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A Parmeggiani

Publications and source records attributed to A Parmeggiani.

At least 109 records · Page 6Linked to original sources
PubMed1987

Effects of the mutation glycine-222----aspartic acid on the functions of elongation factor Tu.

We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu. The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster. Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗
PubMed1987

Properties of a genetically engineered G domain of elongation factor Tu.

The G domain of elongation factor Tu (EF-Tu), representing the N-terminal half of the factor according to its three-dimensional model traced at high resolution, has been isolated by genetic manipulation of tufA and purified to homogeneity. The G domain, whose primary structure shares homology with the eukaryotic protein p21, is capable of supporting the basic activities of the intact molecule (guanine nucleotide binding in 1:1 molar ratio and GTPase activity). However, it is no longer exposed to the allosteric mechanisms regulating EF-Tu. The G-domain complexes with GTP and GDP display similar K'd values in the microM range, in contrast to EF-Tu that binds GDP much more tightly than GTP. Its GTPase shows the characteristics of a slow turnover reaction (0.1 mmol X sec-1 X mol-1 of G domain), whose rate closely corresponds to the initial hydrolysis rate of EF-Tu X GTP in the absence of effectors and lies in the typical range of GTPase of the p21 protein. Of the EF-Tu ligands only the ribosome displays a clear effect enhancing the G-domain GTPase. Our results suggest that the middle and C-terminal domain play an essential role in regulating the activity of the N-terminal domain of the intact molecule as well as in the interactions of EF-Tu with aminoacylated tRNA, elongation factor Ts, and kirromycin. With the isolation of the G domain of EF-Tu, a model protein has been constructed for studying and comparing common characteristics of the guanine nucleotide-binding proteins.

Binding Sites↗
PubMed1986

Characterization of the elongation factors from calf brain. 1. Purification, molecular and immunological properties.

This work describes a method for the purification of the elongation factors (EF) from calf brain. The elongation factor responsible for the binding of aminoacyl-tRNA to the ribosome is found in this organ as a light form (EF-1 alpha) and as a component of heavy, polydispersed aggregates (EF-1H). EF-1 beta, the factor enhancing the EF-1 alpha GDP/GTP exchange, is part of EF-1H and of smaller aggregates. The fraction of EF-1 alpha and EF-1 beta not associated with EF-1H, and EF-2 have been purified to homogeneity after several chromatographic steps. EF-1H consists of many proteins; among them, EF-1 alpha, EF-1 beta and an EF-1 gamma-like protein represent three of the major components. This conclusively shows that EF-1H from calf brain is not a polydispersed aggregate of only EF-1 alpha. EF-1 beta has also been purified to homogeneity from EF-1H. The property of EF-1 beta to aggregate with other proteins suggests that this factor plays an important role in the organization of EF-1H. The relative molecular mass of the purified factors have been determined as: EF-1 alpha, 50,000; EF-1 beta, 30,000; the EF-1 gamma-like component, 49,000; EF-2, 85,000. Some cross-reactivity with the antibodies against the prokaryotic counterparts has been shown for EF-1 alpha, EF-1 beta and EF-2 by functional and immuno-precipitation methods, suggesting the existence of structural homologies.

Animals↗
PubMed1986

Characterization of the elongation factors from calf brain. 2. Functional properties of EF-1 alpha, the action of physiological ligands and kirromycin.

