Regulation of vascular smooth muscle tone by N-terminal region of caldesmon. Possible role of tethering actin to myosin.
AuthorsLee YH, Gallant C, Guo H, Li Y, Wang CA, Morgan KG
JournalJ Biol Chem
PubMed ID10652307
'To assess the functional significance of tethering actin to myosin by caldesmon in the regulation of smooth muscle contraction, we investigated the effects of synthetic peptides, containing the myosin-binding sequences in the N-terminal region of caldesmon, on force directly recorded from single permeabilized smooth muscle cells of ferret portal vein. ... More
Reduction precedes cytidylyl transfer without substrate channeling in distinct active sites of the bifunctional CDP-ribitol synthase from Haemophilus influenzae.
AuthorsZolli M, Kobric DJ, Brown ED
JournalBiochemistry
PubMed ID11305920
'CDP-ribitol synthase is a bifunctional reductase and cytidylyltransferase that catalyzes the transformation of D-ribulose 5-phosphate, NADPH, and CTP to CDP-ribitol, a repeating unit present in the virulence-associated polysaccharide capsules of Haemophilus influenzae types a and b [Follens, A., et al. (1999) J. Bacteriol. 181, 2001]. In the work described here, ... More
The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins.
AuthorsZhang B, Zhang Y, Wang Z, Zheng Y
JournalJ Biol Chem
PubMed ID10843989
'The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which ... More
The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding.
'Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to ... More
The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates.
AuthorsEhmann DE, Shaw-Reid CA, Losey HC, Walsh CT
JournalProc Natl Acad Sci U S A
PubMed ID10688898
'Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS). In this work, the 142-kDa four-domain protein EntF was bisected into two double-domain fragments: a 108-kDa condensation and adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein (PCP) ... More
Changes in gene expression profiles in developing B cells of murine bone marrow.
AuthorsHoffmann R, Seidl T, Neeb M, Rolink A, Melchers F
JournalGenome Res
PubMed ID11779835
'Gene expression profiles of five consecutive stages of mouse B cell development were generated with high-density oligonucleotide arrays from as few as 2 x 10(4) ex vivo isolated and flow-cytometrically purified cells. Between 2.8% and 6.8% of all genes change on differentiation from one cellular stage to the next by ... More
Assembly of tubulin by classic myelin basic protein isoforms and regulation by post-translational modification.
AuthorsHill CM, Libich DS, Harauz G
JournalBiochemistry
PubMed ID16342957
'Myelin basic protein (MBP), a highly cationic protein that maintains the structure of the myelin sheath, associates with tubulin in vivo. The in vitro assembly of tubulin by MBP was examined here using several assays. The unmodified C1 component of 18.5 kDa bovine MBP (bC1) assembled tubulin into microtubules in ... More
Effects of phosphorylation and mutation R145G on human cardiac troponin I function.
AuthorsDeng Y, Schmidtmann A, Redlich A, Westerdorf B, Jaquet K, Thieleczek R
JournalBiochemistry
PubMed ID11724573
'We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin ... More
Substrate conformational restriction and CD45-catalyzed dephosphorylation of tail tyrosine-phosphorylated Src protein.
AuthorsWang D, Esselman WJ, Cole PA
JournalJ Biol Chem
PubMed ID12181320
'Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83-533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565-1268), we have analyzed the kinetic behavior of dephosphorylation. A ... More
Characterization of a vacuolar pyrophosphatase in Trypanosoma brucei and its localization to acidocalcisomes.
AuthorsRodrigues CO, Scott DA, Docampo R
JournalMol Cell Biol
PubMed ID10523660
'Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH(4)Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the ... More
The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth.
'The ability of Legionella pneumophila to grow and cause disease in the host is completely dependent on a type IV secretion system known as the Dot/Icm complex. This membrane-spanning apparatus translocates effector molecules into host cells in a process that is poorly understood but that is known to require the ... More
Kinetics of the phosphorylation of Na,K-ATPase by inorganic phosphate detected by a fluorescence method.
