Vincenzo Bocchini

A fluorimetric method for the estimation of the critical micelle concentration of surfactants

Abstract The present work investigates the possibility of a rapid estimation of critical micelle concentration (cmc) of surfactants by means of soluble fluorescent probes. The effect of nonionic or differently charged surfactants on the fluorescent properties of the anionic 8-anilino-1-naphtalenesulfonic acid magnesium salt (ANS) or cationic rhodamine 6G has been investigated. The possibility of cmc evaluation depends on the appropriate selection of the dye-detergent couple. ANS has to be used with anionic surfactants; on the other hand, rhodamine 6G has to be used with cationic detergents. Both ANS and rhodamine 6G have been proved to be effective with either zwitterionic or nonionic surfactants. Plots of ANS fluorescence increase or rhodamine 6G decrease vs surfactant concentration give two straight lines whose intersection indicates the cmc of the detergent. Under all these conditions the fluorescent probe does not interfere with the micellization process. Excitation of the fluorescent probes at the isosbestic point does not affect the evaluation of the cmc of the detergent. The method applies for linear or steroid surfactants and is independent of the cmc value within a wide range of concentrations.

The crystal structure of Sulfolobus solfataricus elongation factor 1α in complex with GDP reveals novel features in nucleotide binding and exchange

The crystal structure of elongation factor 1α from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF‐1α·GDP) at 1.8 Å resolution is reported. As already known for the eubacterial elongation factor Tu, the SsEF‐1α·GDP structure consists of three different structural domains. Surprisingly, the analysis of the GDP‐binding site reveals that the nucleotide–protein interactions are not mediated by Mg2+. Furthermore, the residues that usually co‐ordinate Mg2+ through water molecules in the GTP‐binding proteins, though conserved in SsEF‐1α, are located quite far from the binding site. [3H]GDP binding experiments confirm that Mg2+ has only a marginal effect on the nucleotide exchange reaction of SsEF‐1α, although essential to GTPase activity elicited by SsEF‐1α. Finally, structural comparisons of SsEF‐ 1α·GDP with yeast EF‐1α in complex with the nucleotide exchange factor EF‐1β shows that a dramatic rearrangement of the overall structure of EF‐1α occurs during the nucleotide exchange.

Relevance of histidine-84 in the elongation factor Tu GTPase activity and in poly(Phe) synthesis: Its substitution by glutamine and alanine

Substitution of His‐84 (→ Gln and → Ala), a residue of the switch II region of E. coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP. The activity in poly(Phe) synthesis and GTP hydrolysis of EF‐Tu H84Q were both reduced to about 35%, as compared to EF‐Tu wt, whereas EF‐Tu H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity. Phe‐tRNAPhe exerted a similar inhibitory effect on the GTPase activity of EF‐Tu wt and EF‐Tu H84Q while abolishing that of EF‐Tu H84A. Ribosomes enhanced the GTPase activity of EF‐Tu H84Q, but not that of EF‐Tu H84A, on which they even seemed to exert an inhibitory effect. The one‐round GTP hydrolysis associated with the EF‐TuH84Q‐dependent binding of Phe‐tRNAPhe to poly(U)‐programmed ribosomes was less efficient than with EF‐Tu wt. Kirromycin stimulated the GTPase activities of both mutants less than EF‐Tu wt. The results of this work do not support a catalytic role of His‐84 in the intrinsic GTPase of EF‐Tu, but they emphasize the importance of its side‐chain for polypeptide synthesis and GTP hydrolysis.

Iron superoxide dismutase from the archaeon Sulfolobus solfataricus : average hydrophobicity and amino acid weight are involved in the adaptation of proteins to extreme environments

The iron-superoxide dismutase in the thermoacidophilic archaeon Sulfolobus solfataricus has a homodimeric structure with a metal content of 0.7 atom of iron per subunit. The enzyme is insensitive to cyanide inhibition, sensitive to inactivation by H2O2 and is the most heat resistant SOD known so far being its half-life 2 h at 100 degrees C. Its primary structure was determined by a profitable combination of advanced mass spectrometry and automated sequence analysis of peptides obtained after cleavage of the purified protein. The enzyme subunit is composed of 210 amino acid residues accounting for a relative molecular mass of 24,112. It does not contain cysteine residues and has a high average of both hydrophobicity and amino acid weight. Vice versa, the hydrophobicity is lower in halophilic SODs. Therefore, it seems that the average hydrophobicity is involved in the adaptation of proteins to extreme environments. The multiple alignment of the primary structure of archaeal and thermophilic eubacterial SODs indicated that archaeal SODs evolved separately from the thermophilic eubacterial SODs and that halophiles originated from a gene different from that of thermophilic archaea.