The properties of EF-1 alpha from calf brain have been investigated and compared with those of EF-Tu. EF-1 alpha binds GDP and GTP in a 1:1 stoichiometry, showing the same affinity for both nucleotides (K'd = 2-4 microM). EF-1 beta strongly enhances the dissociation rate of the EF-1 alpha X GDP complex and to a lesser extent of the EF-1 alpha X GTP complex. Aminoacyl-tRNA (aa-tRNA) stabilized EF-1 alpha X GTP much less efficiently than the EF-Tu X GTP complex. Unlike EF-Tu, EF-1 alpha sustains the binding of aa-tRNA to the ribosome also in the presence of GDP or in the absence of any nucleotide, though to a lesser degree than with GTP. Kirromycin enhances the dissociation rate of both EF-1 alpha X GTP and EF-1 alpha X GDP but especially that of the latter. This effect results in an increase of the exchange rate of the EF-1 alpha-bound nucleotide with free nucleotides. Although in this regard the effect of kirromycin mimics that of EF-1 beta, the antibiotic is incapable of increasing the EF-1 alpha X GDP/GTP exchange rate when aa-tRNA and ribosomes are present. Therefore, unlike EF-1 beta, kirromycin cannot enhance the rate of poly(Phe) synthesis. On the other hand, the failure of kirromycin to induce a GTP-like conformation of EF-1 alpha X GDP, as in the case of EF-Tu X GDP, explains its inability to inhibit peptide bond formation in the eukaryotic system.

Animals↗
PubMed1986

Characterization of the elongation factors from calf brain. 3. Properties of the GTPase activity of EF-1 alpha and mode of action of kirromycin.

The GTPase activity of purified EF-1 alpha from calf brain has been studied under various experimental conditions and compared with that of EF-Tu. EF-1 alpha displays a much higher GTPase turnover than EF-Tu in the absence of aminoacyl-tRNA (aa-tRNA) and ribosomes (intrinsic GTPase activity); this is due to the higher exchange rate between bound GDP and free GTP. Also the intrinsic GTPase of EF-1 alpha is enhanced by increasing the concentration of monovalent cations, K+ being more effective than NH+4. Differently from EF-Tu, aa-tRNA is much more active than ribosomes in stimulating the EF-1 alpha GTPase activity. However, ribosomes strongly reinforce the aa-tRNA effect. In the absence of aa-tRNA the rate-limiting step of the GTPase turnover appears to be the hydrolysis of GTP, whereas in its presence the GDP/GTP exchange reaction becomes rate-limiting, since addition of EF-1 beta enhances turnover GTPase activity. Kirromycin moderately inhibits the intrinsic GTPase of EF-1 alpha; this effect turns into stimulation when aa-tRNA is present. Addition of ribosomes abolishes any kirromycin effect. The inability of kirromycin to affect the EF-1 alpha/guanine-nucleotide interaction in the presence of ribosomes shows that, differently from EF-Tu, the EF-1 alpha X GDP/GTP exchange reaction takes place on the ribosome.

Animals↗
PubMed1986

Mechanism of ribosomal translocation. Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis.

The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions. A burst of GTP hydrolysis, with a kapp value greater than 30 s-1 (20 degrees C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid. The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid. E. coli tRNAPhe, but not N-acetylphenylalanyl-tRNAPhe (N-AcPhe-tRNAPhe), stimulates the GTPase when bound in the P site. If the A site of poly(U)-programmed ribosomes, carrying tRNAPhe in the P site, is occupied by N-AcPhe-tRNAPhe, the burst of Pi discharge is replaced by a slow GTP hydrolysis. Since, under these conditions, N-AcPhe-tRNAPhe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.

Escherichia coli↗
PubMed1985

Plasma and skeletal muscle free amino acids in type I, insulin-treated diabetic subjects.

Insulin-dependent diabetes mellitus (IDDM) induces plasma amino acid (AA) abnormalities, including low alanine and high branched-chain (BCAA). While insulin treatment restores plasma AA pattern, proline, methionine, valine, isoleucine, and total BCAA remain elevated in skeletal muscle intracellular water. This suggests that the restoration of plasma AA concentrations is not a satisfactory index of recovered AA metabolism in IDDM.

Adult↗
PubMed1985

Human pancreatic polypeptide and somatostatin in chronic renal failure.