AuthorsApell HJ, Roudna M, Corrie JE, Trentham DR
JournalBiochemistry
PubMed ID8718885
'Phosphorylation by Pi of the Na,K-ATPase from rabbit kidney in the absence of Na+ ions but in the presence of Mg2+ ions has been studied. In the absence of K+ ions, unphosphorylated and phosphorylated states induce different fluorescence levels in the membrane-bound styryl dye RH421, and hence transitions between the ... More
Iodide and bromide inhibit Ca(2+) uptake by cardiac sarcoplasmic reticulum.
AuthorsKargacin GJ, Ali Z, Zhang SJ, Pollock NS, Kargacin ME
JournalAm J Physiol Heart Circ Physiol
PubMed ID11247773
'Recent studies indicate that the Ca(2+) permeability of the sarcoplasmic reticulum (SR) can be affected by its anionic environment. Additionally, anions could directly modulate the SR Ca(2+) pump or the movement of compensatory charge across the SR membrane during Ca(2+) uptake or release. To examine the effect of anion substitution ... More
Influence of divalent cations on nucleotide exchange and ATPase activity of chloroplast coupling factor 1.
AuthorsDigel JG, Moore ND, McCarty RE
JournalBiochemistry
PubMed ID9860834
'The ATPase activity of the catalytic part of ATP synthases is inhibited by free Mg2+, even though MgATP is the substrate. Here we show that the inhibition of the MgATPase activity of chloroplast coupling factor 1 deficient in its epsilon subunit (CF1-epsilon) by Mg2+ is complex. The hydrolysis of MgATP ... More
Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI-BchD complex.
AuthorsGibson LC, Jensen PE, Hunter CN
JournalBiochem J
PubMed ID9882621
'The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchD and bchH genes. These genes have been expressed in Escherichia coli ... More
ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex.
AuthorsBienengraeber M, Alekseev AE, Abraham MR, Carrasco AJ, Moreau C, Vivaudou M, Dzeja PP, Terzic A
JournalFASEB J
PubMed ID11023978
'ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and ... More
Kinetics of myosin subfragment-1-induced condensation of G-actin into oligomers, precursors in the assembly of F-actin-S1. Role of the tightly bound metal ion and ATP hydrolysis.
AuthorsFievez S, Pantaloni D, Carlier MF
JournalBiochemistry
PubMed ID9305975
'In a low ionic strength buffer and in the absence of free ATP, the interaction of G-actin (G) with myosin subfragment-1 (S1) leads to the formation of arrowhead-decorated F-actin-S1 filaments, through a series of elementary steps. The initial formation of GS and G2S complexes is followed by their condensation into ... More
Studies in humans on the mechanism of potent spermicidal and apoptosis-inducing activities of vanadocene complexes.
AuthorsD'Cruz OJ, Vassilev A, Uckun FM
JournalBiol Reprod
PubMed ID10727263
'We previously demonstrated that bis-cyclopentadienyl (Cp) complexes of vanadium(IV) (vanadocenes) are potent spermicidal and apoptosis-inducing agents. To gain further insight into the structure-function relationships controlling these two properties of vanadocenes, we have synthesized analogues in which the bis-Cp rings were substituted with one or five electron-donating methyl groups. The three ... More
Allosteric regulation of SecA: magnesium-mediated control of conformation and activity.
AuthorsGold VA, Robson A, Clarke AR, Collinson I
JournalJ Biol Chem
PubMed ID17416585
'In bacteria, the SecA protein associates with a ubiquitous protein channel SecYEG where it drives the post-translational secretion of pre-proteins across the plasma membrane. The high-resolution structures of both proteins have been determined in their resting states; however, the mechanism that couples ATP hydrolysis to active transport of substrate proteins ... More
A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems.
