Search PubMed⌕ Search

Biomedical subjects

C L Tsou

Publications and source records attributed to C L Tsou.

At least 37 records · Page 2Linked to original sources
PubMed1996

Phosphorylation by a G protein-coupled kinase inhibits signaling and promotes internalization of the monocyte chemoattractant protein-1 receptor. Critical role of carboxyl-tail serines/threonines in receptor function.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of chemotactic cytokines and signals via activation of a G protein-coupled seven-transmembrane domain receptor to mediate chemotaxis. Monocyte activation is limited by desensitization and internalization of the MCP-1R, but these mechanisms are not well understood. In this study, we show that the type B MCP-1R (MCP-1RB/CCR2B) is rapidly phosphorylated and internalized in response to nanomolar concentrations of MCP-1. Co-expression of CCR2B in Xenopus oocytes with beta-adrenergic receptor kinase 2 (beta ark2), but not beta ark1 or rhodopsin kinase, specifically blocked receptor activation by MCP-1. Mutation of serine (Ser) and threonine (Thr) residues in the terminal carboxyl-tail of the receptor, which are potential targets of beta ark-mediated phosphorylation, prevented inhibition of receptor activation by beta ark2 in microinjected oocytes. Finally, a construct in which multiple Ser and Thr residues in the carboxyl-tail were changed to alanine significantly prolonged the agonist-dependent intracellular calcium flux and inhibited receptor internalization in transfected human embryonic kidney (HEK)-293 cells. These studies demonstrate that phosphorylation of Ser and Thr residues in the carboxyl-tail of CCR2B mediates receptor desensitization and internalization and may serve to limit the chemotactic response of leukocytes to MCP-1 and related chemokines.

Amino Acid Sequence↗
PubMed1996

Sequential unfolding of adenylate kinase during denaturation by guanidine hydrochloride.

The unfolding of adenylate kinase in GuHCl of increasing concentrations has been followed by a combination of different methods. Molecular packing was measured by size-exclusion chromatography (SEC), exposure of buried Tyr residues by second- derivative spectra, loss of secondary structure by circular dichroism in the far-ultraviolet and the decrease in surface hydrophobicity by ANS binding. The conformational changes of adenylate kinase as followed by the above methods depend differently on GuHCl concentration. The concentrations of GuHCl at which 50% changes as measured by the above four methods occur are 0.3, 0.46, 0.64 and 0.64 M, respectively. SEC measurements show that with increasing GuHCl concentrations, the process of unfolding of adenylate kinase involves two slowly interconvertible intermediate stages, I1, and I2, the last is in a more advanced state of unfolding but is still more compact than the fully unfolded state, U, as indicated by their elution volumes in the SEC profile. There is also evidence to suggest that both the intermediates I1 and I2 may contain additional intermediary components in rapid equilibrium as indicated by the gradual shift of both peaks in the SEC elution profile. A sequential mechanism is suggested for the unfolding of adenylate kinase with increasing guanidine hydrochloride concentrations.

Adenylate Kinase↗
PubMed1996

Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site.

It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028-7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulfonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and functional expression of murine JE (monocyte chemoattractant protein 1) and murine macrophage inflammatory protein 1alpha receptors: evidence for two closely linked C-C chemokine receptors on chromosome 9.

We have isolated cDNA clones that encode two closely related, murine C-C chemokine receptors. Both receptors are members of the G-protein-coupled, seven-transmembrane domain family of receptors and are most closely related to the human monocyte chemoattractant protein 1 receptor. Expression of each of the receptors was detected in murine monocyte/macrophage cell lines, but not in nonhematopoietic lines. Expression of these receptors in Xenopus oocytes revealed that one receptor signaled in response to low nanomolar concentrations of murine JE, whereas the second receptor was activated by murine macrophage inflammatory protein (MIP) 1alpha and the human chemokines MIP-1beta and RANTES. Binding studies revealed high affinity binding of radiolabeled mJE to the mJE receptor and murine MIP-1alpha to the second receptor. Chromosomal localization indicated that the two receptor genes were clustered within 80 kilobases of each other on mouse chromosome 9. Creation of receptor chimeras suggested that the amino terminus was critically involved in mediating signal transduction and ligand specificity of the mJE receptor, but not the mMIP-1alpha receptor. The identification and cloning of two functional murine chemokine receptors provides important new tools for investigating the roles of these potent cytokines in vivo.

Amino Acid Sequence↗
PubMed1995

Activation of chicken liver dihydrofolate reductase in concentrated urea solutions.

