Kokumi taste perception is functional in a model carnivore, the domestic cat (Felis catus).

Kokumi taste is a well-accepted and characterised taste modality and is described as a sensation of enhancement of sweet, salty, and umami tastes. The Calcium Sensing Receptor (CaSR) has been designated as the putative kokumi taste receptor for humans, and a number of kokumi-active ligands of CaSR have been discovered recently with activity confirmed both in vivo and in vitro. Domestic cats (Felis catus) are obligate carnivores and accordingly, their diet is abundant in proteins, peptides, and amino acids. We hypothesised that CaSR is a key taste receptor for carnivores, due to its role in the detection of different peptides and amino acids in other species. Using in silico, in vitro and in vivo approaches, here we compare human CaSR to that of a model carnivore, the domestic cat. We found broad similarities in ligand specificity, but differences in taste sensitivity between the two species. Indeed our in vivo data shows that cats are sensitive to CaCl2 as a kokumi compound, but don't show this same activity with Glutathione, whereas for humans the reverse is true. Collectively, our data suggest that kokumi is an important taste modality for carnivores that drives the palatability of meat-derived compounds such as amino acids and peptides, and that there are differences in the perception of kokumi taste between carnivores and omnivores.

Citrate synthase from Synechocystis is a distinct class of bacterial citrate synthase.

Citrate synthase (CS, EC 2.3.3.1) catalyses the initial reaction of the tricarboxylic acid (TCA) cycle. Although CSs from heterotrophic bacteria have been extensively studied, cyanobacterial CSs are not well-understood. Cyanobacteria can produce various metabolites from carbon dioxide. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a cyanobacterium used to synthesize metabolites through metabolic engineering techniques. The production of acetyl-CoA-derived metabolites in Synechocystis 6803 has been widely examined. However, the biochemical mechanisms of reactions involving acetyl-CoA in Synechocystis 6803 are poorly understood. We characterised the CS from Synechocystis 6803 (SyCS) and compared its characteristics with other bacterial CSs. SyCS catalysed only the generation of citrate, and did not catalyse the cleavage of citrate. It is suggested that SyCS is not related to the reductive TCA cycle. The substrate affinity and turnover number of SyCS were lower than those of CSs from heterotrophic bacteria. SyCS was activated by MgCl2 and CaCl2, which inhibit various bacterial CSs. SyCS was not inhibited by ATP and NADH; which are typical feedback inhibitors of other bacterial CSs. SyCS was inhibited by phosphoenolpyruvate and activated by ADP, which has not been reported for CSs from heterotrophic bacteria. Thus, SyCS showed unique characteristics, particularly its sensitivity to effectors.

One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides.

Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, that is pH 7-8, temperature 25-35°C and co-factor concentrations of 5-20 mM MgCl2 . The maximum reaction rate of GDP-mannose achieved was 2.7 µM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP-mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc)2 -Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc)2 -Man1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP-mannose regenerating cascade and can further be used to study coupling of the GDP-mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell-free production of LLOs as precursors for in vitro glycoengineering of proteins.

Magnesium Chloride promotes Osteogenesis through Notch signaling activation and expansion of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are osteoblasts progenitors and a variety of studies suggest that they may play an important role for the health in the field of bone regeneration. Magnesium supplementation is gaining importance as adjuvant treatment to improve osteogenesis, although the mechanisms involving this process are not well understood. The objective of this study was to investigate the effects of magnesium on MSC differentiation. Here we show that in rat bone marrow MSC, magnesium chloride increases MSC proliferation in a dose-dependent manner promoting osteogenic differentiation and mineralization. These effects are reduced by 2-APB administration, an inhibitor of magnesium channel TRPM7. Of note, magnesium supplementation did not increase the canonical Wnt/ß-catenin pathway, although it promoted the activation of Notch1 signaling, which was also decreased by addition of 2-APB. Electron microscopy showed higher proliferation, organization and maturation of osteoblasts in bone decellularized scaffolds after magnesium addition. In summary, our results demonstrate that magnesium chloride enhances MSC proliferation by Notch1 signaling activation and induces osteogenic differentiation, shedding light on the understanding of the role of magnesium during bone regeneration.

SV40 in vitro packaging: a pseudovirion gene delivery system.

