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J T Sparrow

Publications and source records attributed to J T Sparrow.

107 records · Page 6Linked to original sources
PubMed1977

Activation of lipoprotein lipase by native and synthetic fragments of human plasma apolipoprotein C-II.

Apolipoprotein C-II (apoC-II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3). The amino acid sequence of the 78 residues of apoC-II has recently been established in this laboratory. To determine the minimal sequence requirements for activation, we have prepared both native and synthetic fragments of apoC-II and tested them for their ability to activate LPL. Cyanogen bromide fragments of apoC-II corresponding to residues 1--9 and 10--59 had little ability to activate LPL. However, the COOH-terminal cyanogen bromide fragment corresponding to residues 60--78 increased hydrolysis 4-fold compared to an average of 9-fold activation for the same concentration of apoC-II. The synthetic peptide containing residues 60--78 prepared by solid-phase techniques enhanced the lipolysis 3-fold. Addition of five residues produced a synthetic fragment 55--78 that enhanced the release of fatty acid 12-fold compared to 13-fold for intact apoC-II. By contrast, the synthetic peptide containing residues 66--78 did not activate. Removal of the three COOH-terminal residues, Gly-Glu-Glu, from fragment 60--78 decreased the ability to activate LPL by greater than 95%. These studies suggest that the maximal activation of LPL by apoC-II requires a minimal sequence contained within residues 55--78.

Amino Acid Sequence↗
PubMed1976

The solid phase synthesis of a protein activator for lecithin-cholesterol acyltransferase corresponding to human plasma apoC-I.

Apolipoprotein C-I, a protein constituent of the very low density lipoproteins of human plasma, consists of a single chain of 57 amino acids. The total synthesis of a protein corresponding to apolipoprotein C-I in physical properties and compositions was accomplished by solid phase techniques employing a modified polystrene incorporating spacer groups between the point of attachment of the first residue and the polymer matrix. The synthetic apoprotein was shown to activate lecithin:cholesterol acyltransferase to the same extent as the native protein. Comparative lipid-binding studies with dimyristoyl phosphatidylcholine gave complexes for native and synthetic apoprotein which floated at the same density after ultracentrifugation in KBr gradients and had virtually the same lipid:protein ratios.

Acyltransferases↗
PubMed1975

Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.

Acyltransferases↗
PubMed1973

Chemical synthesis and biochemical properties of peptide fragments of apolipoprotein-alanine.

Apolipoprotein-alanine is an apolipoprotein isolated from very-low-density lipoproteins of human plasma. This protein contains 79 amino acids and binds phosphatidyl choline. Four fragments of this molecule corresponding to sequence positions 41-79 (I), 48-79 (II), 55-79 (III), and 61-79 (IV) have been synthesized by standard solid-phase methods. The resulting peptides were cleaved from the resin and deblocked with liquid HF, then purified by chromatography on Sephadex G-50 and DEAE-cellulose. Each purified peptide eluted as a single, symmetrical peak, exhibited a single band on polyacrylamide gel electrophoresis, and gave an amino-acid analysis in good agreement with the theoretical value. Circular dichroism studies indicated that only fragments I and II became more helical in the presence of phosphatidyl choline and significantly inhibited the reactivation of delipidated beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30), an enzyme that requires phosphatidyl choline for activity. When subjected to ultracentrifugation at density 1.064 g/ml in the presence of phosphatidyl choline, fragments I, II, III, and IV floated to the top of the tube to the extent of 85, 50, 13, and 9%, respectively. These results indicate that residues 55-79 do not contain the minimum determinants required for the binding of phospholipid. However, extension of the peptide's N-terminus by seven residues produces a molecule that does bind phosphatidyl choline.

Alanine↗
PubMedDate not supplied

Substitution of Ser61----Gly61 in human apolipoprotein C-II does not alter its activation of lipoprotein lipase.

Lipoprotein lipase (LpL) activity is enhanced by apolipoprotein C-II (apoC-II), a 79 amino acid residue peptide. The minimal apoC-II sequence required for activation of LpL resides between residues 56-79. To determine the possible role of an acyl-apoC-II intermediate involving Ser61 in enzyme catalysis, a synthetic peptide of apoC-II containing residues 56-79 was synthesized and compared to the corresponding peptide with serine at position 61 being substituted with glycine. With two different LpL assay systems, both peptides enhanced enzyme activity. Since glycine does not contain a hydroxyl group, these results rule out the possibility that an acyl-apoC-II intermediate with Ser61 is required for enzyme activation.

Amino Acids↗
PubMedDate not supplied

The application of an improved solid phase synthetic technique to the delineation of an antigenic site of apolipoprotein A-II.

Previous studies have shown that the antigenic sites of human plasma high-density apolipoprotein A-II (apoA-II) are separate from their lipid-binding determinants in human high density lipoproteins (HDL). A specific radioimmunoassay has shown that three distinct antigenic sites are located in residues 4-23, 31-46, and 56-77; these studies suggested that an antigenic site might be restricted to residues 60-77 in the 56-77 fragment. To further delineate this site, we have developed a solid phase radioimmunoassay technique using an improved solid support on which selected sequences of peptides were synthesized, deprotected with HF, and the resulting peptidyl-resins tested for their capability of binding purified 125I-anti-apoA-II antibodies. Amino acid analyses and solid phase sequence analyses were performed to verify the sequence of the synthetic peptide on the solid support. Using this technique, 125I-anti-apoA-II antibodies had achieved 50% of maximal binding when residues 61-77 were attached to the solid support. The maximal binding was achieved by the addition of one more residue, Leu60, thus confirming our suggestion that a major antigenic site is located in residues 60-77. The binding to the peptidyl-resin was inhibited by a synthetic fragment corresponding to residues 60-77 indicating that the antibodies were specifically bound to the resin.

