Linear free energy relationships in the intrinsic and GTPase activating protein-stimulated guanosine 5'-triphosphate hydrolysis of p21ras

Controlling the hydrolysis rate of GTP bound to guanine nucleotide binding proteins is crucial for the right timing of many biological processes. Theoretical, structural, and functional studies have demonstrated that in p21ras the substrate of the reaction, GTP itself, plays a central role by acting as the base catalyst. This substrate-assisted reaction mechanism was analyzed with the help of linear free energy relationships (LFERs). Here we present experimental data that further support the proposed mechanism. We extend the LFER analysis to a wide range of oncogenic as well as nontransforming Ras mutants. It is illustrated that almost all Ras variants follow the observed LFER and thus also the same reaction path. Further, the reduced GTPase reaction rate that characterizes the oncogenic effect of many of the p21 mutants found in human tumors seems to be a consequence of a slightly reduced pKa of the gamma-phosphate group of bound GTP. Factors causing a pKa deviation of just 0.5 unit are enough to slow the intrinsic GTPase reaction rate significantly, and the system may exhibit as a consequence of this an oncogenic potential. Interestingly, we also found oncogenic mutations that do not follow the regular LFER. This suggests that the oncogenic effect of distinct Ras mutants has a different physical origin. The results presented might aid in the design of drugs aimed at reactivating the GTPase reaction of many oncogenic p21ras mutants. We also analyzed the stimulated GTPase reaction of p21ras by the GTPase activating protein (GAP) and the GTPase reaction of Rap1A, a Ras-related GTP binding protein, with similar approaches. The corresponding results indicate that the GAP-stimulated GTPase as well as the Rap1A-catalyzed reaction seem to follow the same substrate-assisted reaction mechanism. However, the correlation coefficient for the GAP-catalyzed reaction is different from the corresponding coefficient for the intrinsic reaction. While the intrinsic reaction exhibits a Br?nsted slope of beta = 2.1, the corresponding value for the GAP-activated reaction is beta = 4.9.     

GDP dissociation inhibitor prevents intrinsic and GTPase activating protein-stimulated GTP hydrolysis by the Rac GTP-binding protein

The majority of the GTP-binding proteins of the Ras superfamily hydrolyze GTP to GDP very slowly. A notable exception to this are the Rac proteins, which have intrinsic GTPase rates at least 50-fold those of Ras or Rho. A protein (or proteins) capable of inhibiting this GTPase activity exists in human neutrophil cytosol. Since Rac appears to exist normally in neutrophils as a cytosolic protein complexed to (Rho)GDI, we examined the ability of (Rho)GDI to inhibit GTP hydrolysis by Rac. (Rho)GDI produced a concentration-dependent inhibition of GTP hydrolysis by Rac1 that paralleled its ability to inhibit GDP dissociation from the Rac protein. Maximal inhibition occurred at or near equimolar concentrations of the GDI and the Rac substrate. The ability of two molecules exhibiting GTPase activating protein (GAP) activity toward Rac to stimulate GTP hydrolysis was also inhibited by the presence of (Rho)GDI. The inhibitory effect of the GDI could be overcome by increasing the GAP concentration to levels equal to that of the GDI. (Rho)GDI weakly, but consistently, inhibited GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) dissociation from Rac1, confirming an interaction of (Rho)GDI with the GTP-bound form of the protein. These data describe an additional activity of (Rho)GDI and suggest a mechanism by which Rac might be maintained in an active form in vivo in the presence of regulatory GAPs.     

Raman difference studies of GDP and GTP binding to c-Harvey ras

The vibrational spectra of phosphate modes for GDP and GTP bound to the c-Harvey p21(ras) protein have been determined using 18O isotope edited Raman difference spectroscopy. A number of the phosphate stretch frequencies are changed upon GDP/GTP binding to ras, and the results are analyzed by ab initio calculations and through the use of empirical relationships that relate bond orders and bond lengths to vibrational frequencies. Bound GDP is found to be strongly stabilized by its interactions, mostly electrostatic, with the active site Mg2+. Bound GTP also interacts with the active site Mg2+ via its beta-phosphate group, as expected on the basis of crystallographic studies of bound GppNp. The angle between the nonbridging P&bondDot;O bonds of the gamma-phosphate of bound GTP increase by about 1-2 degrees compared to its solution value, thus bringing about a geometry that is closer to planar for these bonds as expected for the putative pentacoordinated transition state geometry of the phosphotransfer reaction. Modeling of the interactions at the nucleotide binding site suggests that the water molecule in-line with the P-O bond is positioned to bring about the change in bond angle. Moreover, a weak fifth bond (about 0.03 vu) appears to be formed between it and the gamma-phosphorus atom of bound GTP with a concomitant weakening of the O-P bond between the GDP leaving group and the gamma-phosphorus atom. Hence, an important role of the active site structure appears to be the strategic positioning of this in-line water. These structural results are consistent with a reaction pathway for GTP hydrolysis in ras of synchronous bond formation between the gamma-phosphorus of GTP and the attacking nucleophile and bond breaking between the gamma-phosphorus and the GDP leaving group.     

Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins

Unlike the alpha subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AIF4-). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AIF4 - in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120GAP and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.     

Conformational change rate-limits GTP hydrolysis: the mechanism of the ATP sulfurylase-GTPase

The fluorescent GTP analogues 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imidotriphosphate) (mGMPPNP) and 3'-O-(N-methylanthraniloyl)-2'-deoxy-GTP (mGTP) were used to demonstrate that an enzyme isomerization precedes and rate-limits beta,gamma-bond cleavage in the catalytic cycle of the ATP sulfurylase-GTPase, from E. coli K-12. The binding of mGMPPNP to the E.AMP.PPi complex of ATP sulfurylase is biphasic, indicating that an isomerization occurs in the binding reaction. The isomerization mechanism was assigned based on the results of the enzyme concentration dependence of the observed rate constants, kobs, for both phases of the binding reaction, and sequential-mixing, nucleotide release experiments. The isomerization occurs after, and is driven by, the addition of mGMPPNP. Values were determined for each of the rate constants associated with the two-step kinetic model used in the interpretation of the results. A comparison of the enzyme concentration dependence of kobs for the hydrolysis and binding reactions reveals that the rate constants for the corresponding steps of these two reactions are extremely similar. The virtually identical rate constants for isomerization and beta, gamma-bond scission strongly suggest that isomerization rate-limits bond breaking. The implications of these finding for GTPase/target interactions and the mechanism of energetic linkage in the ATP sulfurylase system are discussed.     

Mapping the transition state for ATP hydrolysis: implications for enzymatic catalysis

BACKGROUND: Phosphoryl transfer, typically involving high energy phosphate donors such as ATP, is the most common class of biological reactions. Despite this, the transition state for phosphoryl transfer from ATP in solution has not been systematically investigated. Characterization of the transition state for the uncatalyzed hydrolysis of ATP would provide a starting point for dissection of enzyme-catalyzed reactions. RESULTS: We examined phosphoryl transfer from ATP, GTP and pyrophosphate to a series of alcohols; these reactions are analogous to the phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. The Br?nsted beta(nucleophile) value of 0.07 is small, indicating that there is little bond formation between the incoming nucleophile and the electrophilic phosphoryl group in the transition state. Coordination of Mg2+ has no measurable effect on this value. The Br?nsted beta(leaving group) value of -1.1 for phosphoryl transfer to water from a series of phosphoanhydrides is large and negative, suggesting that the bond between phosphorous and the leaving group oxygen is largely broken in the transition state. CONCLUSIONS: Uncatalyzed hydrolysis of ATP in solution occurs via a dissociative, metaphosphate-like transition state, with little bond formation between nucleophile and ATP and substantial cleavage of the bond between the gamma-phosphoryl moiety and the ADP leaving group. Bound Mg2+ does not perturb the dissociative nature of the transition state, contrary to proposals that enzyme-bound metal ions alter this structure. The simplest expectation for phosphoryl transfer at the active site of enzymes thus entails a dissociative transition state. These results provide a basis for analyzing catalytic mechanisms for phosphoryl transfer.     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Nucleotidase activities of the 26 S proteasome and its regulatory complex