AuthorsWebb MR
JournalProc Natl Acad Sci U S A
PubMed ID1534409
'A spectrophotometric method for the measurement of inorganic phosphate (P(i)) has been developed by using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This substrate gives an absorbance increase at 360 nm on phosphorolysis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-1.cm-1. ... More
Dual roles of Gln137 of actin revealed by recombinant human cardiac muscle alpha-actin mutants.
AuthorsIwasa M, Maeda K, Narita A, Maéda Y, Oda T,
JournalJ Biol Chem
PubMed ID18515362
'The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln(137) to understand the relationships between two activities: the conformational changes relevant to the G- to F-actin ... More
The Escherichia coli fadK (ydiD) gene encodes an anerobically regulated short chain acyl-CoA synthetase.
AuthorsMorgan-Kiss RM, Cronan JE
JournalJ Biol Chem
PubMed ID15213221
'We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources. This beta-oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX ... More
Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. coli: ligand-dependent quenching of enzyme intrinsic fluorescence.
AuthorsKierdaszuk B, Modrak-Wójcik A, Shugar D,
JournalBiophys Chem
PubMed ID9108686
'Steady-state and time-resolved emission spectroscopy was applied to a study of the binary and ternary complexes of pure E. coli purine nucleoside phosphorylase (PNP) with phosphate (Pi; a substrate) and a close non-substrate analogue (sulfate; SA). The quenching of enzyme fluorescence by Pi was bimodal, best described by two modified ... More
Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release.
AuthorsWebb MR, Hunter JL
JournalBiochem J
PubMed ID1445214
'The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) ... More
Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase.
AuthorsSazinsky MH, Mandal AK, Argüello JM, Rosenzweig AC
JournalJ Biol Chem
PubMed ID16495228
'The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The ... More
Oligomerization of Rac1 gtpase mediated by the carboxyl-terminal polybasic domain.
AuthorsZhang B, Gao Y, Moon SY, Zhang Y, Zheng Y
JournalJ Biol Chem
PubMed ID11134022
'The Rho family GTPase Rac1 mediates a variety of signal transduction processes leading to activation of NADPH oxidase, actin cytoskeleton reorganization, transcription activation, and stimulation of DNA synthesis. In this study, Rac1 was found to form a reversible monomer and oligomer in both the GDP- and GTP-bound states in vitro ... More
GTP-yeast actin.
AuthorsWen KK, Yao X, Rubenstein PA
JournalJ Biol Chem
PubMed ID12191996
'Because of the apparently greater conformational flexibility of yeast versus muscle actin and the ability of other members in the actin protein superfamily to efficiently use both ATP and GTP, we assessed the ability of yeast actin to function with GTP. Etheno-ATP exchange studies showed that the binding of GTP ... More
High-molecular-weight polypeptide substrates for phospholysine phosphatases.
The detection and characterization of the phosphatases responsible for the dephosphorylation of N-phosphoryl groups, such as phospholysine, in proteins has been frustrated in large measure by a lack of suitable substrates for the assay of these enzymes. Herein we describe the preparation of phospholysine-containing amino acid homo- and heteropolymers by ... More
Phosphohistidine and phospholysine phosphatase activities in the rat: potential protein-lysine and protein-histidine phosphatases?
AuthorsWong C, Faiola B, Wu W, Kennelly PJ
JournalBiochem J
PubMed ID8257415
We have detected phosphohistidine and phospholysine phosphatase activities in rat tissue extracts using partially phosphorylated, high-molecular-mass (> 10 kDa) polymers of histidine and lysine as substrates. Multiple phosphohistidine- and phospholysine-specific phosphatases were present in these extracts based on observed differences in heat stability, sensitivity to bivalent metal ions and thiol ... More
Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors.
AuthorsTan YC, Wu H, Wang WN, Zheng Y, Wang ZX
JournalAnal Biochem
PubMed ID12423633
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 ... More
Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis.
AuthorsOberdorfer G, Binter A, Ginj C, Macheroux P, Gruber K,
JournalJ Biol Chem
PubMed ID22810238
Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, ... More
Photometric microtiter assay of inorganic phosphate in the presence of acid-labile organic phosphates.