Amino Acid Sequence↗
PubMedDate not supplied

Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100.

Apolipoprotein (apo) B-100, the major protein component in low density lipoprotein (LDL), is the ligand that binds to the LDL receptor. It is important in the metabolism of LDL and elevated plasma levels of LDL-apo B are strongly associated with increased risk of coronary artery disease. Although apo B-100 is of great clinical and biological importance its primary structure has defied chemical elucidation, mainly because of its enormous size, insolubility, and tendency to aggregate. Less than 5% of the apo B-100 sequence has been reported, despite the efforts of many laboratories over the past twenty years. Here we report the complete amino acid sequence of human apo B-100 as deducted by sequence analysis of complementary DNA clones; 2,366 of the 4,536 residues were also confirmed by direct sequencing of apo B-100 tryptic peptides. The distribution of trypsin-accessible and -inaccessible peptides of the protein on LDL is non-random and they can be grouped into 5 hypothetical domains. Of 20 potential N-glycosylation sites identified in the sequence, 13 were found by direct peptide sequencing to be glycosylated, and 4 unglycosylated. Examination of the primary structure of apo B-100 reveals that it contains a large number of long (greater than 70 residues) internal repeats and an even larger number of shorter ones, suggesting that the apo B-100 sequence was derived largely from internal duplications. Finally, using synthetic peptides of a specific region of apo B-100, we have identified a potential LDL receptor-binding domain (residues 3,345-3,381) which can bind to the LDL receptor and suppress 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase activities in cultured human fibroblasts.

Amino Acid Sequence↗
PubMedDate not supplied

Human Lp(a): regions in sequences of apoproteins similar to domains in signal transduction proteins.

The major apoproteins of Lp(a)--apo(a) and apo B-100--are linked by only one intermolecular disulfide bond. This linkage has been suggested to be located between apo(a) Cys4057 and apo B-100 Cys3734. Several studies, however, have suggested other noncovalent interactions between different regions of apo(a) and apo B-100. One possible mechanism for these interactions may involve the apo(a) proline-rich interkringle regions that share sequence similarities with the proline-rich regions of Src homology 3 (SH3) domain-binding proteins such as 3BP-1. SH3 and SH2 domains, and their respective ligands, proline-rich regions, and phosphotyrosine motifs, are noncatalytic segments common to signal transduction proteins. Therefore, we used sequence comparison algorithms and molecular modeling programs to identify corresponding SH3 and SH2 candidate regions as well as potential phosphotyrosine sites in the apo B-100 sequence. Six SH2 and 16 SH3 candidate regions, along with 21 potential phosphotyrosine sites, are contained in the apo B-100 sequence. In Lp(a), these regions of apo B-100 may be involved in the noncovalent, protein-protein interactions between apo(a) and apo B-100. The presence of candidate SH3 and SH2 regions in apo B-100, and potential phosphotyrosine sites in apo B-100, apo(a), and apo A-I, suggests an alternative signaling pathway unrelated to the known B/E receptor.

Journal Article↗
PubMedDate not supplied

Improvements to the TMSBr method of peptide resin deprotection and cleavage: application to large peptides.

The original trimethylsilyl bromide (TMSBr) method of peptide resin deprotection and cleavage has been modified for convenience and applicability to larger peptides. Equal amounts of a 66-residue test peptide resin were cleaved by the standard hydrogen fluoride (HF) procedure, the original TMSBr method and the modified TMSBr method. The peptide profile from the original TMSBr cleavage procedure showed multiple products and a lower overall yield. In contrast, the modified TMSBr procedure gave high yields of crude products comparable in purity to those obtained by HF cleavage.

Amino Acid Sequence↗
PubMedDate not supplied

Large-pore polydimethylacrylamide resin for solid-phase peptide synthesis: applications in Fmoc chemistry.

We have synthesized a hydrophilic crosslinked aminoalkyl polydimethylacrylamide-beaded support upon which peptides have been assembled using standard Fmoc chemistry in automated batch-wise equipment. The resin was prepared by the free radical-initiated co-polymerization of N,N-dimethylacryl-amide, N,N'-bisacrylyl-1,3-diaminopropane and a functional monomer N-methacrylyl-1,3-diaminopropane hydrochlorid. After coupling of N-alpha-tert-butyloxycarbonyl-glycine (Boc-glycine), amino acid analyses gave resin loading capacities of 0.66 mmol/g. The resulting polymer was highly swollen by polar solvents including aqueous buffers and had an exclusion limit of 50 kDa for soluble proteins. This resin was found to be an excellent support for peptide synthesis using Fmoc chemistry. Typical purities of crude peptides were 80%-95%, including sequences that failed on conventional polystyrene resins.

Acrylamides↗