as a specific acyl-enzyme hydrolase. A similar catalytic mechanism is suggested for other protein co-factors of hydrolytic enzymes.

Amino Acid Sequence↗
PubMed1983

A simplified approach to resonance energy transfer in membranes, lipoproteins and spatially restricted systems.

A general model is developed to simulate dipole-dipole resonance energy transfer in spatially restricted systems. At low concentrations of acceptor molecule, the overall quantum yield of a donor population can be defined quantitatively in terms of transfer to multiple defined acceptor regions. Energy transfer at higher acceptor concentrations can be approximated by assuming an exponential dependence of relative quantum yield on the acceptor concentrations. Through geometrical manipulations, this algorithm has been applied using an electronic calculator to systems in which donor-acceptor interaction is limited by unique steric restriction on donor and acceptor distribution within lipid aggregates. The systems that have been analyzed include monomolecular films, bilayer membranes, small discoidal lipid-protein complexes and plasma lipoproteins. The observed energy transfer from N-(2-naphthyl)-23,24-dinor-5-cholen-22-amide-3 beta-ol to N-dansyldimyristoylphosphatidylethanolamine in a dimyristoylphosphatidylcholine bilayer agrees with that predicted by this model.

Dimyristoylphosphatidylcholine↗
PubMed1983

Epitopes associated with a synthetic hepatitis B surface antigen peptide.

A synthetic peptide (SP1), corresponding to the amino acid residues 122 through 137 of the major polypeptide derived from hepatitis B surface antigen (HBsAg), subtype ayw, was analyzed for the presence of the major epitopes of HBsAg. Both a cyclic form, produced by introduction of an intrachain disulfide bond, and a linear form of the peptide were characterized. A panel of monoclonal antibodies with defined specificity for the cross-reactive group a antigenic determinant(s) and for the y and w subtype specificities was used for this analysis. The cyclic, but not the linear, form of SP1 reacted with five of 14 anti-a monoclonal antibodies, demonstrating that the cyclic peptide contains a conformation-dependent a epitope. Only one anti-a antibody was found to react with both cyclic and linear forms of SP1. Because SP1 failed to react with the remaining 8 anti-a monoclonal antibodies, it was concluded that the a antigenic reactivity associated with HBsAg contains an additional epitope(s) unrelated to that expressed on SP1. Both cyclic and linear SP1 reacted with three of three anti-y monoclonal antibodies, indicating that a sequential y epitope is also present on SP1; no w reactivity was detected. Analysis of the idiotypes associated with the monoclonal antibodies showed those that combined with cyclic SP1 also inhibited the binding of a common human anti-HBs (CHBs) idiotype with its rabbit anti-idiotype serum, whereas a monoclonal antibody that did not react with the cyclic SP1 epitope failed to inhibit the CHBs idiotype-anti-idiotype reaction. Thus, the conformational a epitope present on cyclic SP1 appears to contain the predominant epitope recognized by humans in response to a natural HBV infection.

Animals↗
PubMed1983

Synthesis of carbon-13-labeled tetradecanoic acids.

The synthesis of tetradecanoic acid enriched with 13C at carbons 1, 3, or 6 is described. The label at the carbonyl carbon was introduced by treating 1-bromotridecane with K13CN (90% enriched) to form the 13C-labeled nitrile, which upon hydrolysis yielded the desired acid. The [3-13C]tetradecanoic acid was synthesized by alkylation of diethyl sodio-malonate with [1-13C]1-bromododecane; the acid was obtained upon saponification and decarboxylation. The label at the 6 position was introduced by coupling the appropriately labeled alkylcadmium chloride with the half acid chloride methyl ester of the appropriate dioic acid, giving the corresponding oxo fatty acid ester. Formation of the tosylhydrazone of the oxo-ester followed by reduction with sodium cyanoborohydride gave the labeled methyl tetradecanoate which, upon hydrolysis, yielded the desired tetradecanoic acid. All tetradecanoic acids were identical to unlabeled analogs as evaluated by gas-liquid chromatography and infrared or NMR spectroscopy. These labeled fatty acids were used subsequently to prepare the correspondingly labeled diacyl phosphatidylcholines.

Carbon Isotopes↗
PubMed1982

Immunogenicity of conjugates and micelles of synthetic hepatitis B surface antigen peptides.