AuthorsDrueckes P, Schinzel R, Palm D
JournalAnal Biochem
PubMed ID8585615
A simple and rapid colorimetric microassay for inorganic phosphate in the mild acid pH range has enabled us to perform extensive enzyme kinetic studies even in the presence of high concentrations of acid-labile substrates. The assay is performed in a 96-well microtiter plate using 30-microliters samples containing between 0.5 and ... More
Orphan kinesin NOD lacks motile properties but does possess a microtubule-stimulated ATPase activity.
AuthorsMatthies HJ, Baskin RJ, Hawley RS
JournalMol Biol Cell
PubMed ID11739796
NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NOD(DTW) and ... More
A continuous spectrophotometric assay for phosphorylase kinase.
AuthorsWang ZX, Cheng Q, Killilea SD
JournalAnal Biochem
PubMed ID8585630
A continuous spectrophotometric assay for the determination of the initial rate of the phosphorylase kinase catalyzed reaction at pH 7.0 is presented. The assay incorporates two coupling enzyme systems: (a) recombinant rabbit skeletal muscle type 1 protein phosphatase catalytic subunit which dephosphorylates the phosphorylase a product of the phosphorylase kinase ... More
A continuous spectrophotometric assay for protein phosphatases.
AuthorsCheng Q, Wang ZX, Killilea SD
JournalAnal Biochem
PubMed ID7785781
A continuous spectrophotometric assay for the determination of protein phosphatase activity is presented. The assay incorporates the coupled enzyme system of Webb (M. R. Webb, 1992, Proc. Natl. Acad. Sci. USA 89, 4884-4887), which used purine nucleoside phosphorylase and the chromophoric substrate 7-methyl-6-thioguanosine for the quantitation of inorganic phosphate. The ... More
The p23 molecular chaperone promotes functional telomerase complexes through DNA dissociation.
AuthorsToogun OA, Zeiger W, Freeman BC
JournalProc Natl Acad Sci U S A
PubMed ID17389357
Telomeres are the composite of short DNA element tandem arrays and heterotypic protein components that protect and maintain chromosomal termini. As proper telomere maintenance requires a multitude of DNA extension events, it is important to understand the factors that modulate telomerase DNA association. Here, we show that the endogenous levels ... More
Rate-limiting guanosine 5'-triphosphate hydrolysis during nucleotide turnover by FtsZ, a prokaryotic tubulin homologue involved in bacterial cell division.
AuthorsRomberg L, Mitchison TJ
JournalBiochemistry
PubMed ID14705956
FtsZ is a prokaryotic tubulin homologue that polymerizes into a dynamic ring during cell division. GTP binding and hydrolysis provide the energy for FtsZ dynamics. However, the precise role of hydrolysis in polymer assembly and turnover is not understood, limiting our understanding of how FtsZ functions in the cell. Here ... More
The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump.
AuthorsWalmsley AR, Zhou T, Borges-Walmsley MI, Rosen BP
JournalJ Biol Chem
PubMed ID10347168
The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis ... More
A kinetic model for the action of a resistance efflux pump.
AuthorsWalmsley AR, Zhou T, Borges-Walmsley MI, Rosen BP
JournalJ Biol Chem
PubMed ID11096086
ArsA is the catalytic subunit of the arsenical pump, coupling ATP hydrolysis to the efflux of arsenicals through the ArsB membrane protein. It is a paradigm for understanding the structure-function of the nucleotide binding domains (NBD) of medically important efflux pumps, such as P-glycoprotein, because it has two sequence-related, interacting ... More
A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd.
Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors ... More
Multiple ATP-hydrolyzing sites that potentially function in cytoplasmic dynein.