The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3-fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed.

Ammonium Sulfate↗
PubMed1995

Inactivation during denaturation of ribonuclease A by guanidinium chloride is accompanied by unfolding at the active site.

Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

Amino Acid Sequence↗
PubMed1995

Expression of two related vacuolar H(+)-ATPase 16-kilodalton proteolipid genes is differentially regulated in a tissue-specific manner.

The 16-kD proteolipid subunit is the principal integral membrane protein of the vacuolar H(+)-ATPase (V-ATPase) complex that forms the proton channel responsible for translocating protons across lipid bilayers. Two degenerate synthetic oligonucleotides, COT11 and COT12, corresponding to highly conserved transmembrane domains in all 16-kD subunits sequenced so far, were used to amplify a partial cDNA of the V-ATPase proteolipid subunit from cotton (Gossypium hirsutum L.) by polymerase chain reaction (PCR). These PCR products were used to isolate two full-length cDNAs from a -3 d postanthesis cotton ovule library. Both clones, CVA16.2 and CVA16.4, consisting of 816 and 895 bp, respectively, encode the 16-kD proteolipid subunit of the V-ATPase. At the nucleotide level, the complete sequences of the two clones show 73.5% identity, but share about 95% identity within the coding region, although the two polypeptides differ by only one amino acid. Comparison of deduced amino acid sequences of the proteolipid subunits revealed that the four transmembrane domains and the two cytosolic extramembrane domains are highly conserved in all eukaryotes. Southern blot analysis of cotton genomic DNA showed that these clones belong to small gene families in related diploid and allotetraploid species. Northern blot analysis suggested that the three major V-ATPase subunits (69, 60, and 16 kD) are coordinately regulated, in part, at the transcriptional level. RNA analysis and reverse-transcription PCR established that 16-kD proteolipid transcripts differentially accumulate in different tissues and increase dramatically in tissues undergoing rapid expansion, particularly in anthers, ovules, and petals. The CVA16.4 proteolipid transcript is the most prevalent of the two proteolipid messages in expanding ovules harvested 10 d post-anthesis. In contrast, the two proteolipid mRNAs accumulate to similar levels in developing petals.

Amino Acid Sequence↗
PubMed1994

Chaperone-like activity of protein disulfide isomerase in the refolding of a protein with no disulfide bonds.

D-Glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) is a protein containing no disulfide bonds; the guanidine HCl-denatured enzyme shows only a limited extent of refolding and reactivation upon dilution, and the enzyme is particularly prone to aggregation during the dilution process. With increasing GAPDH concentration, reactivation decreases and aggregation increases. The presence of protein disulfide isomerase in the dilution mixture markedly increases reactivation of GAPDH and at the same time prevents the aggregation of GAPDH as shown by light-scattering measurements. It is suggested that upon dilution, denatured GAPDH is faced with two competing processes of correct folding and assembly to yield the native enzyme and non-productive association of the partially refolded species to form aggregates. Independent of the isomerase activity as no disulfide bond is present in GAPDH, protein disulfide isomerase assists the refolding of GAPDH to its active state by suppressing aggregation in a way closely similar to the action of chaperones.

Disulfides↗
PubMed1994

Inactivation precedes changes in allosteric properties and conformation of D-glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase during denaturation by guanidinium chloride.

It has been shown that inactivation of several enzymes precedes overall conformational changes of the enzyme molecules as a whole during denaturation [Tsou (1993) Science, 262, 380-381]. However, the relation between inactivation, loss of allosteric properties of oligomeric enzymes and unfolding of the enzyme molecule during denaturation remain little explored. These have now been compared for D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6-bisphosphatase (FruP2ase) during denaturation by guanidinium chloride (GdmCl). GAPDH is completely inactivated at 0.3 M GdmCl but at this GdmCl concentration it still binds NAD+ with negative co-operativity. At 0.4 M GdmCl, inactivation of FruP2ase reaches completion whereas its allosteric properties, including the heterotropic effect of AMP inhibition and K+ activation with positive co-operativity, are only partially affected. Much higher GdmCl concentrations are required to bring about unfolding of the overall structures of both enzymes.

Adenosine Monophosphate↗
PubMed1994

Association and dissociation of protein disulfide isomerase.

Purified protein disulfide isomerase, homogeneous by SDS-PAGE, can be separated into two components by PAGE and by gel filtration. These two components, with the same amino-acid composition as well as N- and C-terminal sequences, are the tetramer and dimer of molecular weight 240 kDa and 120 kDa, respectively. The specific activity of the dimer is twice that of the tetramer. At 4 degrees C and pH 7.5 the purified dimer associates and the tetramer dissociates, both slowly and partially, to form a dimer-tetramer mixture. Treatment with dithiothreitol has only a minor effect on the dissociation of the tetramer indicating that the association is not through disulfide formation between the protomers. By prolonged treatment with 1% Triton X-100 or in strong salt solutions the tetramer dissociates to the dimer, but further dissociation to the monomer can only be effected in SDS or guanidine hydrochloride. These results suggest that apart from hydrogen bonds, hydrophobic forces and ionic interactions are mainly involved in the association of the protomers.

Binding Sites↗
PubMed1993

A comparison of Zn(II) and Co(II) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme.

The kinetics of reconstitution of apoacylase with either Zn(II) or Co(II) and the inactivation of the Co(II) reconstituted enzyme by 1,10-phenanthroline (OP) has been studied by following the substrate reaction continuously in presence of the metal ion or OP respectively. Although the native Zn(II)-containing and the Co(II)-reconstituted enzymes have closely similar Michaelis constants and maximal velocities, the kinetics for both the inactivation by OP and the reconstitution of the apoenzyme with the metal ions differs considerably. For Co(II), both the inactivation by OP and the reconstitution show simple kinetics, but for Zn(II), the inhibition by OP is a multi-phasic process [Wang, Wu, Wang, Zhou and Tsou (1992) Biochem. J. 281, 285-290], and the kinetics of reconstitution is also much more complicated. Both the native and the Co(II)-reconstituted enzymes are inhibited by excess of Zn(II), but not by Co(II). The inhibition by Zn(II) in excess and the reconstitution of the apoenzyme with Zn(II) are co-operative processes. The inhibition by Zn and its effect on the fluorescence emission of 1-anilinonaphthalene-8-sulphonic acid bound to the native enzyme indicate multiple Zn(II)-binding sites.

Amidohydrolases↗
PubMed1993

Inactivation before significant conformational change during denaturation of papain by guanidine hydrochloride.

During denaturation by GuHCl, papain shows a rapid decrease in activity with increasing concentrations of the denaturant followed by an intermediate stage of relatively little change from 1 to 2 M before complete inactivation at 4 M GuHCl. At GuHCl concentrations lower than 2 M, enzyme activity is more sensitive to GuHCl than noticeable conformation changes as followed by fluorescence and CD measurements. Kinetics of GuHCl inactivation were studied by following the substrate reaction in the presence of denaturant and the apparent rate constants thus obtained were found to be only slightly higher than those for conformational changes. However, apparent inactivation rate constants obtained in the presence of saturating concentration of substrate are actually inactivation constants for the ES complex. The inactivation rates at different substrate concentrations were, therefore, followed and the microscopic inactivation rate constants for the free enzyme obtained (Tsou, C.L. (1988) Adv. Enzymol. 61, 381-436). It was found that substrate protects strongly against inactivation and at the same GuHCl concentration, the inactivation rate of the free enzyme is 100-fold higher than that of unfolding. The above results show that the activity of papain is more sensitive to GuHCl than its overall conformation and like the enzymes previously studied in this laboratory, its active site is more flexible than the enzyme molecule as a whole.

Dose-Response Relationship, Drug↗
PubMed1993

Inactivation precedes conformation change during thermal denaturation of adenylate kinase.

During the thermal denaturation of rabbit muscle adenylate kinase, the extents and rates of both unfolding and aggregation are dependent on protein concentration. Under identical conditions, inactivation takes place at a lower temperature than noticeable conformational changes and aggregation as measured by fluorescence, second derivative absorption spectroscopy, far ultraviolet circular dichroism and light scattering. Kinetics of inactivation can be resolved into two phases and at the same protein concentrations, the unfolding and aggregation rates are about one order of magnitude slower than the fast phase and approximately the same as the slow phase rate of the inactivation reaction between 35 and 60 degrees C. This is in general accord with the suggestion made previously that the active site of this enzyme is situated in a region more flexible than the molecule as a whole (Tsou, C.L. (1986) Trends Biochem. Sci. 11, 427-429). The inactivated enzyme cannot be reactivated by cooling and standing at 4 degrees C but can be over 80% reactivated by cooling and first standing in 3 M guanidine hydrochloride followed by diluting out the denaturant.

Adenylate Kinase↗
PubMed1993

Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride.

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

Animals↗