A cyclic peptide containing the amino acid sequence 122 through 137 of the major hepatitis B surface antigen (HBsAg) polypeptide was synthesized. The immunogenicity of this synthetic peptide, aggregated in micelles or covalently coupled to tetanus toxoid, was assessed in mice. Antibodies against HBsAg (anti-HBs) were obtained with both preparations, administered either in saline suspension or adsorbed on aluminum gel. The peptide-tetanus toxoid conjugate was more immunogenic than the peptide micelles, producing high levels of specific anti-HBs.

Animals↗
PubMed1982

Apolipoprotein/lipid interactions: studies with synthetic polypeptides.

The understanding of complex interactions which occur in the serum lipoproteins has been greatly aided by using peptide synthesis to obtain fragments of the apolipoproteins which are unobtainable by other means. The results from lipid-binding studies with these synthetic materials have generally supported the amphipathic helical hypothesis of Segrest et al. for the interaction of phospholipid with the apolipoprotein. However, CD results from these same experiments suggest that the amphipathic helices may not be as large as originally proposed. The contribution of other protein structural features, e.g. beta-sheets and beta-turns, to lipid binding has not been systematically investigated. The importance of hydrophobicity to lipid-protein interaction is strongly supported by the experimental data. Indeed, there is preliminary evidence that the hydrophobic residues positioned beneath the paired acidic and basic residues on the amphipathic helix are extremely critical to the interaction with phospholipid. The role of charged residues in binding is less clear and needs further investigation. The importance of the structural features previously mentioned can be elucidated through the synthesis of appropriately substituted peptides. However, the final proof of the protein structural features involved in protein-lipid interaction must await x-ray diffraction analysis and detailed NMR measurements. As more peptides are synthesized and studied, the authors feel that the complexities of lipid transport and metabolism will be better understood. The surface properties of peptide fragments of the apoproteins are presently being investigated and could lead to important findings on the exchange of apoproteins between lipoprotein classes. The interactions of synthetic peptides with the enzymes which control lipid synthesis and degradation have increased the understanding of protein-protein and protein-lipid interactions which control these important processes. The ability of a synthetic peptide to accelerate lipolysis in an apoC-II deficient lipoprotein offers the potential for treating these patients with synthetic material to reduce their hypertriglyceridemia. The ability to model the amphipathic helix opens new vistas for the study of the role of hydrophobicity, peptide length, helix potential, and charged residues in lipid binding. The observation of Pownall et al. and Yokayama et al. that phospholipid-cholesterol complexes of these model peptides can serve as substrates for LCAT suggests several exciting avenues for further study of cholesterol metabolism and transport. As these studies increase knowledge of lipid transport, the potential exists to intervene therapeutically with potent synthetic lipid-binding peptides to reduce serum cholesterol or to remove cholesterol from arterial lesions.

Amino Acid Sequence↗
PubMed1981

Kinetics and mechanism of association of human plasma apolipoproteins with dimyristoylphosphatidylcholine: effect of protein structure and lipid clusters on reaction rates.

We have used a series of lipid-associating proteins with similar pI's and with molecular weights between 2280 and 28000 to study the mechanism of lipid-protein association. All of these polypeptides spontaneously associate with dimyristoylphosphatidylcholine (DMPC) to give quasi-discrete products. The reaction of the apoproteins with unsaturated lecithins is slow, if it occurs at all. Our data support the Kanehisa-Tsong cluster model [Kanehisa & Tsong (1978) J. Am. Chem. Soc. 100, 424] of lipid permeability in many of its qualitative aspects. These are (a) that the rate of lipid- protein association increases with decreasing polypeptide molecular weight, (b) that there is a small temperature dependence for the rate of association of small peptides with DMPC but with large polypeptides the temperature at which association with lipid is rapid is confined to the solid leads to fluid transition temperature (Tc) of DMPC, and (c) that the rate is asymmetric about Tc, with the change in the rate with respect to temperature below Tc being greater than at T greater than Tc. In addition, we have shown that unfolded monomeric proteins with a large number of exposed hydrophobic residues associate with DMPC faster than self-associated and/or folded proteins. Our data suggest that the association of some of the apoproteins with phospholipids is subject to kinetic control.