AuthorsTakahashi Y, Edamatsu M, Toyoshima YY
JournalProc Natl Acad Sci U S A
PubMed ID15326307
Cytoplasmic dynein is a minus-end-directed microtubule motor involved in numerous essential processes within eukaryotic cells, such as nuclear segregation and trafficking of intracellular particles. The motor domain of the dynein heavy chain comprises six tandemly linked AAA (ATPase associated with diverse cellular activities) modules (AAA1-AAA6). The first four modules include ... More
Structure of the BH domain from graf and its implications for Rho GTPase recognition.
AuthorsLongenecker KL, Zhang B, Derewenda U, Sheffield PJ, Dauter Z, Parsons JT, Zheng Y, Derewenda ZS
JournalJ Biol Chem
PubMed ID10982819
Cellular signaling by small G-proteins is down-regulated by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis. The GTPase regulator associated with focal adhesion kinase (Graf) exhibits GAP activity toward the RhoA and Cdc42 GTPases, but is only weakly active toward the closely related Rac1. We determined the crystal ... More
Formins regulate actin filament flexibility through long range allosteric interactions.
AuthorsBugyi B, Papp G, Hild G, Lõrinczy D, Nevalainen EM, Lappalainen P, Somogyi B, Nyitrai M
JournalJ Biol Chem
PubMed ID16490788
The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence ... More
A continuous spectrophotometric method for the determination of glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis.
AuthorsSergienko EA, Srivastava DK
JournalAnal Biochem
PubMed ID7810877
We offer a "continuous" spectrophotometric method for the determination of the glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis. This method relies on a coupled enzyme procedure, involving purine nucleoside phosphorylase and its chromophoric substrate, 2-amino-6-mercapto-7-methyl ribonucleoside (7-methyl-6-thioguanosine (MTGuo)), for the estimation of inorganic phosphate (M. R. Webb, Proc. ... More
The rate of activation by calmodulin of isoform 4 of the plasma membrane Ca(2+) pump is slow and is changed by alternative splicing.
AuthorsCaride AJ, Elwess NL, Verma AK, Filoteo AG, Enyedi A, Bajzer Z, Penniston JT
JournalJ Biol Chem
PubMed ID10575008
A reconstitution system allowed us to measure the ATPase activity of specific isoforms of the plasma membrane Ca(2+) pump continuously, and to measure the effects of adding or removing calmodulin. The rate of activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 ... More
Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation.
AuthorsZhang B, Zheng Y
JournalJ Biol Chem
PubMed ID9748241
The Rho family GTPases are tightly regulated between the active GTP-bound state and the inactive GDP-bound state in a variety of signal transduction processes. Here the Rho family members Cdc42, Rac2, and RhoA were found to form reversible homodimers in both the GTP- and the GDP-bound states. The homophilic interaction ... More
Possible involvement of hyphal phosphatase in phosphate efflux from intraradical hyphae isolated from mycorrhizal roots colonized by Gigaspora margarita.
AuthorsKojima T, Saito M
JournalMycol Res
PubMed ID15323242
We developed a method for separating physiologically active intraradical hyphae of arbuscular mycorrhizal (AM) fungi from mycorrhizal roots, allowing the hyphae to be used for physiological and biochemical experiments. In the present study, the phosphate efflux from the intraradical hyphae in vitro was examined in relation to hyphal phosphatase activity. ... More
F-actin-like ATPase activity in a polymerization-defective mutant yeast actin (V266G/L267G).
AuthorsYao X, Rubenstein PA
JournalJ Biol Chem
PubMed ID11328808
Polymerization increases a low level G-actin ATPase activity yielding ADP-P(i) F-actin and then ADP F-actin following release of P(i). By monitoring P(i) release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG. In this mutant, two hydrophobic residues at the tip of ... More
Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin.
The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were ... More
INF2 Is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization.
AuthorsChhabra ES, Higgs HN
JournalJ Biol Chem
PubMed ID16818491
Formin proteins modulate both nucleation and elongation of actin filaments through processive movement of their dimeric formin homology 2 (FH2) domains with filament barbed ends. Mammals possess at least 15 formin genes. A subset of formins termed "diaphanous formins" are regulated by autoinhibition through interaction between an N-terminal diaphanous inhibitory ... More
Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b.
