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Biomedical subjects

W Cho

Publications and source records attributed to W Cho.

69 records · Page 4Linked to original sources
PubMed1996

Interactions of annexin V with phospholipid monolayers.

To understand the mechanism of annexin V-membrane interactions, we measured the interaction of human recombinant annexin V with phospholipid monolayers with differing head group and acyl group structures. Annexin V interacted with anionic phospholipid monolayers via non-specific electrostatic interactions, which was highly dependent on the surface pressure of monolayer with a sharp maximum. The unique surface pressure dependence of the annexin V-monolayer binding is strikingly similar to that observed for the binding of Ca2+ to anionic phospholipid monolayers, which indicates that the annexin V-bound Ca2+ binds two phospholipids at the membrane surface and that factors governing the Ca(2+)-phospholipid complex formation regulate the overall annexin V-Ca(2+)-membrane interactions.

Annexin A5↗
PubMed1996

Mutagenesis of residues adjacent to transmembrane prolines alters D1 dopamine receptor binding and signal transduction.

Proline is highly conserved in the presumed transmembrane alpha-helices of seven-transmembrane helix-containing, G protein coupled receptors. Unique properties of this imino acid have led to speculations of structural and perhaps dynamic importance for seven-transmembrane helix-containing receptor function. To avoid potentially deleterious consequences of proline-directed mutagenesis, substitutions were made in the X residue of X-Pro peptide bonds (where X is the residue on the amino-terminal side of proline), which may influence static geometries and potential agonist-induced conformational changes at the X-Pro peptide bond. In the fifth helix, Ile205 was substituted with either an alanine (I205A) or a tyrosine (I205Y). Similarly, in the sixth helix, Leu286 was substituted with either an alanine (L286A) or a tyrosine (L286Y). Mutant I205A demonstrated subtle changes in D1 pharmacology and signal transduction. The I205Y and L286Y mutations produced comparatively drastic impairments in both binding and signal transduction. Remarkably, the L286A mutation resulted in constitutive activity characterized by elevated basal signal transduction and increased agonist potencies. In addition, (R)-(+)-SCH23390, a classical antagonist at the wild-type D1 receptor, behaved as a partial agonist at L286A. This is the first report of a constitutively active receptor resulting from this point mutation and the first report of a constitutively active mutant dopamine receptor. These results are discussed in terms of binding pocket geometry and potential mechanisms of signal transduction.

Amino Acid Sequence↗
PubMed1995

Isolation of small polarized bile duct units.

Fragments of small interlobular bile ducts averaging 20 microns in diameter can be isolated from rat liver. These isolated bile duct units form luminal spaces that are impermeant to dextran-40 and expand in size when cultured in 10 microM forskolin for 24-48 hr. Secretion is Cl- and HCO3- dependent and is stimulated by forskolin > dibutyryl cAMP > secretion but not by dideoxyforskolin, as assessed by video imaging techniques. Secretin stimulates Cl-/HCO3- exchange activity, and intraluminal pH increases after forskolin administration. These studies establish that small polarized physiologically intact interlobular bile ducts can be isolated from rat liver. These isolated bile duct units should be useful preparations for assessing the transport properties of small bile duct segments, which are the primary site of injury in cholestatic liver disorders, known as "vanishing bile duct syndromes."

1-Methyl-3-isobutylxanthine↗
PubMed1995

A structure-function study of bovine pancreatic phospholipase A2 using polymerized mixed liposomes.

A new combinatorial approach that includes the genetic variation of protein structure and the chemical modification of phospholipid structure in polymerized mixed liposomes was used to delineate the structure-function relationships in the interfacial catalysis of bovine pancreatic phospholipase A2 (PLA2). Based on previous structural and mutational studies, several bovine PLA2 mutants were generated in which a positive charge of putatively important lysyl side chains was reversed (K10E, K53E, K56E, and K116E) or neutralized (K56Q and K116Q). Kinetic parameters of bovine wild type and mutant PLA2s determined using polymerized mixed liposomes consisting of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine (or -phosphoglycerol) and 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol showed that Lys-53 is involved specifically in the interaction with a substrate bound in the active site. Also, these results showed that Lys-10 and Lys-116 are involved in the interaction of bovine PLA2 with anionic interfaces but not in the interaction with the active site-bound substrate. In particular, Lys-116 makes more significant contribution than Lys-10 by approximately 1.0 kcal/mol to the binding to anionic interfaces. Most importantly, Lys-56 was shown to participate in the interaction with both the active site-bound substrate and anionic interfaces. These findings establish Lys-56 and Lys-116 as essential residues for the binding of bovine pancreatic PLA2 to anionic interfaces. Lastly, our structure-function analysis based on the use of polymerized mixed liposomes was further supported by equilibrium binding measurements of these proteins using 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol polymerized liposomes and by kinetic analyses using monomeric substrates, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine and -phosphoglycerol.