Apolipoproteins↗
PubMed1981

Mechanism of lipid-protein interaction in the plasma lipoproteins: identification of a lipid-binding site in apolipoprotein A-II.

Apolipoprotein A-II (apoA-II) is a dimeric 77-residue apoprotein of human high-density lipoproteins. Previous studies indicate that residues 56--77 in the apoprotein do not bind phospholipid whereas residues 47--77 form a complex with dimyristoylphosphatidylcholine (DMPC). To further delineate the lipid-binding region between residues 47 and 77, we have prepared synthetic fragments of apoA-II corresponding to residues 54--77, 52--77, and 50--77 and have tested each fragment for its ability to interact with vesicles of DMPC. The interaction of the fragments was determined by changes in secondary structure as measured by circular dichroism and by isolation of peptide--DMPC complexes by ultracentrifugation in density gradients of KBr. By these criteria, only fragment 50--77 binds DMPC; there is an increase in alpha helicity from 17% to 41% when the fragment associates with lipid. Since the 56--77 fragment does not associate with phospholipid, we propose that the addition of residues Thr-Pro-Leu-Ile-Lys-Lys (corresponding to residues 50--55) to the 56--77 fragment gives the peptide the necessary sequence information for lipid binding. To further identify the important amino acid residues in the region of 50--55, we have substituted Leu-Ile with Ala-Ala. This substitution totally abolishes the lipid-binding capacity of the 50--77 fragment. On the other hand, substitution of Lys-Lys with Ser-Ser does not alter the lipid-binding capacity of the peptide. We conclude that residues 50--55 are important in lipid binding and that the hydrophobic center formed by Leu-Ile plays an important role.

Amino Acid Sequence↗
PubMed1981

The use of reversed-phase high-performance liquid chromatography with radial compression for the analysis of peptide and protein mixtures.

This report describes the separation of peptide and protein mixtures on a C18- microparticulate support which was packed in a polyethylene cartridge and subjected to radial compression of ca. 2600 p.s.i. The purpose of the radial compression was to minimise inhomogeneities in the column packing, in particular in the region of the column wall and end fittings. The effectiveness of this new chromatographic system was demonstrated by the efficient separation of the following mixtures: the C-apolipoproteins isolated from human very low density lipoproteins; the polymorphs of apolipoprotein A-I; the tryptic fragments of apolipoprotein C-II; the complex mixture generated by partial proteolysis of apolipoprotein B; the tryptic fragments of 3H- and 14C-labelled beta-chain of murine IA alloantigen and the tryptic fragments of carboxymethylated lambda chain isolated from human immunoglobulin G. The separated peaks were identified by amino acid analysis, radioactivity counting and in the last example by amino acid sequence determination. The mobile phase consisted of 1% aqueous solution of triethylammonium phosphate, pH 3.2 with acetonitrile or isopropanol as the organic modifier.

Amino Acids↗
PubMed1981

Use of mixed-mode, high-performance liquid chromatography for the separation of peptide and protein mixtures.

The packing material recently introduced for use in a radial compression chamber, with a partially flexible cartridge, has a low C18-coating (5% w/s) combined with an absence of secondary capping. This report demonstrates that such a support, which contains significant concentrations of both free silanol and hydrocarbon groups, can allow a mixed-mode separation to occur via adsorption and reversed-phase separation mechanisms. In any given separation, the predominant mechanism depends both on the nature of the sample and the mobile phase. For efficient peptide and protein separations, it was necessary to suppress most silanol group interactions by the use of a mobile phase which contained a high concentration of an amine phosphate, e.g., 0.17 M triethylammonium phosphate, pH 3.2. In addition, it was necessary to deactivate further the silanol groups by an initial column wash of at least 20 column volumes of methanol. Samples which contained strongly basic groups, for example the guanidino group of arginine, can still exhibit poor separation efficiencies on such a support. These problems were largely overcome, however, with the use of isopropanol as an organic modifier. If these precautions were followed, the packing material gave excellent selectivities in the separation of closely related materials, as well as allowing increased sample capacities. These observations will be supported by an examination of the chromatographic properties of a range of small peptides, the C-apolipoprotein mixture present in human very-low-density lipoproteins and the purification of an 8-mg sample of a synthetic pentadecapeptide in a single chromatographic run.

Apolipoproteins↗