Nuclear extracts of Spodoptera frugiperda (Sf9) insect cells infected with baculoviruses encode the simian virus 40 (SV40) major coat protein (VP1). As described in this protocol, these extracts are able to package supercoiled plasmid DNA or RNA interference (RNAi) sequences in the presence of MgCl(2), CaCl(2), and ATP, thus forming SV40 pseudovirions in vitro. Such packaging has numerous advantages over other viral and nonviral delivery systems. Specifically, it provides a wide host range and high transduction efficiency. The only major disadvantage of this system is low expression per transduced cell observed in vitro, likely a result of DNA trapped in the cytoplasmic compartment of the cell that does not enter the cell nucleus.

Stabilization of ribozyme-like cis-noncoding rRNAs induces apoptotic and nonapoptotic death in lung cells.

Bidirectional non-protein-coding RNAs are ubiquitously transcribed from the genome. Convergent sense and antisense transcripts may regulate each other. Here, we examined the convergent cis-noncoding rRNAs (nc-rRNAs) in A5 and E9 lung cancer models. Sense nc-rRNAs extending from rDNA intergenic region to internal transcribed spacer of around 10 kb in length were identified. nc-rRNAs in sense direction exhibited in vitro characteristics of ribozymes, namely, degradation upon incubation with MgCl(2) and stabilization by complementary oligonucleotides. Detection of endogenous cleavage-ligation products carrying internal deletion of hundreds to thousands nucleotides by massively parallel sequencing confirmed the catalytic properties. Transfection of oligonucleotides pairing with antisense nc-rRNAs stabilized both target and complementary transcripts, perturbed rRNA biogenesis, and induced massive cell death via apoptotic and/or nonapoptotic mechanisms depending on cell type and treatment. Oligonucleotides targeting cellular sense transcripts are less responsive. Spontaneously detached cells, though rare, also showed accumulation of nc-rRNAs and perturbation of rRNA biogenesis. Direct participation of nc-rRNAs in apoptotic and nonapoptotic death was demonstrated by transfection of synthetic nc-rRNAs encompassing the rDNA promoter. In sum, convergent cis-nc-rRNAs follow a feed-forward mechanism to regulate each other and rRNA biogenesis. This opens an opportunity to disrupt rRNA biogenesis, commonly upregulated in cancers, via inhibition of ribozyme-like activities in nc-rRNAs.

Hasllibacter halocynthiae gen. nov., sp. nov., a nutriacholic acid-producing bacterium isolated from the marine ascidian Halocynthia roretzi.

A Gram-negative, aerobic, ovoid to rod-shaped bacterial strain, KME 002(T) was isolated from a marine ascidian, Halocynthia roretzi, off the coast of Gangneung, Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that this strain belonged to the family Rhodobacteraceae in the class Alphaproteobacteria and was closely related to the type strains of Dinoroseobacter shibae, Roseovarius crassostreae and Pseudoruegeria aquimaris with 95.0, 94.7 and 94.5% 16S rRNA gene sequence similarities, respectively. KME 002(T) was an obligately halophilic bacterium requiring 1 to 5% (w/v) NaCl, with an absolute requirement for magnesium chloride for growth. Cells were motile by means of a single polar flagellum and showed budding fission. The predominant cellular fatty acid of the isolate was C(18:1)ω7c and Q-10 was the major ubiquinone. The DNA G+C content of the strain was 71.6 mol%. The major secondary metabolites from cultures in liquid medium were cholic acid derivatives, including 3α,12α-hydroxy-3-keto-glycocholanic acid, 12-hydroxy-3-keto-glycocholanic acid, nutriacholic acid and deoxycholic acid. These characteristics determined in this polyphasic study suggest that strain KME 002(T) represents a novel species in a new genus of the family Rhodobacteraceae. The name Hasllibacter halocynthiae gen. nov., sp. nov. is proposed for this isolate, and the type strain is KME 002(T) (=JCM 16214(T)=KCCM 90082(T)).

Biochemical characterization of an ecto-ATP diphosphohydrolase activity in Candida parapsilosis and its possible role in adenosine acquisition and pathogenesis.

In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.

A novel biochromatographic Oatp2 column to study the transmembrane transport of statins.