A NAD(P)H Oxidase Isolated from the Archaeon Sulfolobus solfataricus Is Not Homologous with Another NADH Oxidase Present in the Same Microorganism BIOCHEMICAL CHARACTERIZATION OF THE ENZYME AND CLONING OF THE ENCODING GENE

A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus. The enzyme is a homodimer with M r 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 °C in the absence or presence, respectively, of 8m urea. The enzyme did not show any reductase activity. TheSsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative βαβ-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47–54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase.

Hydrolysis of GTP by the elongation factor Tu·kirromycin complex: Specific action of monovalent cations

Gernot SANDER, Martina OKONEK, Jean-Bernard CRECHET, Richard IVELL*, Vincenzo BOCCHINI’ and Andrea PA~EGGI~I Laboratoire de 3iochimie, Ecole Po~~technique ~~aboratoire associk no. 240 du Centre National de la Rech~ehe ~ie~ti~q~e~, 91128 Palaiseau Cedex, France and *I Cottedra di Chimica Biologica e Cattedm di Chimica, IIa Facoltd di~edi~ina e Chbwgia, Universitd delli Studi, Via Sergiio Pans% 5, 80131 Napoli, Italy

Protein-encoding genes in the sulfothermophilic archaea Sulfolobus and Pyrococcus

A number of unrelated protein-encoding genes from sulfothermophilic archaea, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Pyrococcus furiosus and Pyrococcus woesei, has been analyzed. In the Sulfolobus genus, the content of A + T is significantly higher than that of C + G and the base usage follows the order, A > T > G > C. In Pyrococcus, the A + T content is also higher than that of C + G, but with lower values; in the order of base usage, G precedes T. The codon usage of these sulfothermophiles has been determined; alternative start codons are frequently used in both genera; codon preferences reflect the rich A + T composition of the corresponding genomes; for both genera the codon bias is particularly evident within the different arginine triplets, where AGA and AGG are predominant. From the similarities in the codon usage, close taxonomic relationships become evident within the Sulfolobus or the Pyrococcus genus; a lower, but significant similarity is also clear between these genera. The synonymous codon usage of these sulfothermophiles shows similarities with that of Saccharomyces cerevisiae and bovine mitochondria, whereas clear divergences are observed with the halophilic archaeal genus, Halobacterium, or the eubacterium, Escherichia coli. The unrelated proteins of the considered sulfothermophiles have been analyzed for the content of hydrophobic residues; the comparison with mesophiles reveals a significant increase in the average hydrophobicity of amino acid residues. This finding could indicate a mechanism of adaptation of proteins in organisms living under extreme environments. It is noteworthy that an opposite trend, i.e. a decreased average hydrophobicity, occurs in unrelated halophilic proteins.

Superoxide dismutase activity in the skin of rats irradiated by He-Ne laser

The activity of the enzyme superoxide dismutase has been measured in the skin of rats irradiated by a lowpower He−Ne laser. The irradiation was performed at the doses of 4, 8 and 20 J/cm2 in a single or repeated treatment. The increase in activity of superoxide dismutase in the skin of irradiated animals was not statistically significant.

Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacterium Sulfolobus solfataricus.

ASulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (λG1–λG6) and one of them (λG4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of theS. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.

Molecular, functional and structural properties of an archaebacterial elongation factor 2.

The elongation factor 2 (aEF-2) from the extreme thermo-acidophilic archaebacterium Sulfolobus solfataricus, is the only cytosolic target protein which is ADP-ribosylated by diphtheria toxin in presence of NAD. Once ADP-ribosylated, aEF-2 is no longer able to sustain poly(Phe) synthesis in vitro. aEF-2 displays a great thermoresistance: at the growth temperature of the archaebacterium, 87 degrees C, its half-life is 3 h. The amino acid sequence of the N-terminal region of aEF-2 has been determined up to residue 22. In the first 15 positions such a sequence is identical to that of EF-2 from Sulfolobus acidocaldarius and very similar to that of EF-2 from other archaebacteria or eukaryotes. The same is true for the primary structure of the peptide containing the ADP-ribosylation site. The fact that the primary structure of EF-2 at the ADP-ribosylation site is highly conserved ensures either the correct recognition of the histidine residue by the enzymes involved in its modification to diphthamide, or the proper interaction with the diphtheria toxin.