AuthorsKatalin Pászty, Alan R. Penheiter§, Anil K. Verma§, Rita Padányi¶, Adelaida G. Filoteo§, John T. Penniston§, and Ágnes Enyedi
JournalJ Biol Chem
PubMed ID12145294
The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform ... More
Impact of profilin on actin-bound nucleotide exchange and actin polymerization dynamics.
AuthorsSelden LA, Kinosian HJ, Estes JE, Gershman LC
JournalBiochemistry
PubMed ID10052948
We have investigated the effects of profilin on nucleotide binding to actin and on steady state actin polymerization. The rate constants for the dissociation of ATP and ADP from monomeric Mg-actin at physiological conditions are 0.003 and 0.009 s-1, respectively. Profilin increases these dissociation rate constants to 0.08 s-1 for ... More
Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b.
AuthorsPenheiter AR, Caride AJ, Enyedi A, Penniston JT
JournalJ Biol Chem
PubMed ID11886854
Tryptophan 1093 resides in the 28-residue calmodulin-binding/autoinhibitory domain of the plasma membrane Ca(2+) pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. ... More
A continuous spectrophotometric assay for aspartate transcarbamylase and ATPases.
AuthorsRieger CE, Lee J, Turnbull JL
JournalAnal Biochem
PubMed ID9056187
A new continuous coupled uv-spectrophotometric assay is described for two phosphate-releasing enzymes, aspartate transcarbamylase and ATPase of herpes simplex virus (HSV). Phosphate release is coupled to the phosphorolysis of the nucleoside analog 7-methylinosine (m7Ino) catalyzed by purine nucleoside phosphorylase. When this reaction is monitored at 291 nm, the coupled assay ... More
Kinetics of inorganic phosphate release during the interaction of p21ras with the GTPase-activating proteins, p120-GAP and neurofibromin.
AuthorsNixon AE, Brune M, Lowe PN, Webb MR
JournalBiochemistry
PubMed ID7492562
The rate of GTP hydrolysis on p21ras is accelerated by approximately 10(5) times by the catalytic domains of GTPase-activating proteins (GAPs), p120-GAP (GAP-344) or neurofibromin (NF1-334). The kinetic mechanism of this activation has been investigated by following the release of inorganic phosphate (Pi), using a fluorescent probe that is sensitive ... More
Cyclophilin residues that affect noncompetitive inhibition of the protein serine phosphatase activity of calcineurin by the cyclophilin.cyclosporin A complex.
AuthorsEtzkorn FA, Chang ZY, Stolz LA, Walsh CT
JournalBiochemistry
PubMed ID8117697
Mutation of three cationic surface residues of human cyclophilin A (hCyPA), R69, K125, and R148, to both anionic and neutral residues left its intrinsic peptidyl-prolyl isomerase (PPIase) activity and cyclosporin A (CsA) binding unaffected, but altered its ability to inhibit the serine phosphatase activity of calcineurin (CN). R69E was 13-fold ... More
Characterization of isolated acidocalcisomes of Trypanosoma cruzi.
AuthorsScott DA, Docampo R
JournalJ Biol Chem
PubMed ID10816577
The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H(+)-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least ... More
A fusion protein between rac and p67phox (1-210) reconstitutes NADPH oxidase with higher activity and stability than the individual components.
AuthorsMiyano K, Ogasawara S, Han CH, Fukuda H, Tamura M
JournalBiochemistry
PubMed ID11705402
Activation of the phagocyte NADPH oxidase, a superoxide-generating enzyme, involves assembly of cytosolic p47(phox), p67(phox), and rac with the membrane-associated cytochrome b(558). Following cell-free activation, enzymatic activity is highly labile [Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529-7538]. ... More
Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate.