In a previous paper, using a biophysical model system to study the passive diffusion of the statin molecules through the cell membrane, our group demonstrated that statins could cross biological membrane by passive diffusion (Sarr et al. [40]). However, in the liver, the uptake of statins would also be mediated by organic anion transporting polypeptides (Oatps) like Oatp2 a member of this family. Thus, a novel biochromatographic approach was developed in our laboratory to study the transmembrane transport of statins and an Oatp2 inhibitor via this carrier family. For this, the Oatp2 protein was immobilized via its amino groups on a chromatographic support using an "in situ" immobilization technique. For the first time, using this novel biochromatographic concept, the effect of magnesium chloride salt (MgCl(2)) on the pharmacomolecule-Oatp2 binding was investigated. It was shown an Mg(2+)-dependent pharmacomolecule-protein association and a potential facilitated diffusion of these pharmacomolecules into biological membrane. This association process was due to the central positive potential pore of the Oatp2. Indeed, at pH 7.4, all the pharmacomolecules studied were ionized (i.e. negatively charged) and so interact with this positive potential pore. However, an increase of the Mg(2+) concentration led a decrease of the pharmacomolecule-Oatp2 association attributed to ion pair formations between the Mg(2+) cation and molecules. Moreover, the decrease of this affinity could be explained by an ion attraction between the Cl(-) anion of the MgCl(2) salt and the positively charged pore of the protein. This novel biochromatographic column could be useful to find a specific reversible inhibitor for these transporters and so open new perspectives to be investigated.

Trapping of a transcription complex using a new nucleotide analogue: AMP aluminium fluoride.

Mechanochemical proteins rely on ATP hydrolysis to establish the different functional states required for their biological output. Studying the transient functional intermediate states these proteins adopt as they progress through the ATP hydrolysis cycle is key to understanding the molecular basis of their mechanism. Many of these intermediates have been successfully 'trapped' and functionally characterised using ATP analogues. Here, we present a new nucleotide analogue, AMP-AlF(x), which traps PspF, a bacterial enhancer binding protein, in a stable complex with the sigma(54)-RNA polymerase holoenzyme. The crystal structure of AMP-AlF(x)*PspF(1-275) provides new information on protein-nucleotide interactions and suggests that the beta and gamma phosphates are more important than the alpha phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AlF(x) establish distinct roles for the conserved catalytic AAA(+) (ATPases associated with various cellular activities) residues, suggesting that AMP-AlF(x) is a powerful new tool to study AAA(+) protein family members and, more generally, Walker motif ATPases.

The role of contact chemoreception in egg-laying behaviour of locusts.

Following selection of an appropriate egg-laying site desert locusts lay their eggs at depths in soil by digging their abdomen into the substrate using rhythmic movements of their abdomen and hard, sclerotised ovipositor valves. We have analysed the role of contact chemoreception on egg-laying behaviour and on the rhythmic digging movements of the valves. All chemicals tested acted aversively and reduced both the duration spent egg-laying and the number of eggs laid, with the concentration at which they became aversive being dependent on whether the chemical was normally present in the diet. Chemicals such as sucrose and a lysine glutamate salt prevented egg-laying only at much higher concentrations than known anti-feedants such as nicotine hydrogen tartrate and hydroquinine. Similarly for animals in which fictive digging movements were induced all chemicals stopped the digging rhythm, with sucrose and sodium chloride inhibiting the rhythm at relatively high concentrations compared to NHT and hydroquinone. We conclude that for both egg-laying behaviour and rhythmic digging that the aversiveness of a chemical rather than its identity per se plays a major role in regulating behaviour.

Methermicoccus shengliensis gen. nov., sp. nov., a thermophilic, methylotrophic methanogen isolated from oil-production water, and proposal of Methermicoccaceae fam. nov.