Animals↗
PubMed1995

Hydrophobic residues of the D2 dopamine receptor are important for binding and signal transduction.

Dopamine receptors belong to the seven transmembrane helix-containing, G protein-coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate-114 in helix 3 and two helix 5 serines (194 and 197) of the D2 receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site-directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D2 receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine-198 with an alanine selectively disrupted [3H]N-0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine-411 or the helix 6 leucine-387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine-411 in helix 7 and leucine-387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D2 receptor.

Amino Acid Sequence↗
PubMed1995

Taiwanese love styles and their association with self-esteem and relationship quality.

This study is an examination of how Lee's (1973, 1977, 1988) love styles are related to Taiwanese attitudes toward romantic relationships. Traditional literature was explored to ascertain Chinese beliefs about love and customs surrounding courtships and marriage. Taiwanese students at the University of Texas completed the Love Attitudes Scale (C. Hendrick & S. Hendrick, 1990), a measure of Lee's love styles. Six factors were extracted, using a principle components analysis, that were similar in many ways to the love styles derived from American samples, but that reflected Chinese beliefs and attitudes. The relationships of Taiwanese love styles to self-esteem and relationship quality (i.e., satisfaction, the probability of breaking up, and the number of conflicts experienced in a month) suggested the significance of Chinese traditions as well as current social changes in Taiwan.

Adolescent↗
PubMed1995

Highly efficient immobilization of phospholipase A2 and its biomedical applications.

A new method for the immobilization of phospholipase A2 (PLA2) has been developed to enhance the activity retention of immobilized PLA2. When PLA2 from the venom of Agkistrodon piscivorus piscivorus was pretreated with 4-nitro-3-octanoyl-oxybenzoic acid to acylate epsilon-amino groups of two lysines (Lys-7 and Lys-10) and the resulting acylated enzyme was covalently coupled onto carbonyldiimidazole-activated cross-linked agarose beads, the immobilized acylated enzyme showed high retention of activity toward various aggregated phospholipids. Toward densely packed phospholipid bilayers, such as large unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the immobilized acylated A. p. piscivorus PLA2 was 25-fold more active than the soluble A. p. piscivorus PLA2. The general applicability of our immobilization protocol was demonstrated by the high retention of activity achieved for the immobilized acylated PLA2 from the venom of Naja naja naja. In particular, full activity retention of the immobilized acylated A. p. piscivorus PLA2 toward phospholipids on the surface of human low density lipoproteins suggests its potential usefulness in a newly developed PLA2-based therapy for hypercholesterolemia.

Acylation↗
PubMed1994

Effects of specific fatty acid acylation of phospholipase A2 on its interfacial binding and catalysis.

Monomeric phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus (App-D49) was treated with 3-acyloxy-4-nitrobenzoic acids to acylate the epsilon-amino groups of two lysines (Lys-7 and Lys-10) in the amino terminal region. Resulting 7,10-diacylated-App-D49s, with acyl groups ranging from lauroyl to palmitoyl, spontaneously aggregated in solution. By contrast, 7,10-dioctanoyl-App-D49 existed as a monomer under the same condition. Kinetic and interfacial binding properties of diacylated enzymes indicated that they catalyzed the hydrolysis at the interface as a monomer. When compared to nonacylated App-D49, diacylated enzymes showed slightly increased activity or decreased activity toward monodispersed 1,2-dibutyryl-sn-glycero-3-phosphocholine, Triton X-100/1,2-dilauroyl-sn-glycero-3-phosphocholine mixed micelles, and small unilamellar vesicles (SUV) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Toward densely-packed liquid-crystalline phospholipid bilayers, such as large unilamellar vesicles (LUV) of POPC, however, diacylated enzymes exhibited a large increase in activity, which reacted up to 250-fold for 7,10-dilauroyl-App-D49 ((kcat/Km)app = (1.0 +/- 0.02) x 10(6) M-1 s-1). Measurements of the penetration of individual diacylated enzymes into 2-oleoyl-3-palmitoyl-sn-glycero-1-phosphocholine (i.e., D-POPC) monolayers indicated that the acyl groups enhanced the interfacial binding of protein by interacting with hydrocarbon moieties of phospholipids and that these hydrophobic interactions remained effective even when the phospholipid packing density was high. Furthermore, fluorometric measurements of the binding of diacylated enzymes to polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine showed that the hydrophobic interactions increased the enzymatic activity toward LUV by accelerating the migration of enzyme molecules to vesicle surfaces. The analysis of the kinetic course of POPC LUV hydrolysis showed that diacylated enzymes as a catalyst were superior to nonacylated App-D49 in that they were not only more catalytically efficient but also able to catalyze more turnovers without being trapped in product-containing vesicles. In summary, the acylation of App-D49 by 3-acyloxy-4-nitrobenzoic acids provides a simple and convenient way of converting the enzyme into a highly active form toward densely-packed liquid-crystalline phospholipid bilayers, which might have potential industrial and biomedical applications.

