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Query: UMLS:C0027960 (mole)
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We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli. We compared these proteins to E. coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs. Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (> 2 GTP per FtsZ per min). All three required magnesium and a monovalent cation for GTP hydrolysis. Previous reports showed that EcFtsZ (and some other species) required potassium. We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az- and TmFtsZ. Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 microg/ml, rose steeply from 50 to 300 microg/ml and plateaued at a constant high value above 300 microg/ml. This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 microg/ml. A good candidate for the active polymer was visualized by negative stain electron microscopy--straight protofilaments and protofilament pairs were seen under all conditions with active GTPase. We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end. As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay. This is the first accurate determination of FtsZ concentration. Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml. This is just above the plateau for full GTPase activity in vitro.
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PMID:FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly. 960 73

From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTP gamma S and [3H]-GDP with association constants of 1.5 x 10(6) M-1 and 0.58 x 10(6) M-1, respectively. The binding of [35S]-GTP gamma S was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl2, bound [35S]-GTP gamma S and [3H]-GDP were exchanged with GTP gamma S most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.
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PMID:Molecular cloning of cDNA for BRab from the brain of Bombyx mori and biochemical properties of BRab expressed in Escherichia coli. 983 23

Transducin is a heterotrimer formed by a fatty acylated alpha-subunit and a farnesylated betagamma-subunit. The role of these two covalent modifications and of adjacent hydrophobic and charged amino acid residues in reversible anchoring at disk model membranes is investigated at different pH values, salt concentrations, and lipid packing densities using the monolayer expansion technique and CD spectroscopy. The heterotrimer only binds if the acetylated alpha-subunit is transformed into its surface-active form by divalent cations. In the presence of salts the alpha(GDP)-subunit, the betagamma-complex, and the heterotrimer bind to POPC monolayers at 30 mN/m, estimated to mimic the lateral packing density of disk membranes, with apparent binding constants of Kapp = (1.1 +/- 0.3) x 10(6) M-1 (reflecting the penetration of the fatty acyl chain together with approximately three adjacent hydrophobic amino acid residues), Kapp = (3.5 +/- 0.5) x 10(6) M-1 (reflecting the penetration of the farnesyl chain), and Kapp = (1.6 +/- 0.3) x 10(6) M-1 (reflecting a major contribution of the alpha(GDP)-subunit with only a minor contribution from the betagamma-complex). The apparent binding constant of the alpha(GTP)-subunit is distinctly smaller than that of the alpha(GDP)-subunit. Binding to negatively charged POPC/POPG (75/25 mole/mole) monolayers is reinforced by 2-3 cationic residues for the betagamma-complex. The alpha-subunit shows no electrostatic attraction and the heterotrimer shows even a slight electrostatic repulsion which becomes the dominating force in the absence of salts.
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PMID:Molecular determinants of the reversible membrane anchorage of the G-protein transducin. 1038 38

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) requires two divalent cations for activity. One cation activates the enzyme through a direct interaction with the protein at site n(1). The second cation, at site n(2), acts in the cation-nucleotide complex that serves as a substrate. The Co(3+)(n(1))-PEPCK and Cr(3+)(n(1))-PEPCK complexes were used to examine the kinetic, mechanistic, and binding properties of the n(2) metal. EPR studies performed on the Co(3+)(n(1))-PEPCK-GTP complex yielded a stoichiometry of 1 mol of Mn(2+) bound per mole of Co(3+)(n(1))-PEPCK-GTP with a K(D) of 5 microM. PRR studies show a significant enhancement for the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complex. A change in enhancement in the presence of PEP suggests that PEP interacts with the second metal ion. The distance between Mn(2+) at site n(2) on PEPCK and the cis and trans protons and the (31)P of PEP are 7.0, 7.5, and 4.8 A, respectively, as measured by high-resolution NMR. PRR studies of the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GTP and Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complexes as a function of frequency (omega(I)) were used to estimate the hydration number of the n(2) metal to be between 0.5 and 0.7. The metal-metal distance for the M(n(1))-PEPCK-M(n(2))-GTP complex is approximately 8.3 A, and the distance for the M(n(1))-PEPCK-M(n(2))-GDP complex is 9.2 A. The change in the metal-metal distance suggests a conformational change at the active site of PEPCK occurs during catalysis. The Co(3+)(n(1))-PEPCK complex was incubated with Co(2+), GTP, and H(2)O(2) to create a doubly labeled and inactive Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex. The Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex was digested by LysC, and two cobalt-containing peptides were purified using RP-HPLC. Amino acid sequencing of the second cobalt-containing peptide points to the region of Tyr57-Lys76 of PEPCK. Asp66, Asp69, and Glu74 are all feasible ligands to the site n(2) metal.
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PMID:Characterization of the second metal site on avian phosphoenolpyruvate carboxykinase. 1068 18

Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.
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PMID:Activation of annexin 7 by protein kinase C in vitro and in vivo. 1127 15

Modification of transducin (T) with iodoacetic acid (IAA) inhibited its light-dependent guanine nucleotide-binding activity. Approximately 1 mol of [(3)H]IAA was incorporated per mole of T. Cys(347), located on the alpha-subunit of T (T(alpha)), was identified as the major labeled residue in the [(3)H]IAA-modified holoenzyme. In contrast, Cys(135) and Cys(347) were modified with [(3)H]IAA in the isolated T(alpha). IAA-modified T was able to bind tightly to photoexcited rhodopsin (R*), but GTP did not promote the dissociation of the complex between alkylated T and R*. In addition, R* protected against the inhibition of T by IAA. A comparable inactivation of T and analogous interactions between T and R* were observed when 2-nitro 5-thiocyanobenzoic acid (NTCBA) was used as the modifying reagent (J. O. Ortiz and J. Bubis, 2001, Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin, Arch. Biochem. Biophys. 387, 233-242). However, while carboxymethylated T was capable of liberating GDP in the presence of R*, NTCBA-modified T was unable to release the guanine nucleotide diphosphate upon incubation with the photoactivated receptor. Thus, IAA-labeling stabilized a T:R* complex intermediate carrying the empty nucleotide pocket conformation of T. On the other hand, NTCBA-modified T seemed to be "locked" in the GDP-bound state of T, even in the presence of R*.
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PMID:Chemical modification of transducin with iodoacetic acid: transducin-alpha carboxymethylated at Cys(347) allows transducin binding to Light-activated rhodopsin but prevents its release in the presence of GTP. 1169 51

Annexin VI (AnxVI) of molecular mass 68-70 kDa belongs to a multigenic family of ubiquitous Ca2+- and phospholipid-binding proteins. In this report, we describe the GTP-binding properties of porcine liver AnxVI, determined with a fluorescent GTP analogue, 2'-(or 3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP). The optimal binding of TNP-GTP to AnxVI was observed in the presence of Ca2+ and asolectin liposomes, as evidenced by a 5.5-fold increase of TNP-GTP fluorescence and a concomitant blue shift (by 17 nm) of its maximal emission wavelength. Titration of AnxVI with TNP-GTP resulted in the determination of the dissociation constant (Kd) and binding stoichiometry that amounted to 1.3 microM and 1:1 TNP-GTP/AnxVI, mole/mole, respectively. In addition, the intrinsic fluorescence of the membrane-bound form of AnxVI was quenched by TNP-GTP and this was accompanied by fluorescence resonance energy transfer (FRET) from AnxVI Trp residues to TNP-GTP. This indicates that the GTP-binding site within the AnxVI molecule is probably located in the vicinity of a Trp-containing domain of the protein. By controlled proteolysis of human recombinant AnxVI, followed by purification of the proteolytic fragments by affinity chromatography on GTP-agarose, we isolated a 35 kDa fragment corresponding to the N-terminal half of AnxVI containing Trp192. On the basis of these results, we suggest that AnxVI is a GTP-binding protein and the binding of the nucleotide may have a regulatory impact on the interaction of annexin with membranes, e.g. formation of ion channels by the protein.
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PMID:GTP-binding properties of the membrane-bound form of porcine liver annexin VI. 1199 96

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k(inact) of 2.7 min(-1) and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP+, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 microM. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD+-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in alpha helices and beta sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.
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PMID:Inactivation of human glutamate dehydrogenase by aluminum. 1462 97

ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, approximately 0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent K(d) of 2-5 microm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5'-(beta,gamma-imido)triphosphate and guanosine 5'-(beta,gamma-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.
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PMID:N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR). 1547 66

The 2',3'-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H(+)-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH(4). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K(i) of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s(-1). The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [(3)H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.
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PMID:Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP. 1666 47

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