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J T Sparrow

Publications and source records attributed to J T Sparrow.

At least 91 records · Page 5Linked to original sources
PubMed1981

Separation of the apoprotein components of human very low density lipoproteins by ion-paired, reversed-phase high performance liquid chromatography.

A number of crude apolipoprotein samples isolated from human very low density lipoproteins (VLDL) were analyzed by reversed phase high performance liquid chromatography. The mobile phase consisted of a 1% solution of the polar ion-pairing reagent triethylammonium phosphate. A slow, nonlinear gradient of acetonitrile (37--42%) was used to elute the apolipoproteins. The order of elution was as follows: apolipoprotein CX, apolipoprotein C-I, apolipoprotein C-III2, apolipoprotein C-III1, apolipoprotein C-IIIQ and apolipoprotein C-II. This order is consistent with the known polarity of the proteins, i.e., the most nonpolar, apolipoprotein C-II, was the last to be eluted, whereas apolipoprotein C-I, with the lowest nonpolar surface area eluted first. The recovery of the individual apolipoproteins was 80--95% and the individual peaks were characterized by amino acid analysis, UV absorption spectra amd chromatography of pure protein standards.

Acetonitriles↗
PubMed1980

Acidic phospholipases in cultured human fibroblasts: deficiency of phospholipase C in Niemann-Pick disease.

Radioactive phosphatidyl choline substrates specifically labeled in the one position or two position fatty acid were used to establish conditions for the detection of acidic phospholipase A1, A2 and C activities in extracts of cultured human fibroblasts. Maximal activity was detected at a pH of 3.0, 4.0 and 5.0 respectively, suggesting that the enzymes are of lysosomal origin. None of the activities were stimulated or inhibited markedly by Ca2+ or EDTA. The A1 and A2 activities, but not the C activity, were inactivated by the sulfhydryl reactive Ellman reagent. All three enzyme activities were in the normal range for cultured fibroblasts which were deficient in acid lipase, indicating that these activities are not attributable to the acid lipase gene product. Phospholipase A activity was deficient in fibroblast extracts from patients with Niemann-Pick disease, types A, B and C. These data suggest either identity or a genetic relationship between sphingomyelinase and phospholipase C. The activities examined were within the normal range in fibroblasts from patients with neuronal ceroid lipofuscinosis, sea blue histiocyte disease and selected uncharacterized degenerative diseases.

Calcium↗
PubMed1980

Magnetic resonance studies of apolipoprotein C-I nitroxide labeled or [13C]methyl enriched at methionine-38.

One of the three proposed lipid-binding regions of the human apolipoprotein C-I (apo-C-I) is an amphipathic helix which extends from residue 33 to residue 53 and includes a single methionine at sequence position 38. The involvement of the sequence around methionine-38 in phospholipid binding has been evaluated with paramagnetic and nuclear reported groups attached to the thiomethyl moiety. This moiety has been spin-labeled with N-(2,2,6,6-tetramethylpiperidinyl-1-oxy)bromoacetamide or 13C enriched with 13CH3I. As determined from its EPR spectrum, the nitroxide at Met-38 of apoC-I had a rotational correlation time (tau C) of 0.22 ns. When dimyristoylphosphatidylcholine (DMPC) was bound to the spin-labeled apoprotein, tau c increased to 0.35 ns, indicating decreased motion for the methionyl side chain. The line width (nu 1/2) and spin--lattice relaxation time (T1) for the thiomethyl resonance of 13C-enriched apoC-I in 10 mM phosphate buffer was 6.0 Hz and 320 ms, respectively. When the protein solution was made 1.6 M in Gdn-HCl, these values changed to 2.6 Hz and 970 ms, respectively. Upon addition of DMPC multilamellar liposomes to [13C]apoC-I in 1.6 M Gdn-HCl, the line width increased to 4.7 Hz and the T1 decreased to 380 ms. These results strongly suggest that methionine-38 of apoC-I resides in a region of the apoprotein which undergoes significant secondary and/or tertiary structural change upon disaggregation/unfolding in Gdn-HCl and upon interaction with phospholipid.