Acylation↗
PubMed1994

A continuous fluorometric assay for phospholipases using polymerized mixed liposomes.

A versatile continuous fluorometric assay for phospholipases A2, C, and D has been developed utilizing polymerized mixed liposomes made of pyrene-containing phospholipids (5 mol%) uniformly inserted in the polymerized liposomes of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol (BLPG) and its derivatives. 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine was used for phospholipase A2 and N-(1-pyrenesulfonyl)-egg phosphatidyl ethanolamine for phospholipases C and D. Fluorescence emission of pyrene moieties in polymerized mixed liposomes was strongly quenched by BLPG molecules and, thus, the hydrolysis of pyrene-containing phospholipids and the subsequent displacement of pyrene moieties from the liposomes resulted in a large increase in fluorescence intensity. All the phospholipases tested selectively and rapidly hydrolyzed the inserted pyrene-containing phospholipids, which were readily monitored by measuring an increase in fluorescence emission at 380 nm. Assay conditions for individual phospholipases were optimized by altering interfacial properties of polymerized liposomes, such as surface charge, and subsequently by changing the chemical structure of hydrolyzable phospholipids. Phospholipase activities were linearly proportional to enzyme concentrations in the range from 0.1 to 50 ng. Specific activity determined for phospholipases from a wide variety of sources ranged from 0.5 to 100 mumol/min/mg. Polymerized mixed liposomes are exceptionally stable against chemical and physical degradation and the assay requires only a small amount of pyrene-containing phospholipids. In addition, the polymerized matrix of BLPG (and its derivatives), due to its inertness to the phospholipase hydrolysis, allows the direct measurement of the equilibrium dissociation constant for a protein-liposome complex.

Fluorometry↗
PubMed1994

Inhibition of human secretory class II phospholipase A2 by heparin.

By means of kinetic analyses using Triton X-100/deoxycholic acid/dilauroylglycerophosphoethanolamine (4:2:1, molar ratio) mixed micelles we examined the effects of heparin on the activity of several phospholipases A2 (PLA2). Heparin avidly bound cationic PLA2s including human secretory class II PLA2 and thereby inhibited their hydrolysis of phospholipids in the mixed micelles. Initial velocity measurements indicated that heparin behaved as a competitive inhibitor for human secretory class II PLA2 and closely related A.h. blomhoffii PLA2 and A.p. piscivorus PLA2. In particular, heparin showed the highest specificity for human secretory class II PLA2. In the absence of deoxycholic acid in mixed micelles, A.h. blomhoffii PLA2 was also strongly inhibited by heparin. The observed inhibition was not due to the interaction of heparin with the active site of PLA2 because heparin did not inhibit the hydrolysis of monomeric substrates by PLA2s. Both kinetic measurements and fluorescence measurements of PLA2-bound 8-anilino-1-naphthalene sulfonate in the presence of varying amounts of heparin showed that a heparin molecule bound about seven molecules of PLA2. When positive charges of four lysines in the amino-terminal region of A.h. blomhoffii PLA2 were neutralized by limited carbamoylation, heparin neither bound the carbamoylated A.h. blomhoffii PLA2 nor inhibited the hydrolysis of Triton X-100/dilauroylglycerophosphocholine mixed micelles by the carbamoylated A.h. blomhoffii PLA2 that retained 50% activity of native A.h. blomhoffii PLA2. Also, heparin did not inhibit the hydrolysis of mixed micelles by 7,10-bis(octanoyl)ated A.p. piscivorus PLA2 in which two lysines in the amino-terminal alpha-helix are acylated. These results indicate that the inhibition of human secretory class II PLA2 and related cationic PLA2s by heparin originates from the interaction of heparin with cationic residues in the amino-terminal region that forms a part of interfacial binding site. In addition, unique structural features of human secretory class II PLA2, together with its unique mode of interaction with heparin, suggest that this PLA2 might have an additional heparin-binding site. Although the heparin-PLA2 binding diminished as the ionic strength of reaction medium increased, the inhibition of human secretory class II PLA2 by heparin remained significant at the physiological ionic strength. An estimated value of inhibition constant (Ki) was 0.1 microM under physiological conditions, which suggests that a normal pharmaceutical dose of heparin might inhibit human secretory class II PLA2 and regulate its biological effects.

