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F Y Yang

Publications and source records attributed to F Y Yang.

At least 19 recordsLinked to original sources
PubMed1998

Gene synthesis by a LCR-based approach: high-level production of leptin-L54 using synthetic gene in Escherichia coli.

Synthetic genes are very useful in genetic and protein engineering. Here we propose a general method for construction of synthetic genes. Short oligonucleotides are joined through ligase chain reaction (LCR) in high stringency conditions to make "unit fragments" which are then fused to form a full-length gene sequence by polymerase chain reaction. The procedure is simple and accurate and does not place constraints on sequence and length. In this report, a recombinant leptin gene was synthesized according to the codon preference of Escherichia coli. Besides, a substitution of the only Met at position 54 for Leu and an addition of a Met at the N-terminus were introduced in the synthetic gene. The gene was cloned in the pQE-31 expression vector and was expressed in E. coli. A large amount of recombinant leptin containing 6 x His tag was produced and purified by Ni-NTA affinity column. Finally, intact leptin-L54 was released after removing the tag by CNBr cleavage at the Met residue.

Animals↗
PubMed1998

Further studies on Zn2+ -mediated domain-domain communication in human erythrocyte band 3.

Human erythrocyte band 3 is purified and reconstituted into vesicles, forming right-side-out proteoliposomes. Zn2+ entrapped inside the proteoliposomes inhibits the anion transport activity of band 3, and removal of the cytoplasmic domain of band 3 is able to diminish Zn2+ inhibition. Thus, the inhibition of activity of band 3 results from the Zn2+ induced conformational change of the cytoplasmic domain, which in turn is transmitted to the membrane domain. The results of intrinsic fluorescence and its quenching by HB and the 35Cl NMR study indicate that the cytoplasmic domain is essential for the conformational change induced by Zn2+. SH-blocking reagents, CH(3)I and GSSG, are used to modify the cytoplasmic domain, where they specifically bind to Cys201 and Cys317. It is observed that the Zn2+ induced inhibition of anion transport activity is blocked. This demonstrates that Cys201 and Cys317 are required in Zn2+ -mediated domain-domain communication.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

A method for uni-directional reconstitution of human erythrocyte glucose transporter.

The glucose transporter of human erythrocytes (Glut1) was reconstituted into soybean phospholipid liposomes by a method of direct incorporation using the nonionic detergent n-octyl beta-D-glucopyranoside. The reconstituted proteoliposomes were proved to be intact and low ionic permeability. Freeze-fracture electron microscopy study showed that the diameter of the proteoliposomes was about 150 +/- 50 nm and the protein was randomly distributed. The kinetic parameters of the reconstituted transporter were: Km =16.23 mmol/L, Vmax = 34.48 nmol/sec x mg protein. Furthermore, about 90% of the glucose transporter in the reconstituted proteoliposomes were orientated inside-out. Until now it is a more efficient method for uni-directional reconstitution of Glut1 with good reproducibility and higher transport activity.

Detergents↗
PubMed1997

The role of ganglioside GM3 in the modulation of conformation and activity of sarcoplasmic reticulum Ca(2+)-ATPase.

Rabbit sarcoplasmic reticulum does contain trace amounts of gangliosides, and the main species is GM3. Incorporation of GM3 into the SR vesicles or addition of it to the soybean phospholipid used for reconstitution of proteoliposomes obviously increased ATP hydrolysis, as well as, Ca2+ uptake activity of sarcoplasmic reticulum Ca(2+)-ATPase. Conformation changes of Ca(2+)-ATPase induced by GM3 were also observed by circular dichroism, intrinsic fluorescence and fluorescence quenching measurements.

Animals↗
PubMed1997

Membrane formation and cellular response on the surface of lenses implanted in rabbit eyes.

PURPOSE: To study the pathogenesis of membrane formation and cellular response on the surface of posterior chamber intraocular lenses (IOLs) implanted in rabbits. SETTING: Department of Histology and Embryology, Pathology, Ophthalmology. The First Military Medical University, Guangzhou, P.R. China. METHODS: Thirty rabbits had extracapsular lens extraction and posterior chamber IOL implantation. The IOLs were removed 4, 7, 15, 30, and 90 days postoperatively. Membrane formation and cellular response on IOL surfaces were evaluated using light (n = 25), transmission (n = 5), and scanning electron (n = 5) microscopy. RESULTS: On 30 IOLs, the incidence of cellular adhesion was 100%. Cellular components comprised macrophages, fibroblast-like cells, epithelioid cells, giant cells, ultralarge giant cells, and lymphocytes. A thin, proteinaceous film was also seen on the surface of the IOLs. The membrane of the IOL surface comprised fibrin, collagen fibrils, macrophages, fibroblast-like cells, giant cells, and fibroblasts. CONCLUSION: The findings of this study might apply to humans because cellular elements and membranes have been reported in humans.

Animals↗
PubMed1997

Sensitivity of Ca2+ transport of mitochondria to reactive oxygen species.