AuthorsGibson KJ, Lorimer GH, Rendina AR, Taylor WS, Cohen G, Gatenby AA, Payne WG, Roe DC, Lockett BA, Nudelman A
JournalBiochemistry
PubMed ID7669755
Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanoic acid (7,8-diaminopelargonic acid, DAPA), CO2, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO2. However, four lines ... More
Uncoupling conformational change from GTP hydrolysis in a heterotrimeric G protein alpha-subunit.
AuthorsThomas CJ, Du X, Li P, Wang Y, Ross EM, Sprang SR
JournalProc Natl Acad Sci U S A
PubMed ID15128951
Heterotrimeric G protein alpha (G alpha) subunits possess intrinsic GTPase activity that leads to functional deactivation with a rate constant of approximately 2 min(-1) at 30 degrees C. GTP hydrolysis causes conformational changes in three regions of G alpha, including Switch I and Switch II. Mutation of G202-->A in Switch ... More
Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay.
AuthorsMelki R, Fievez S, Carlier MF
JournalBiochemistry
PubMed ID8810908
ATP and GTP are hydrolyzed during self-assembly of actin and tubulin, respectively. It is known that nucleotide is hydrolyzed on the polymer in two consecutive steps, chemical cleavage of the gamma-phosphate followed by the slower release of Pi. This last step has been shown to play a crucial role in ... More
Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.
AuthorsBrune M, Hunter JL, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID8031761
A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant ... More
A continuous visible spectrophotometric assay for aspartate transcarbamylase.
AuthorsWedler FC, Ley BW, Moyer ML
JournalAnal Biochem
PubMed ID8074305
A continuous spectrophotometric method for assaying ATCase activity has been devised that couples the production of inorganic phosphate from the ATCase-catalyzed reaction to the phosphorolysis reaction catalyzed by purine nucleoside phosphorylase, using a chromophoric nucleotide analogue, methylthioguanosine (MESG). This latter reaction results in a change in extinction coefficient of 11,000 ... More
Alteration of a nonconserved active site residue in the chemotaxis response regulator CheY affects phosphorylation and interaction with CheZ.
AuthorsSilversmith RE, Smith JG, Guanga GP, Les JT, Bourret RB
JournalJ Biol Chem
PubMed ID11278903
CheY is a response regulator in the well studied two-component system that mediates bacterial chemotaxis. Phosphorylation of CheY at Asp(57) enhances its interaction with the flagellar motor. Asn(59) is located near the phosphorylation site, and possible roles this residue may play in CheY function were explored by mutagenesis. Cells containing ... More
The plasma membrane calcium pump displays memory of past calcium spikes. Differences between isoforms 2b and 4b.
AuthorsCaride AJ, Penheiter AR, Filoteo AG, Bajzer Z, Enyedi A, Penniston JT
JournalJ Biol Chem
PubMed ID11514555
To understand how the plasma membrane Ca(2+) pump (PMCA) behaves under changing Ca(2+) concentrations, it is necessary to obtain information about the Ca(2+) dependence of the rate constants for calmodulin activation (k(act)) and for inactivation by calmodulin removal (k(inact)). Here we studied these constants for isoforms 2b and 4b. We ... More
Kinetic characterization of enzyme forms involved in metal ion activation and inhibition of myo-inositol monophosphatase.
AuthorsStrasser F, Pelton PD, Ganzhorn AJ
JournalBiochem J
PubMed ID7733900
Activation and inhibition of recombinant bovine myo-inositol monophosphatase by metal ions was studied with two substrates, D,L-inositol 1-phosphate and 4-nitrophenyl phosphate. Mg2+ and Co2+ are essential activators of both reactions. At high concentrations, they inhibit hydrolysis of inositol 1-phosphate, but not 4-nitrophenyl phosphate. Mg2+ is highly selective for inositol 1-phosphate ... More
Characterization of the interactions between the small GTPase Cdc42 and its GTPase-activating proteins and putative effectors. Comparison of kinetic properties of Cdc42 binding to the Cdc42-interactive domains.