Apolipoprotein C-I↗
PubMed1980

Activation of lecithin:cholesterol acyltransferase by a synthetic model lipid-associating peptide.

We have synthesized a model lipid-associating peptide of 20 residues (LAP-20) and studied its association with the phospholipid dimyristoyl phosphatidylcholine (DMPC) and its activation of the plasma enzyme lecithin:cholesterol acyl-transferase (EC 2.3.1.43). The lipid-associating behavior of LAP-20 is similar to that of well-characterized native plasma apolipoproteins after which it was modeled. Upon forming an isolated complex with DMPC, LAP-20 exhibits a large blue-shift in its intrinsic fluorescence, converts from a random coil to an alpha -helix, and changes turbid multilamellar structures of DMPC into small complexes that are optically clear. Addition of 2 mol % cholesterol does not detectably alter the structure or properties of the complex. The cholesterol-containing complexes of LAP-20 and DMPC are substrates for LCAT, having an activity 65% of that of complexes composed of DMPC, cholesterol, and the natural activator, apolipoprotein A-I. These findings suggest that the LCAT-activating regions of apoA-I may be confined to relatively short sequences that contain a lipid-binding determinant.

Apolipoprotein A-I↗
PubMed1979

Very low density lipoprotein. Removal of Apolipoproteins C-II and C-III-1 during lipolysis in vitro.

In this study we have investigated the effects of very low density lipoprotein (VLDL) lipolysis on the removal of radiolabeled apolipoprotein C-II and apolipoprotein C-III-1 from in vitro lipolyzed lipoproteins. Lipolysis was carried out in vitro using lipoprotein lipase purified from bovine milk, and mixtures with or without plasma. Lipoproteins were isolated by ultracentrifugation and by gel filtration. Labeled apo-C-II and apo-C-III-1 distributed among plasma lipoproteins, predominantly VLDL and high density lipoprotein (HDL). Lipolysis induced transfer of apo-C-II and apo-C-III-1 from VLDL to HDL. The transfer was proportional to the extent of triglyceride hydrolysis, and similar for the two apoproteins. The apo-C-II/apo-C-III-1 radioactivity ratio did not change in either VLDL or the fraction of d greater than 1.006 g/ml during the progression of the lipolytic process. Similar observations were recorded while using plasma-devoid lipolytic systems. Gel filtration of incubation mixtures, on 6% agarose, revealed that the removal of labeled apo-C molecules from VLDL is not a consequence of either centrifugation or high salt concentration. These results suggest that there is no preferential removal of apo-C-II or apo-C-III-1 from lipolyzed VLDL particles. They further indicate that the ratio of apo-C-II to apo-C-III-1 does not regulate the extent of lipolysis of different VLDL particles, at least in VLDL isolated from normolipidemic humans.

Animals↗
PubMed1979

Apolipoprotein A-II: chemical synthesis and biophysical properties of three peptides corresponding to fragments in the amino-terminal half.

Three peptide fragments of apolipoprotein A-II corresponding to residues 17--31, 12--31, and 7--31 have been synthesized by solid-phase techniques and purified to apparent homogeneity. Each of these fragments contains residues 18--30, a region previously proposed to possess potential amphipathic helical properties. Secondary structural changes of these synthetic fragments accompanying their interaction with phospholipid have been studied by circular dichroism. The magnitude of this interaction has been evaluated from the yields and stoichiometry of lipid-protein complexes isolated by density gradient ultracentrifugation. Fragment 17--31, the smallest peptide containing the proposed amphipathic helix, did not interact with dimyristoylphosphatidylcholine (DMPC) single bilayer vesicles at 24 degrees C; upon addition of DMPC, no ellipticity change could be detected nor could a stable lipid-peptide complex be isolated. However, fragments 12--31 and 7--31 did interact with phosphilipid; in the absence of lipid, both fragments had primarily disordered structures, but when isolated as DMPC-peptide complexes, both fragments possessed increased helical structure. The phospholipid:peptide molar ratio was 14:1 for fragment 12--31 and 27:1 for fragment 7--31. Studies of space-filling models of these fragments suggest that hydrophobicity and/or length are important properties of phospholipid binding apoproteins.