The relationship between Ca2+ transport and energy transduction of myocardial mitochondria in the presence of reactive oxygen species was investigated. Following treatment with oxygen free radicals [superoxide(O2.-) or hydroxyl radical (.OH)], lipid free radicals in myocardial mitochondrial membrane could be detected by using the method of EPR spin trap. Simultaneously there were obvious alterations in the free Ca2+ ([Ca2+]m) in the mitochondrial matrix; the physical state of membrane lipid; the efficiency of oxidative phosphorylation (ADP/O); the value of the respiratory control ratio (RCR); and the membrane potential of the inner membrane of myocardial mitochondria. If the concentrations of reactive oxygen species were reduced by about 30%, the alterations in the physical state of the membrane lipid and energy transduction of myocardial mitochondria were not observed, but the changes in Ca2+ homeostasis remained. We conclude that Ca2+ transport by myocardial mitochondria is more sensitive to agents such as O2.- or OH, etc. than are oxidation phosphorylation and the respiratory chain.

Animals↗
PubMed1997

Lipids may not be involved in the recognition of apocytochrome C during its transportation.

It was shown that apocytochrome c was less insertion into monolayers from outer mitochondrial membrane lipids than into those from microsomal membrane lipids; the alpha-helix content of apocytochrome c induced by small unilamellar vesicles prepared from outer mitochondrial membrane lipids was less than by those from microsomal membrane lipids; the import efficiency of apocytochrome c into large unilamellar vesicles from outer mitochondrial membrane lipids was also lower than into those from microsome membrane lipids. No specific affinity between apocytochrome c and outer mitochondrial membrane lipids could be found. Import of apocytochrome c across the intact mitochondria, sealed outer mitochondrial membrane and microsome membrane vesicles was compared. Results showed that apocytochrome c was accumulated only in mitochondria, but not the other two kinds of vesicles.

Animals↗
PubMed1996

V92A mutation altered the folding propensity of chicken apocytochrome c and its interaction with phospholipids.

Chicken apocytochrome c has been shown to possess a much stronger tendency to fold spontaneously in aqueous solution than the equivalent enzyme from other species. In the present work, the amino acid that determines its folding ability was elucidated by site-directed mutagenesis. Wild-type chicken apocytochrome c and three mutants V92A, S103A, and V92A/S103A were expressed in Escherichia coli. The wild-type apoprotein and S103A exhibited the same folding property during dialysis renaturation processes as that chemically prepared from chicken cytochrome c, while those containing V92A mutation did not. Quantitative studies by 2,2,2-trifluoroethanol (TFE) and sodium perchlorate (NaClO4) titration demonstrated that the V92A mutation decreased the helix content that could be induced and confirmed that valine 92 is the major determinant of the folding propensity of chicken apocytochrome c. Furthermore, CD spectra, turbidity measurements, and a translocation assay on a model membrane system showed that the V92A mutation also drastically altered the conformation of apocytochrome c after being incorporated into lipid bilayer and decreased the aggregation of phospholipid vesicles after association of the apoprotein, thus rendering the molecule more competent for translocation across the membrane. Our results showed that a single amino acid substitution could radically alter the folding propensity of an unfolded polypeptide chain and thus influence the conformation following its insertion into phospholipid bilayer.

Animals↗
PubMed1996

Effect of ganglioside GM3 on the activity and conformation of reconstituted Ca2+-ATPase.

Trace amounts of gangliosides were found in rabbit skeletal muscle sarcoplasmic reticulum and their main part was shown, by high performance thin layer chromatography. to be GM3. Addition of GM3 to the soybean phospholipids used for reconstitution of proteoliposomes markedly increased ATP hydrolysis as well as Ca2+ uptake activity of sarcoplasmic reticulum Ca2+-ATPase incorporated into the proteoliposomes. Conformation changes of Ca2+-ATPase induced by GM3 were also observed by intrinsic fluorescence and circular dichroism measurements.

Adenosine Triphosphate↗
PubMed1996

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes.

The role of a transmembrane Ca2+ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca2+ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca2+ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca2+ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibitions is observed at 0.25 mM Ca2+. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca2+ gradient by activation of Ca(2+)-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca(2+)-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particle can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca2+ gradient.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1996

Effect of transmembrane Ca2+ gradient on the coupling of beta-adrenergic receptors and adenylyl cyclase.

In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of beta-AR and adenylyl cyclase, beta-AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2+ gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated beta-AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of beta-AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled beta-AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted beta-AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated beta-AR via Gs.

Adenylyl Cyclases↗
PubMed1996

Se-mediated domain-domain communication in band 3 of human erythrocytes.