A thermophilic, methylotrophic methanogen, strain ZC-1(T), was isolated from the Shengli oilfield, China. Cells of strain ZC-1(T) were motile cocci, 0.7-1.0 microm in diameter and always occurred in clusters of two to four cells. Lysis-susceptibility experiments and analysis of transmission electron micrographs of strain ZC-1(T) suggested the presence of a proteinaceous cell wall. Strain ZC-1(T) used methanol, methylamine and trimethylamine as substrates for methanogenesis. Optimal growth, with a doubling time of around 5 h, occurred at pH 6.0-6.5, 65 degrees C, 0.3-0.5 M NaCl and 0.05-0.20 M MgCl(2). The DNA G+C content of this organism was 56 mol%. Analysis of 16S rRNA gene sequence and the inferred amino acid sequence of the mcrA gene of strain ZC-1(T) indicated that it is related specifically to members of the family Methanosaetaceae (90.6 and 76.6 % sequence similarity, respectively). However, strain ZC-1(T) failed to grow with acetate as substrate for methanogenesis, which is a special characteristic of the family Methanosaetaceae. Based on these phenotypic and phylogenic characteristics, strain ZC-1(T) is proposed to represent a novel genus and species, for which the name Methermicoccus shengliensis gen. nov., sp. nov. is proposed. The type strain is ZC-1(T) (=CGMCC 1.5056(T)=DSM 18856(T)). Methermicoccaceae fam. nov. is also proposed.

Altererythrobacter epoxidivorans gen. nov., sp. nov., an epoxide hydrolase-active, mesophilic marine bacterium isolated from cold-seep sediment, and reclassification of Erythrobacter luteolus Yoon et al. 2005 as Altererythrobacter luteolus comb. nov.

A novel marine bacterium, strain JCS350(T), was isolated from marine sediment samples collected from a cold-seep area. The 16S rRNA gene sequence of the isolate showed high similarity to that of Erythrobacter luteolus SW-109(T) (95.9 % sequence similarity). Lower 16S rRNA gene sequence similarities were shown to other members of the genus Erythrobacter (94.6-95.4 %) and members of the genus Porphyrobacter (94.5-95.2 %). Phylogenetic analysis with all members of the family Erythrobacteraceae and several members of the family Sphingomonadaceae revealed that the isolate formed a phyletic line with [Erythrobacter] luteolus that was distinct from other members of the family Erythrobacteraceae. The dominant fatty acids of strain JCS350(T) were 18 : 1omega7c, 16 : 1omega7c and cyclopropane 17 : 0. The major respiratory quinone was ubiquinone 10. The DNA G+C content was 54.5 mol%. The isolate did not contain bacteriochlorophyll a. Optimal growth required the presence of 2 % (w/v) NaCl with either 0.18 % CaCl(2) or 0.59 % MgCl(2), at pH 6.5 and at 35 degrees C. On the basis of the evidence of this polyphasic taxonomic study, strain JCS350(T) should be classified in a novel genus and species in the family Erythrobacteraceae, for which the name Altererythrobacter epoxidivorans gen. nov., sp. nov. is proposed. The misclassified species [Erythrobacter] luteolus is transferred to the new genus as Altererythrobacter luteolus comb. nov. The type strain of Altererythrobacter epoxidivorans is JCS350(T) (=KCCM 42314(T) =JCM 13815(T)) and the type strain of Altererythrobacter luteolus is SW-109(T) (=KCTC 12311(T) =JCM 12599(T)).

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration.

Previous studies have shown that inhibition of L-type Ca(2+) current (I(Ca)) by cytosolic free Mg(2+) concentration ([Mg(2+)](i)) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I(Ca), rat cardiac myocytes and tsA201 cells expressing L-type Ca(2+) channels were whole cell voltage-clamped with patch pipettes in which [Mg(2+)] ([Mg(2+)](p)) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca(2+) channels (alpha(1C)/beta(2A)/alpha(2)delta), increasing [Mg(2+)](p) from 0.2 mM to 1.8 mM decreased peak I(Ca) by 76 +/- 4.5% (n = 7). Mg(2+)-dependent modulation of I(Ca) was also observed in cells loaded with ATP-gamma-S. With 0.2 mM [Mg(2+)](p), manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP(2A)) produced large changes in I(Ca) amplitude; however, with 1.8 mM [Mg(2+)](p), these same manipulations had no significant effect on I(Ca). With mutant channels lacking principal PKA phosphorylation sites (alpha(1C/S1928A)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) had only small effects on I(Ca). However, when channel open probability was increased by alpha(1C)-subunit truncation (alpha(1CDelta1905)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) greatly reduced peak I(Ca). Correspondingly, in myocytes voltage-clamped with pipette PP(2A) to minimize channel phosphorylation, increasing [Mg(2+)](p) produced a much larger reduction in I(Ca) when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg(2+) modulates the extent to which channel phosphorylation regulates I(Ca). This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation.

Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride.

Inhibition of ATPase activity of Escherichia coli ATP synthase by magnesium fluoride (MgFx) was studied. Wild-type F(1)-ATPase was inhibited potently, albeit slowly, when incubated with MgCl(2), NaF, and NaADP. The combination of all three components was required. Reactivation of ATPase activity, after removal of unbound ligands, occurred with half-time of approximately 14 h at 22 degrees C and was quasi-irreversible at 4 degrees C. Mutant F(1)-ATPases, in which catalytic site residues involved in transition state formation were modified, were found to be resistant to inhibition by MgFx. The data demonstrate that MgFx in combination with MgADP behaves as a tight-binding transition state analog in E. coli ATP synthase.

Insulin signaling meets vesicle traffic of GLUT4 at a plasma-membrane-activated fusion step.

A hypothesis that accounts for most of the available literature on insulin-stimulated GLUT4 translocation is that insulin action controls the access of GLUT4 vesicles to a constitutively active plasma-membrane fusion process. However, using an in vitro fusion assay, we show here that fusion is not constitutively active. Instead, the rate of fusion activity is stimulated 8-fold by insulin. Both the magnitude and time course of stimulated in vitro fusion recapitulate the cellular insulin response. Fusion is cell cytoplasm and SNARE dependent but does not require cell cytoskeleton. Furthermore, insulin activation of the plasma-membrane fraction of the fusion reaction is the essential step in regulation. Akt from the cytoplasm fraction is required for fusion. However, the participation of Akt in the stimulation of in vitro fusion is dependent on its in vitro recruitment onto the insulin-activated plasma membrane.

Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A.

Polyphosphate:AMP phosphotransferase (PAP) has been identified as an enzyme that catalyzes the phosphorylation of AMP with inorganic polyphosphates [poly(P)] as phosphate donors. We found that the purified PAP of Acinetobacter johnsonii 210A has poly(P) synthetic activity. The PAP catalyzes the dephosphorylation of ADP and processively synthesizes poly(P) of 200 to 700 residues. Comparatively lower concentrations of MgCl(2) (20 mM) were required to obtain optimum poly(P) synthetic activity, whereas higher concentrations of MgCl(2) (100 mM) were necessary for optimum PAP activity. ADP is preferred over GDP as a phosphate donor for poly(P) synthesis. The K(m) and V(max) values for ADP in the poly(P) synthetic activity of PAP were 8.3 mM and 55 micromol min(-1) mg(-1), respectively. We concluded that the PAP of A. johnsonii 210A is a novel type of poly(P) kinase that uses ADP and GDP as substrates.

Expression, purification, and biochemical characterization of Chk, a soluble protein tyrosine kinase.

CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.

MIC channels are inhibited by internal divalent cations but not ATP.

TRPM7 channels are nonselective cation channels that possess a functional alpha-kinase domain. It has been proposed that heterologously expressed TRPM7 channels are activated (Runnels et al., 2001) or inhibited (Nadler et al., 2001) by dialyzing the cell with millimolar levels of ATP. The endogenous correlate of TRPM7 has been identified in T-lymphocytes and RBL (rat basophilic leukemia) cells and named MagNuM (for Mg(2+)-nucleotide-inhibited metal) or MIC (for Mg(2+)-inhibited cation). Here, we report that internal Mg(2+) rather than MgATP inhibits this current. Cytoplasmic MgATP, supplied by dialysis at millimolar concentrations, effectively inhibits only when a weak Mg(2+) chelator is present in the pipette solution. Thus, MgATP acts as a source of Mg(2+) rather than a source of ATP. Using an externally accessible site within the pore of the MIC channel itself as a bioassay, we show that equimolar MgCl(2) and MgATP solutions contain similar amounts of free Mg(2+), explaining the fact that numeric values of Mg(2+) and MgATP concentrations necessary for complete inhibition are the same. Furthermore, we demonstrate that Mg(2+) is not unique in its inhibitory action, as Ba(2+), Sr(2+), Zn(2+), and Mn(2+) can substitute for Mg(2+), causing complete inhibition. We conclude that MIC current inhibition occurs simply by divalent cations.

Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors.

A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).