Amino Acid Sequence↗
PubMed1979

A review of the unique features of HDL apoproteins.

The human plasma high density lipoproteins (HDL) are a heterogeneous ensemble of five proteins associated with both neutral and polar lipids. The sequence of all five proteins are known. ApoA-I and apoA-II are the major protein components; apoC-I, apoC-II and apoC-III are the minor protein components. All these apoproteins spontaneously recombine with phospholipids to give stable lipid-protein complexes and freely exchange between the two major HDL subclasses, HDL2 and HDL3. In addition, ApoC-I, apoC-II, and apoC-III exchange between HDL and very low density lipoproteins. Furthermore, certain HDL apoproteins are activators for plasma enzymes that are important in lipid metabolism. ApoA-I and apoC-I activate lecithin/cholesterol acyltransferase; apoC-II is an activator of lipoprotein lipase. The regions of apoC-I and apoC-II that are involved in the activation of these enzymes have been localized with synthetic peptides. Studies of synthetic and native fragments of apoA-II, apoC-I, apoC-II, and apoC-III as well as model lipid-binding peptides have identified specific regions with structural features common to lipid-binding proteins. These special properties, which include helical potential, sequences with a critical amphipathic length, and high hydrophobicity of the nonpolar side of the amphipathic helix, are the determinants of HDL structure and metabolism.

Amino Acid Sequence↗
PubMed1979

A convenient synthesis of phosphatidylcholines: acylation of sn-glycero-3-phosphocholine with fatty acid anhydride and 4-pyrrolidinopyridine.

A high-yield synthesis of saturated, unsaturated, and short chain phosphatidylcholines from sn-glycero-3-phosphocholine is described. The procedure offers advantages over other reported procedures for the synthesis of phosphatidylcholine in that the large-scale synthesis and purification can be achieved in a minimum time. The procedure utilizes 4-pyrrolidinopyridine as a catalyst and moderate amounts of fatty acid anhydride (2 mol eq. of fatty acid anhydride per mol of OH) in a 1:1 mixture of benzene-dimethylsulfoxide (DMSO) at 40 degrees--42 degrees C (oilbath) for 2--5 hr. At the end of the reaction, the phosphatidylcholine can be purified in the usual manner or by using a Waters Prep LC/500 with a radially compressed silica gel column eluted with chloroform-methanol-water 60:30:4. At a flow rate of 200 ml/min, the phospholipid elutes in 10--15 min, depending on the chain length and unsaturation.

Acylation↗
PubMed1978

Lecithin:cholesterol acyltransferase activation and lipid binding by synthetic fragments of apolipoprotein C-I.

Peptide fragments of apolipoprotein C-I (apoLP-C-I) have been synthesized by solid phase methodology. After purification, each peptide showed the correct amino acid analysis and was a single band by polyacrylamide gel electrophoresis. In density gradient ultracentrifugation with vesicles of dimyristoyl phosphatidylcholine, peptide fragments 32-57, 24-57, and 17-57 formed stable complexes while 39-57 did not. With mixed vesicles of dimyristoyl phosphatidylcholine-cholesterol 20 micrometer of the fragments 32-57, 24-57 and 17-57 stimulated lecithin:cholesterol acyltransferase (LCAT) activity 50, 60, and 100%, respectively, of the value found for apolipoprotein C-I, while fragment 39-57 was inactive. The results indicate that residues 17-57 contain the structural requirements for LCAT activation by apoLP-C-I, and that residues 32-57 represent one of the major phospholipid-binding regions of apoLP-C-I.

Amino Acid Sequence↗
PubMed1977

Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage.

We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.

Amino Acid Sequence↗