Na2SeO3 could affect the anion flux of Band 3 of inside-out erythrocyte membrane vesicles (IOVs). Such effect was believed to be based on the interaction of SH groups of Band 3 with Na2SeO3. This effect could be eliminated when the cytoplasmic domain of Band 3 was proteolytically removed by trypsin. This suggested that SH groups in the cytoplasmic domain were involved in such interaction. Measurement of the pH dependence of intrinsic fluorescence intensity provided evidence that conformational changes of Band 3 occurred as a consequence of interaction with selenite. KI quenching of intrinsic fluorescence of Band 3 could also show that there was a conformational change in the cytoplasmic domain of Band 3 after reaction with Na2SeO3. Such conformational change in turn could be transmitted to the membrane domain of Band 3 monitored by quenching of intrinsic fluorescence of Band 3 using hypocrellin B (HB) (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China). It is suggested that the cytoplasmic domain of Band 3 is not necessary for its anion flux, but is essential for the regulation (e.g., by Se) of its active site located at the membrane domain, and hence, it may provide evidence of communication between the cytoplasmic domain and the membrane domain of Band 3.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1995

Effect of propensity of hexagonal II phase formation on the activity of mitochondrial ubiquinol-cytochrome c reductase and H(+)-ATPase.

The propensity of hexagonal II phase formation plays an important role in the activity of mitochondrial ubiquinol-cytochrome c reductase or H(+)-ATPase. The respiratory control ratio of reconstituted ubiquinol-cytochrome c reductase or the ATP-induced membrane potential of reconstituted H(+)-ATPase became higher as the non-bilayer phospholipid phosphatidylethanolamine content of proteoliposomes increased. The highest respiratory control ratio or ATP-induced membrane potential was obtained in the case of 60-80% phosphatidylethanolamine-containing proteoliposomes. Dioleoylphosphatidylethanolamine could significantly enhance the respiratory control ratio of ubiquinol-cytochrome c reductase and ATP-induced membrane potential of H(+)-ATPase, while no obvious change could be observed when dielaidoylphosphatidylethanolamine was used. The bilayer to hexagonal II phase transition temperature of ubiquinol-cytochrome c reductase-containing proteoliposomes reconstituted with phosphatidylcholine+phosphatidylethanolamine increases with decreasing content of phosphatidylethanolamine. Several additives such as the bilayer stabilizers, cholesterol 3-sulfate and carbobenzoxy-D-Phe-L-PheGly, or hexagonal II phase-forming promoters, such as diolein or eicosane, can decrease or increase the activity of these two enzyme complexes.

Adenosine Triphosphate↗
PubMed1995

Effect of transmembrane Ca2+ gradient on Gs function.

Gs and adenylate cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with or without 1000-fold transmembrane Ca2+ gradient. Obtained results showed that Gs activities of both binding GTP gamma S and stimulating adenylate cyclase were the highest in proteoliposomes, with a transmembrane Ca2+ gradient similar to the physiological situation and the lowest while the transmembrane Ca2+ gradient was in the inverse direction. Such a difference could be diminished following the dissipation of the transmembrane Ca2+ gradient by A23187. Time-resolved fluorescence anisotropy of diphenylhexatriene (DPH) has been used to compare the physical state of phospholipids among those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for higher membrane fluidity, which may favor Gs function with higher GTP-binding activity and stimulation of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1995

Transmembrane Ca2+ gradient and function of membrane proteins.

This review will focus on the recent advance in the study of effect of transmembrane Ca2+ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca2+ gradient and sarcoplasmic reticulum Ca(2+)-ATPase; 2. Effect of transmembrane Ca2+ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca2+ gradient may play an important role in modulating the conformation and activity of SR Ca(2+)-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.

Animals↗
PubMed1995

Zn(2+)-mediated domain-domain communication in human erythrocyte band 3.

Zn2+ could inhibit the anion transport activity of spectrin-stripped inside-out human erythrocyte membrane vesicles (IOVs). Removal of the cytoplasmic domain from Band 3 by trypsin could eliminate Zn2+ inhibition. The location of a Zn(2+)-binding site was confirmed by atomic absorbance spectrometry. The results of time-resolved fluorescence and intrinsic fluorescence quenching by KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China) showed that the cytoplasmic domain is necessary for the Zn(2+)-induced conformational changes of the whole molecule as well as the membrane domain of Band 3. It is suggested that Zn2+ induced a conformational change in the cytoplasmic domain of Band 3, which in turn was transmitted to the membrane domain, resulting in an inhibition of activity of Band 3. Such long-range conformational changes may imply that the cytoplasmic domain is poised to function as a cytosolic arm in order to modulate the structure of the membrane domain of Band 3.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1995

Cytoplasmic Ca2+ inhibits the glucose transporter of human erythrocytes.

The effect of Ca2+ on the glucose transporter of human erythrocytes was investigated. The results showed that extracellular Ca2+ had no effect. But, the glucose transport of erythrocytes was markedly inhibited due to the increase in cytoplasmic Ca2+ concentration by addition of ionophore A23187. The Ca2+ inhibition exhibited a dose-dependent manner with an apparent half maximal concentration of 250 microM and could not be recovered by 10 mM EGTA. Unlike Ca2+, Mg2+ did not affect the glucose transporter.

Calcimycin↗