The 26 S proteasome can be assembled from the multicatalytic protease (or 20 S proteasome) and a large multisubunit regulatory complex in an ATP-dependent reaction. The 26 S proteasome and its regulatory complex were purified from rabbit reticulocytes for characterization of their nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phosphate. The Km values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 microM for ATP and CTP, 50 microM for GTP, and 100 microM for UTP; Km values for nucleotide hydrolysis by the regulatory complex are 2-4-fold higher for each nucleotide. Several ATPase inhibitors (erythro-9-[3-(2-hydroxynonyl)]adenine, oligomycin, ouabain, and thapsigargin) had no effect on ATP hydrolysis by either complex whereas known inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmaleimide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM but only GTP and UTP hydrolysis was significantly reduced at 50 microM NEM. The 15 microM Km for ATP hydrolysis by the 26 S proteasome is virtually identical to the observed Km of 12 microM ATP for Ub-conjugate degradation. Although nucleotide hydrolysis is required for protein degradation by the 26 S proteasome, nucleotide hydrolysis and peptide bond cleavage are not strictly coupled. Substrate specificity constants (kcat/Km) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation. Further evidence that nucleotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibited peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP or UTP hydrolysis by the 26 S enzyme.     

Functional consequences of monoglucosylation of Ha-Ras at effector domain amino acid threonine 35

Monoglucosylation of low molecular mass GTPases is an important post-translational modification by which microbes interfere with eukaryotic cell signaling. Ha-Ras is monoglucosylated at effector domain amino acid threonine 35 by Clostridium sordellii lethal toxin, resulting in a blockade of the downstream mitogen-activated protein kinase cascade. To understand the molecular consequences of this modification, effects of glucosylation on each step of the GTPase cycle of Ras were analyzed. Whereas nucleotide binding was not significantly altered, intrinsic GTPase activity was markedly decreased, and GTPase stimulation by the GTPase-activating protein p120(GAP) and neurofibromin NF-1 was completely blocked, caused by failure to bind to glucosylated Ras. Guanine nucleotide exchange factor (Cdc25)-catalyzed GTP loading was decreased, but not completely inhibited. A dominant-negative property of modified Ras to sequester exchange factor was not detectable. However, the crucial step in downstream signaling, Ras-effector coupling, was completely blocked. The Kd for the interaction between Ras.GTP and the Ras-binding domain of Raf was 15 nM, whereas glucosylation increased the Kd to >1 mM. Because the affinity of Ras.GDP for Raf (Kd = 22 microM) is too low to allow functional interaction, a glucose moiety at threonine 35 of Ras seems to block completely the interaction with Raf. The net effect of lethal toxin-catalyzed glucosylation of Ras is the complete blockade of Ras downstream signaling.     

Transient kinetic studies on the interaction of Ras and the Ras-binding domain of c-Raf-1 reveal rapid equilibration of the complex

Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.     

GTPase activity of Rab5 acts as a timer for endocytic membrane fusion

The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport. Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein. To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of [alpha(32)P]-XTP hydrolysis in situ during endosome fusion in vitro. Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis. Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5. Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.     

Differences in self reported morbidity by educational level: a comparison of 11 western European countries

Raman and infrared spectra were examined for guanosine 5'-diphosphate (GDP) and guanosine 5'-triphosphate (GTP) in aqueous solution. The vibrational modes were assigned on the basis of isotopic frequency shifts and relative intensities in the Raman and infrared spectra. The observed frequency shifts on 18O isotope labeling made it possible to identify the bands from each phosphate group (alpha, beta, gamma). Frequency shifts were observed as Mg2+ complexes with GDP and GTP. The results suggested that Mg2+ binds to GDP in a bidentate manner to the alpha, beta P[symbol: see text]O bonds and in a tridentate manner to the alpha, beta and gamma P[symbol: see text]O bonds of Mg.GTP. The results indicate that structure of Mg2+ coordinated to GTP in aqueous solution differs somewhat to that found for Mg.ATP.     

Reconstitution of beta2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells--high coupling efficiency in a beta2-adrenoceptor-G(s alpha) fusion protein

In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.     

Synthesis, structure, and antiproliferative activity of selenophenfurin, an inosine 5'-monophosphate dehydrogenase inhibitor analogue of selenazofurin

The synthesis and biological activity of selenophenfurin (5-beta-D-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivative. Deprotected ethyl 5-beta-D-ribofuranosylselenophene-3-carboxylate (12 beta) was converted into selenophenfurin by ammonolysis. The structure of 12 beta was determined by 1H- and 13C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.     

GTP hydrolysis is not important for Ypt1 GTPase function in vesicular transport

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37 degrees C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.     

Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras

Ras-related GTP-binding proteins function as molecular switches which cycle between GTP-bound 'on'- and GDP-bound 'off'-states. GTP hydrolysis is the common timing mechanism that mediates the return from the 'on' to the 'off'-state. It is usually slow but can be accelerated by orders of magnitude upon interaction with GTPase-activating proteins (GAPs). In the case of Ras, a major regulator of cellular growth, point mutations are found in approximately 30% of human tumours which render the protein unable to hydrolyse GTP, even in the presence of Ras-GAPs. The first structure determination of a GTPase-activating protein reveals the catalytically active fragment of the Ras-specific p120GAP (ref. 2), GAP-334, as an elongated, exclusively helical protein which appears to represent a novel protein fold. The molecule consists of two domains, one of which contains all the residues conserved among different GAPs for Ras. From the location of conserved residues around a shallow groove in the central domain we can identify the site of interaction with Ras x GTP. This leads to a model for the interaction between Ras and GAP that satisfies numerous biochemical and genetic data on this important regulatory process.     

Three-dimensional structure of p21H-ras and its implications

The three-dimensional structure of the H-ras oncogene product p21 has been determined in both its active, GTP-bound and its inactive, GDP-bound forms. This has supplied a wealth of information on the mode of binding of guanine nucleotides, on the mechanism of the GTPase reaction and on the conformational change of the protein which accompanies GTP hydrolysis. The structural analysis has also given clues to the interaction of p21 with the regulatory proteins GAP (GTPase Activating Protein) and nucleotide exchange factor. The three-dimensional structures of oncogenic mutants of p21 have also been determined and can nicely explain different biochemical and biological behaviour of these mutant proteins.     

Zinc phosphating of 6061-Al alloy using REN as additive

Zinc phosphate coating formed on 6061-A1 alloy was studied with the help of electrochemical measurements, Fourier Transform Infrared （FTIR）, and Scanning Electron Microscopy （SEM）, after dipping it in phosphating solutions containing different concentrations of Rare Earth Nitrate （REN）. REN, which acted as an accelerator in the phosphating solution, could catalyze the surface reaction and accelerate the phosphating process. REN mainly enabled the P in the phosphate coating to exist in the form of PO4^3- and promoted the hydrolysis of phosphatic acid in a liquid layer at the cathodes. This resulted in the evolution of H2 at the cathodes, which increased the local pH value and in turn drove the precipitation of the phosphate coating. Additionally, REN was adsorbed on the surface of the aluminum substrates to form a gel during the phosphating process. These gel particles were good crystal seeds, which helped to form phosphate crystal nuclei and possess the function of a nucleation agent that could decrease the phosphate crystal size. The corrosion resistance of the formed zinc phosphate coatings was improved.     

Zinc phosphating of 6061-Al alloy using REN as additive

Zinc phosphate coating formed on 6061-Al alloy was studied with the help of electrochemical measurements, Fourier Transform Infrared (FTIR), and Scanning Electron Microscopy (SEM), after dipping it in phosphating solutions containing different concentrations of Rare Earth Nitrate (REN). REN, which acted as an accelerator in the phosphating solution, could catalyze the surface reaction and accelerate the phosphating process. REN mainly enabled the P in the phosphate coating to exist in the form of PO43- and promoted the hydrolysis of phosphatic acid in a liquid layer at the cathodes. This resulted in the evolution of H2 at the cathodes, which increased the local pH value and in turn drove the precipitation of the phosphate coating. Additionally, REN was adsorbed on the surface of the aluminum substrates to form a gel during the phosphating process. These gel particles were good crystal seeds, which helped to form phosphate crystal nuclei and possess the function of a nucleation agent that could decrease the phosphate crystal size. The corrosion resistance of the formed zinc phosphate coatings was improved.     

The structure and interactions of Ca(2+)-ATPase

Electron crystallographic studies on membrane crystals of Ca(2+)-ATPase reveal different patterns of ATPase-ATPase interactions depending on enzyme conformation. Physiologically relevant changes in Ca2+ concentration and membrane potential affect these interactions. Ca2+ induced difference FTIR spectra of Ca(2+)-ATPase triggered by photolysis of caged Ca2+ are consistent with changes in secondary structure and carboxylate groups upon Ca2+ binding; the changes are reversed during ATP hydrolysis suggesting that a phosphorylated enzyme form of low Ca2+ affinity is the dominant intermediate during Ca2+ transport. A two-channel model of Ca2+ translocation is proposed involving the membrane-spanning helices M2-M5 and M4, M5, M6 and M8 respectively, with separate but interacting Ca2+ binding sites.