The effect of propranolol on cAMP concentration in the rat preoptic region during the wake-sleep cycle

In control conditions preoptic cAMP concentration during wakefulness was significantly higher than during synchronized sleep. No differences in nucleotide concentration were observed in the cerebral cortex. Propranolol decreases brain cAMP concentration. This change was associated with the suppression of the difference observed between wakefulness and synchronized sleep in the preoptic region.     

Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain

The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Determinants of Ras proteins specifying the sensitivity to yeast Ira2p and human p120-GAP.

Human and Saccharomyces cerevisiae Ras proteins and their regulators GAP (GTPase activating protein)and GEF (guanine nucleotide exchange factor) display structural similarities and are functionally interchangeable in vivo and in vitro, indicating that the molecular mechanism regulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly specific for yeast Ras proteins and cannot stimulate the GTPase of mammalian Ras. This study searches for the reasons for the different sensitivity to Ira2p of human H-ras p21 and yeast Ras2p. Construction of H-ras/Ras2p chimaeras showed that Gly18 of Ras2p (Ala11 of H-ras p21) is an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more crucial determinant was found to be the 89-102 region of Ras2p (82-95 of H-ras p21) including the distal part of strand beta4, loop L6 and the proximal part of helix alpha3. It was possible to construct Ras2p's resistant to Ira2p but still sensitive to human p120-GAP and, conversely, a H-ras p21 sensitive to Ira2p. This work helps clarify specific aspects of the conserved molecular mechanism of interaction between Ras proteins and their negative GAP regulators.     

Asparagine-135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket

This work studies the structure-function relationships of Asn¹³⁵, a residue situated in the GTP binding pocket of elongation factor Tu (EF-Tu). For this purpose we constructed EF-TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF-TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn¹³⁵ side-chain does not recognize the exocyclic keto group of the guanine base. However, EF-TuN 135D/D 138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa-tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn¹³⁵ is important for the correct folding of the nucleotide binding site and that EF-Tu·kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native conformation of this site.     

A mouse CDC25-like product enhances the formation of the active GTP complex of human ras p21 and Saccharomyces cerevisiae RAS2 proteins.

GDP-dissociation stimulators (GDSs) are the key element for the regeneration of the active state of ras proteins, but despite intensive investigations, little is so far known about their functional and structural properties, particularly in mammals. A growing number of genes from various organisms have been postulated to encode GDSs on the basis of sequence similarity with the Saccharomyces cerevisiae CDC25 gene, whose product acts as a GDS of RAS proteins. However, except for CDC25 and the related SDC25 C-domain, no biochemical evidence of ras GDS activity for these CDC25-like proteins has yet been available. We show that the product of a recently isolated mouse CDC25-like gene (CDC25Mm) can strongly enhance (more than 1000 times) the GDP release from both human c-Ha-ras p21 and yeast RAS2 in vitro. As a consequence, the CDC25Mm induces a rapid formation of the biologically active Ras.GTP complex. This GDS is much more active on the GDP than on the GTP complex and has a narrow substrate specificity, since it was found to be inactive on several ras-like proteins. The mouse GDS can efficiently substitute for yeast CDC25 in an in vitro adenylylcyclase assay on RAS2 cdc25 yeast membranes. Our results show that a cloned GDP to GTP exchange factor of mammalian ras belongs to the novel family of CDC25-like proteins.     

Function of sulfhydryl groups in ribosome-elongation factor G reactions. Assignment of guanine nucleotide binding site to elongation factor G.

Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes.