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1.  Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells 
BMC Biochemistry  2002;3:32.
The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) – dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors.
In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs), and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1.
Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.
PMCID: PMC140315  PMID: 12509223
2.  Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides 
BMC Biochemistry  2002;3:31.
Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report.
In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail) each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C) and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present.
We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.
PMCID: PMC139967  PMID: 12453307
3.  Inhibition of the MEK1/ERK pathway reduces arachidonic acid release independently of cPLA2 phosphorylation and translocation 
BMC Biochemistry  2002;3:30.
The 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear.
MEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126.
These results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2.
PMCID: PMC130032  PMID: 12370087
anisomycin; arachidonic acid; calcium; cytosolic phospholipase A2; ERK; fluorescent proteins; phosphorylation; MEK; U0126; translocation
4.  The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator 
BMC Biochemistry  2002;3:29.
Endoplasmic reticulum retention of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) mutants and their rapid degradation is the major cause of cystic fibrosis (CF). An important goal is to understand the mechanism of how the misfolded proteins are recognized, retained, and targeted for degradation.
Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R) domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of ΔF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant ΔF508 CFTR.
These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR.
PMCID: PMC130031  PMID: 12361483
5.  MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function 
BMC Biochemistry  2002;3:28.
Three macromolecular assemblages, the lid complex of the proteasome, the COP9-Signalosome (CSN) and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function.
We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects.
The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1-removing activity.
PMCID: PMC129983  PMID: 12370088
6.  Specificity of DNA triple helix formation analyzed by a FRET assay 
BMC Biochemistry  2002;3:27.
A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis.
We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 × 105 and 2.3 × 106 M-1. Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg2+ions whereas it is inhibited by K+ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 × 104 and 8.1 × 104 min-1 M-1. Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation.
We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA.
PMCID: PMC128820  PMID: 12323077
7.  Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels 
BMC Biochemistry  2002;3:26.
Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model.
Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits.
These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability.
PMCID: PMC128819  PMID: 12350235
8.  Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH 
BMC Biochemistry  2002;3:25.
One of the signatures of Alzheimer's disease is the accumulation of aggregated amyloid protein, Aβ, in the brain. Aβ arises from cleavage of the Amyloid Precursor protein by β and γ secretases, which present attractive candidates for therapeutic targeting. Two β-secretase candidates, ASP-1 and ASP-2, were identified as aspartic proteases, both of which cleave the amyloid precursor at the β-site. These are produced as immature transmembrane proteins containing a pro-segment.
ASP-2 expressed in HEK293-cells cleaved the Swedish mutant amyloid precursor at different β-sites at different pHs in vitro. Recent reports show that furin cleaves the pro-peptide of ASP-2, whereas ASP-1 undergoes auto-catalysis. We show that purified recombinant ASP-2 cleaves its own pro-peptide at ph 5 but not pH 8.5 as seen by mass spectrometry, electrophoresis and N-terminal sequencing.
We suggest that ASP-2 processing as well as activity are influenced by pH, and hence the cellular localisation of the protein may have profound effects on the production of Aβ. These factors should be taken into consideration in the design of potential inhibitors for these enzymes.
PMCID: PMC126479  PMID: 12204094
9.  Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 
BMC Biochemistry  2002;3:24.
The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the β-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 were used to define a domain conferring interaction with the signaling scaffold protein, RACK1.
Truncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM).
The RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal β-propeller blades of RACK1 in a manner akin to the interaction of the helical G-γ signal transducing protein with the β-propeller protein, G-β.
PMCID: PMC126212  PMID: 12193273
Rolipram; Protein kinase C binding protein; signalling scaffold; cyclic AMP
10.  A ~35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP 
BMC Biochemistry  2002;3:23.
The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested.
Partially purified fractions containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. However, further purification revealed the presence of a putative 35 kDa insect cell protein (p35) which was found responsible for the DNA binding activity. A close match to the 9/11 bases of the ARS consensus sequence was sufficient for p35 binding activity. A DNA fragment from the human c-myc origin region containing yeast ACS like elements also showed p35 binding activity.
We have identified a Spodoptera frugiperda protein with ATP dependent DNA binding activity to ACS like elements. ACS like elements have been reported to be essential for ORC binding and replication initiation in yeast but their role in higher eukaryotes still remains elusive. Like the ARS consensus sequence elements of yeast, ACS like elements found in c-myc and lamin beta 2 origin regions may play similar roles in replication and indicate a conserved role for this DNA motif among eukaryotes.
PMCID: PMC122095  PMID: 12186657
11.  Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p 
BMC Biochemistry  2002;3:22.
SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes.
We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity.
Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates.
PMCID: PMC128837  PMID: 12167173
12.  Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine 
BMC Biochemistry  2002;3:21.
Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.
The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent.
It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.
PMCID: PMC117796  PMID: 12149129
13.  Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases 
BMC Biochemistry  2002;3:20.
The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised.
We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 × 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 × 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell.
Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.
PMCID: PMC117780  PMID: 12121577
14.  Molecular cloning and tissue distribution of mammalian L-threonine 3-dehydrogenases 
BMC Biochemistry  2002;3:19.
In mammals, L-threonine is an indispensable amino acid. The conversion of L-threonine to glycine occurs through a two-step biochemical pathway involving the enzymes L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase. The L-threonine 3-dehydrogenase enzyme has been purified and characterised, but the L-threonine 3-dehydrogenase gene has not previously been identified in mammals.
Transcripts for L-threonine 3-dehydrogenase from both the mouse and pig are reported. The ORFs of both L-threonine dehydrogenase cDNAs encode proteins of 373 residues (41.5 kDa) and they share 80% identity. The mouse gene is located on chromosome 14, band C. The amino-terminal regions of these proteins have characteristics of a mitochondrial targeting sequence and are related to the UDP-galactose 4-epimerases, with both enzyme families having an amino-terminal NAD+ binding domain. That these cDNAs encode threonine dehydrogenases was shown, previously, by tiling 13 tryptic peptide sequences, obtained from purified L-threonine dehydrogenase isolated from porcine liver mitochondria, on to the pig ORF. These eukaryotic L-threonine dehydrogenases also have significant similarity with the prokaryote L-threonine dehydrogenase amino-terminus peptide sequence of the bacterium, Clostridium sticklandii. In murine tissues, the expression of both L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase mRNAs were highest in the liver and were also present in brain, heart, kidney, liver, lung, skeletal muscle, spleen and testis.
The first cloning of transcripts for L-threonine dehydrogenase from eukaryotic organisms are reported. However, they do not have any significant sequence homology to the well-characterised Escherichia coli L-threonine dehydrogenase.
PMCID: PMC117216  PMID: 12097150
15.  Insertion of a small peptide of six amino acids into the β7–β8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities 
BMC Biochemistry  2002;3:18.
HIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the β7-β8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001). Deletion or alanine substitution of four amino acid residues of this loop in the p51 subunit severely impaired DNA binding and catalytic activities of the enzyme. To further examine the role of this loop in HIV-1 RT, we have increased its size such that the six amino acids loop sequences are repeated in tandem and examined its impact on the dimerization process and catalytic function of the enzyme.
The polymerase and the RNase H activities of HIV-1 RT carrying insertion in the β7-β8 loop of both the subunits (p66INS/p51INS) were severely impaired with substantial loss of DNA binding ability. These enzymatic activities were restored when the mutant p66INS subunit was dimerized with the wild type p51. Glycerol gradient sedimentation analysis revealed that the mutant p51INS subunit was unable to form stable dimer either with the wild type p66 or mutant p66INS. Furthermore, the p66INS/p66INS mutant sedimented as a monomeric species, suggesting its inability to form stable homodimer.
The data presented herein indicates that any perturbation in the β7-β8 loop of the p51 subunit of HIV-1 RT affects the dimerization process resulting in substantial loss of DNA binding ability and catalytic function of the enzyme.
PMCID: PMC117134  PMID: 12086585
16.  Low solubility of unconjugated bilirubin in dimethylsulfoxide – water systems: implications for pKa determinations 
BMC Biochemistry  2002;3:17.
Aqueous pKa values of unconjugated bilirubin are important determinants of its solubility and transport. Published pKa data on an analog, mesobilirubin-XIIIα, studied by 13C-NMR in buffered solutions containing 27 and 64 vol% (C2H3)2SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pKa values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pKa, 8.12 and 8.44, and its aqueous solubility. A thermodynamic analysis, using this solubility and a published solubility in DMSO, suggested that the systems used to measure 13C-NMR shifts were highly supersaturated. This expectation was assessed by measuring the residual concentrations of bilirubin in the supernatants of comparable DMSO-buffer systems, after mild centrifugation to remove microprecipitates.
Extensive sedimentation was observed from numerous systems, many of which appeared optically clear. The very low supernatant concentrations at the lowest pH values (4.1-5.9) were compatible with the above thermodynamic analysis. Extensive sedimentation and low supernatant concentrations occurred also at pH as high as 7.2.
The present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pKa values. Many of the mesobilirubin systems in the 13C-NMR studies were probably supersaturated, contained microsuspensions, and were not true solutions. This, and previously documented errors in pH determinations that caused serious errors in pKa values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pKa estimates for mesobilirubin from the 13C-NMR studies.
PMCID: PMC116679  PMID: 12079498
17.  Identification of critical residues in loop E in the 5-HT3ASR binding site 
BMC Biochemistry  2002;3:15.
The serotonin type 3 receptor (5-HT3R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.
Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.
Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.
PMCID: PMC117120  PMID: 12079500
18.  Functional group interactions of a 5-HT3R antagonist 
BMC Biochemistry  2002;3:16.
Lerisetron, a competitive serotonin type 3 receptor (5-HT3R) antagonist, contains five functional groups capable of interacting with amino acids in the 5-HT3R binding site. Site directed mutagenesis studies of the 5-HT3AR have revealed several amino acids that are thought to form part of the binding domain of this receptor. The specific functional groups on the ligand that interact with these amino acids are, however, unknown. Using synthetic analogs of lerisetron as molecular probes in combination with site directed mutagenesis, we have identified some of these interactions and have proposed a model of the lerisetron binding site.
Two analogs of lerisetron were synthesized to probe 5-HT3R functional group interactions with this compound. Analog 1 lacks the N1 benzyl group of lerisetron and analog 2 contains oxygen in place of the distal piperazine nitrogen. Both analogs show significantly decreased binding affinity to wildtype 5-HT3ASRs. Mutations at W89, R91, Y142 and Y152 produced significant decreases in binding compared to wildtype receptors. Binding affinities of analogs 1 and 2 were altered only by mutations at W89, and Y152.
Based on the data obtained for lerisetron and analogs 1 and 2, we have proposed a tentative model of the lerisetron binding pocket of the 5-HT3ASR. According to this model, The N-benzyl group interacts in a weak interaction with R91 while the benzimidazole group interacts with W89. Our data support an interaction of the distal amino nitrogen with Y142 and Y152.
PMCID: PMC116678  PMID: 12079499
19.  Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals 
BMC Biochemistry  2002;3:14.
Expression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator.
Utilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins.
We have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals.
PMCID: PMC116586  PMID: 12069692
20.  The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I 
BMC Biochemistry  2002;3:13.
Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain). The 67 kDa N-terminal domain (Top67) has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism.
Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA. The two domains had similar affinities to ssDNA. However, only Top67 could bind dsDNA with high affinity. DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme. DNA rejoining also did not require the presence of the ZD domain.
We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage. However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils.
PMCID: PMC115839  PMID: 12052259
21.  Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site 
BMC Biochemistry  2002;3:12.
Profilin is a small cytoskeletal protein which interacts with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate (PI(4,5)-P2). Crystallography, NMR and mutagenesis of vertebrate profilins have revealed the amino acid residues that are responsible for the interactions with actin and poly(L-proline) peptides. Although Arg88 of human profilin I was shown to be involved in PI(4,5)-P2-binding, it was suggested that carboxy terminal basic residues may be involved as well.
Using site directed mutagenesis we have refined the PI(4,5)-P2 binding site of human profilin I. For each mutant we assessed the stability and studied the interactions with actin, a proline-rich peptide and PI(4,5)-P2 micelles. We identified at least two PI(4,5)-P2-binding regions in human profilin I. As expected, one region comprises Arg88 and overlaps with the actin binding site. The second region involves Arg136 in the carboxy terminal helix and neighbours the poly(L-proline) binding site. In addition, we show that adding a small protein tag to the carboxy terminus of profilin strongly reduces binding to poly(L-proline), suggesting local conformational changes of the carboxy terminal α-helix may have dramatic effects on ligand binding.
The involvement of the two terminal α-helices of profilin in ligand binding imposes important structural constraints upon the functions of this region. Our data suggest a model in which the competitive interactions between PI(4,5)-P2 and actin and PI(4,5)-P2 and poly(L-proline) regulate profilin functions.
PMCID: PMC116585  PMID: 12052260
22.  The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes 
BMC Biochemistry  2002;3:11.
Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus.
Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF) 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF) 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F.
Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.
PMCID: PMC116439  PMID: 12028592
23.  Hisactophilin is involved in osmoprotection in Dictyostelium 
BMC Biochemistry  2002;3:10.
Dictyostelium cells exhibit an unusual stress response as they protect themselves against hyperosmotic stress. Cytoskeletal proteins are recruited from the cytosolic pool to the cell cortex, thereby reinforcing it. In order to gain more insight into the osmoprotective mechanisms of this amoeba, we used 1-D and 2-D gel electrophoresis to identify new proteins that are translocated during osmotic shock.
We identified hisactophilin as one of the proteins that are enriched in the cytoskeletal fraction during osmotic shock. In mutants lacking hisactophilin, viability is reduced under hyperosmotic stress conditions. In wild type cells, serine phosphorylation of hisactophilin was specifically induced by hypertonicity, but not when other stress conditions were imposed on cells. The phosphorylation kinetics reveals a slow accumulation of phosphorylated hisactophilin from 20–60 min after onset of the hyperosmotic shock condition.
In the present study, we identified hisactophilin as an essential protein for the osmoprotection of Dictyostelium cells. The observed phosphorylation kinetics suggest that hisactophilin regulation is involved in long-term osmoprotection and that phosphorylation occurs in parallel with inactivation of the dynamic actin cytoskeleton.
PMCID: PMC115871  PMID: 11996675
24.  Expression of monolysocardiolipin acyltransferase activity is regulated in concert with the level of cardiolipin and cardiolipin biosynthesis in the mammalian heart 
BMC Biochemistry  2002;3:9.
Monolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation.
In rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil in the drinking water for 34 days, cardiac cardiolipin content was decreased 29% (p < 0.025) and this was associated with a 32% decrease (p < 0.025) in cardiolipin synthase and a 35% reduction (p < 0.025) in MLCL AT activities. Streptozotocin-induced diabetes or hyperinsulinemia in rats did not affect cardiac cardiolipin content nor MLCL AT and cardiolipin synthase activities. Finally, cardiolipin content, MLCL AT and cardiolipin synthase activities were unaltered during murine P19 teratocarcinoma cell differentiation into cardiac myocytes. In all models, phospholipase A2 activities were unaltered compared with controls.
We propose a general model in which the expression of MLCL AT activity is regulated in concert with the biosynthesis and level of cardiolipin in the heart.
PMCID: PMC113744  PMID: 12019031
25.  Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate 
BMC Biochemistry  2002;3:8.
Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant.
Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 μM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values.
The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.
PMCID: PMC111190  PMID: 12014993

Results 1-25 (32)