Human pancreatic polypeptide is the only hormone so far reported which clearly suppresses somatostatin release, suggesting that this peptide may have a role in controlling somatostatin secretion from the gut and pancreas. In this study endogenous high circulating human pancreatic polypeptide concentrations in patients with chronic renal failure do not decrease somatostatin circulating levels. The reduced clearance rate of somatostatin in chronic renal failure may partially account for the normal circulating levels of somatostatin observed in our patients with respect to controls. Renal insufficiency may, itself, induce an increase in some gastrointestinal peptides capable of stimulating somatostatin secretion.

Humans↗
PubMed1984

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.

Amino Acids↗
PubMed1983

Transfer ribonucleic acid deprived of the C-C-A 3'-extremity can interact with elongation factor Tu.

In this work we describe that uncharged tRNAs or modified tRNAs lacking all or part of the C-C-A end (i.e., tRNA minus pCpCpA, tRNA minus pA, and tRNA minus A) can still influence the GTPase activity of the elongation factor Tu (EF-Tu), thus showing that, besides the aminoacylated 3'-end, other regions of the aa-tRNA interact with EF-Tu. The existence of an interaction between EF-Tu and truncated tRNAs was also confirmed by examining the dissociation of the EF-Tu-GTP complex: the rate of this reaction is decreased upon addition of tRNAVal1 minus pCpCpA. The effect on the EF-Tu GTPase activity of tRNAs deprived of the C-C-A 3'-end is still evident in the presence of C-C-A-aa. The stimulatory pattern obtained with C-C-A-Val at 5 mM MgCl2 is decreased upon addition of tRNAVal1 minus pCpCpA, tRNAVal1 minus pA, or tRNAVal1 minus A. This shows that the effect of the aminoacylated C-C-A 3'-end can be influenced via EF-Tu by the remaining regions of the tRNA, after cleavage of a bond in the 3'-extremity. However, also with an excess of tRNAVal1 minus pCpCpA over C-C-A-Val, no "aa-tRNA-like" effect, i.e., no inhibition of the EF-Tu GTPase, was obtained, suggesting that, upon binding with EF-Tu, a specific conformational change in the aa-tRNA molecule also takes place, regulating the expression of the GTPase activity. Our results unequivocally show that different regions of the aa-tRNA are needed for a coordinated interaction with EF-Tu.

Base Sequence↗
PubMed1983

Interaction between the different domains of aminoacyl-tRNA and the elongation-factor-Tu x kirromycin complex.

In this work, we have studied the effect of aa-tRNA and derived 3' aminoacylated fragments on the EF-Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes. The aa-tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C-A-Val and Val-tRNA Val1 3' half molecule, as well as a purification method for short 3' aminoacylated fragments based on the amino group charge, were newly developed for this work. Aminoacyl-adenosine was found to be able to stimulate the EF-Tu x kirromycin GTPase, but only to a very small extent. Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A-Val much less than C-A-Val less than C-C-A-Val less than 3' valyladenosine dodecanucleotide much less than Val-tRNA Val1 3' half molecule less than Val-tRNA Val1. The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF-Tu x kirromycin and the stimulation by aa-tRNA, its 3' half molecule and even more by its 3' short fragments. The effect of aa-tRNA and derived 3' fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5-30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa-tRNA and, to a lesser extent, also the effect of the C-C-A-aa as well as the basic activity of the EF-Tu x kirromycin GTPase. The extent of the stimulation by aa-tRNA, and even more by C-C-A-aa, depends on the nature of the amino acid. Among the aminoacyl side chains tested (Arg-, Phe-, Val-, Met-, Leu-, Lys-) arginine was found to be the most active and leucine the least. Our results show that (a) the 3' aminoacylated extremity is of prime importance for the stimulation of the EF-Tu GTPase, (b) in the 3' extremity there are critical sequences for the interaction with EF-Tu and (c) other domains of the aa-tRNA molecule are capable of influencing this reaction: one of the most important is the region including the T psi C loop and stem.

Cations↗
PubMed1983

Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu.

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule. The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val. The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys). In the absence of ribosomes and at low [Mg2+], the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs. Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu. The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.

Escherichia coli↗