AuthorsZhang B, Wang ZX, Zheng Y
JournalJ Biol Chem
PubMed ID9268338
The small GTPase Cdc42 interacts with multiple factors to transduce diverse intracellular signals. The factors that preferentially recognize the GTP-bound, active state of Cdc42 include a panel of GTPase-activating proteins (GAPs), the Cdc42/Rac interactive binding (CRIB) motif-containing molecules, and the RasGAP domain containing IQGAP1 and IQGAP2. In the present study, ... More
The distinct functional properties of the nucleotide-binding domain of ATP7B, the human copper-transporting ATPase: analysis of the Wilson disease mutations E1064A, H1069Q, R1151H, and C1104F.
AuthorsMorgan CT, Tsivkovskii R, Kosinsky YA, Efremov RG, Lutsenko S
JournalJ Biol Chem
PubMed ID15205462
Copper transport by the P(1)-ATPase ATP7B, or Wilson disease protein (WNDP),1 is essential for human metabolism. Perturbation of WNDP function causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the WNDP nucleotide-binding domain (N-domain), where they are predicted to disrupt ATP ... More
Interaction of Rac1 with GTPase-activating proteins and putative effectors. A comparison with Cdc42 and RhoA.
AuthorsZhang B, Chernoff J, Zheng Y
JournalJ Biol Chem
PubMed ID9535855
The intrinsic GTPase activity of the Rho family GTP-binding protein Rac1 is drastically stimulated upon interaction with its GTPase-activating proteins (GAPs) and is significantly inhibited when coupled to certain effector targets such as the p21-activated kinases (PAKs) and IQGAPs. Here we have characterized the interaction of Rac1 with a panel ... More
The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases.
AuthorsZhou B, Wang ZX, Zhao Y, Brautigan DL, Zhang ZY
JournalJ Biol Chem
PubMed ID12082107
The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and ... More
Reaction mechanism of pyridoxal 5'-phosphate synthase. Detection of an enzyme-bound chromophoric intermediate.
Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 ... More
Characterization of isolated acidocalcisomes from Toxoplasma gondii tachyzoites reveals a novel pool of hydrolyzable polyphosphate.
AuthorsRodrigues CO, Ruiz FA, Rohloff P, Scott DA, Moreno SN
JournalJ Biol Chem
PubMed ID12379647
Toxoplasma gondii tachyzoites were fractionated by modification of an iodixanol density gradient method previously used for acidocalcisome isolation from Trypanosoma cruzi epimastigotes. Fractions were characterized using electron microscopy, x-ray microanalysis, and enzymatic markers, and it was demonstrated that the heaviest (pellet) fraction contains electron-dense vacuoles rich in phosphorus, calcium, and ... More
ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits.
AuthorsJensen PE, Gibson LC, Hunter CN
JournalBiochem J
PubMed ID10085236
Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation. The activation step requires ATP and two of the subunits, ChlI and ChlD (I and D respectively), and it has ... More
Structural determinants required for the interaction between Rho GTPase and the GTPase-activating domain of p190.
AuthorsLi R, Zhang B, Zheng Y
JournalJ Biol Chem
PubMed ID9407060
The Rho family small GTP-binding proteins are subjected to regulation by Rho GTPase-activating proteins (GAPs) in the course of transmitting diverse intracellular signals. To understand the mechanism of GAP-catalyzed GTP hydrolysis of Rho GTPases, we have studied the interaction between RhoA and p190, the RasGAP binding phosphoprotein which has been ... More
Regulation of RhoA GTP hydrolysis by the GTPase-activating proteins p190, p50RhoGAP, Bcr, and 3BP-1.
AuthorsZhang B, Zheng Y
JournalBiochemistry
PubMed ID9548756
The small GTP-binding protein RhoA becomes inactivated by hydrolyzing bound GTP to GDP through its intrinsic GTPase activity which is further stimulated by a family of Rho GTPase-activating proteins (GAPs). Here we have compared the kinetics of interaction between recombinant RhoA and the RhoGAP domains of p190, p50RhoGAP, Bcr, and ... More