Amino Acid Sequence↗
PubMed1993

Use of polymerized mixed liposomes to study interactions of phospholipase A2 with membranes.

Polymerized liposomes of thiol-based phospholipids, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (BLPC) and -phosphoglycerol (BLPG) were used to study interactions of several phospholipases A2 (PLA2) with membranes. Large liposomes (an average diameter of 100 +/- 10 nm) prepared from BLPC or BLPG were readily hydrolyzed by PLA2. Once polymerized, however, these liposomes were resistant to the PLA2 hydrolysis. When liposomes were prepared from a mixture of 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphocholine (pyrene-PC) (5 mol%) and BLPC, fluorescence measurements of resulting polymerized mixed liposomes showed that the pyrene-PC molecules exist solely as monomers without forming a patch and were selectively hydrolyzed by PLA2. Progress of the hydrolysis can be readily monitored by measuring the change in fluorescence emission at 380 nm in the presence of bovine serum albumin. Rapid and selective hydrolysis of inserted phospholipids in polymerized mixed liposomes supports the notion that facile migration of a phospholipid substrate from membrane to the active site of enzyme is a critical step in the catalysis of PLA2. On the basis of these findings, various combinations of polymerized mixed liposomes were prepared and their hydrolysis by PLA2 measured. When compared to the substrate specificity of PLA2s determined using Triton X-100/phospholipid mixed micelles, results from polymerized mixed liposomes indicate that electrostatic interactions between the interfacial binding site of PLA2 and membrane surfaces play an important role in the determination of substrate specificity of PLA2 and in the regulation of PLA2 activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis↗
PubMed1988

The chemical basis for interfacial activation of monomeric phospholipases A2. Autocatalytic derivatization of the enzyme by acyl transfer from substrate.

A basic monomeric phospholipase A2 from the venom of the American water moccasin, Agkistrodon piscivorus piscivorus, undergoes Ca2+-dependent, autocatalytic acylation during the course of hydrolysis of both model and natural phospholipid substrates. Acylation occurs at 2 lysine residues, Lys-7 and Lys-10, in the NH2-terminal alpha-helical segment of the enzyme, and when both positions are fully derivatized, the stable bisacylphospholipase A2 becomes a dimer in solution. The acylated enzyme is fully activated toward monomolecular layers of lecithins. Similar studies applied to the monomeric phospholipases A2 from porcine pancreas and from the venom of Agkistrodon contortrix contortrix also showed irreversible activation of the enzymes by substrate with the same kinetic consequences and formation of dimers. Acylation thus enables these enzymes to overcome the lag period observed under such conditions with native monomeric phospholipases, a phenomenon referred to as interfacial activation. Activation of the enzyme by acylation potentiates the phospholipase for interfacial recognition via formation of a dimeric enzyme. The naturally occurring phospholipase A2 dimer from Crotalus atrox venom displays no lag in the hydrolysis of lecithin monolayers nor does it undergo substrate level acylation. These facts support our proposal that dimerization concomitant with acylation is responsible for the large rate enhancements seen in the hydrolysis of aggregated phospholipids by monomeric phospholipases. Our findings demonstrate for the first time a chemical mechanism for interfacial activation of and interfacial recognition by phospholipases A2.

Amino Acid Sequence↗
PubMed1984

A new class of phospholipases A2 with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding.

We report here the discovery of a new class of phospholipases A2 in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine. Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its beta-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop. The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding. Indeed, although the Lys-49 phospholipases A2 show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium. Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all. These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases A2. Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase A2 catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.

Amino Acid Sequence↗