Catalysis of amide proton exchange by the molecular chaperones GroEL and SecB.

Hydrogen-deuterium exchange of 39 amide protons of Bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones GroEL and SecB. Both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent. Such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones. Subsequent collapse of unfolded barnase to the exchange-protected folding intermediate was markedly slowed in the presence of GroEL or SecB. Thus, both chaperones have the potential to correct misfolding in proteins by annealing.

Folding of barnase in the presence of the molecular chaperone SecB.

SecB is a molecular chaperone dedicated to interact exclusively with proteins destined for translocation across membranes. We find that SecB interacts with barnase during its folding in a similar manner to its interaction with GroEL. On mixing acid-denatured barnase with SecB in a stopped-flow spectrofluorimeter under conditions that favour refolding, we observe a series of fluorescence changes, corresponding to the binding of the denatured protein and the subsequent refolding of multiply and singly bound forms. The different phases were assigned using a combination of kinetics and mutant proteins. The refolding of barnase when bound to SecB is strongly retarded but never blocked. Multiply bound barnase is less tightly bound and refolds with a higher rate constant than singly bound barnase. Up to 4 mol of denatured barnase bind to 1 mol of tetrameric SecB.

Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105.

Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

Importance of electrostatic interactions in the rapid binding of polypeptides to GroEL.

The question of how chaperones rapidly bind non-native proteins of very different sequence and function has been examined by determining the effect of ionic strength on the refolding of barnase on GroEL, and on the thermal denaturation of barnase in the presence of GroEL and SecB. Both chaperones bind the denatured state of barnase, so lowering the T(m) value. The refolding of barnase in the presence of GroEL is multiphasic, the slowest phase corresponding to the refolding of a singly bound molecule of barnase in the complex with GroEL. The fastest phase is related to the association of barnase and GroEL. At high ratios of GroEL to barnase and low ionic strength (less than 200 mM) this fast phase corresponds to the observed rate of binding. The rate of association of barnase and GroEL was found to be highly dependent on ionic strength, and at high ionic strength (greater than 600 mM) the majority of barnase molecules escaped binding and refolded free in solution. The data are consistent with an initial, transient, ionic interaction between barnase and GroEL, before hydrophobic binding occurs, allowing diffusion-controlled association and slow dissociation of unfolded polypeptide.

Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants.

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.

Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1.

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.

Structures of thermolabile mutants of human glutathione transferase P1-1.

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.

Ethanol reduces asymmetry of visual rCBF responses.

Visual regional CBF (rCBF) responses were measured in 10 healthy male subjects before and after an ethanol dose of 1 g/kg body weight. This dose induces well-established cerebral vasodilatation. However, significant bilateral occipital increases were found in both conditions. Apparently, the coupling between neuronal activity and rCBF is preserved following ethanol. The occipital and posterior parietal flow increases were, however, larger on the right than the left side in the sober state. During inebriation the asymmetry disappeared, possibly representing a more undifferentiated processing of visual information. We propose that ethanol causes a reduced inhibition of the left posterior cortex and a reduction of right-hemisphere information processing.

Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase.

Human class Alpha glutathione transferase (GST) A1-1 has been subjected to site-directed mutagenesis of a Tyr residue conserved in all classes of cytosolic GSTs. The change of Tyr8----Phe lowers the specific activities with three substrates to 2-8% of the values for the wild-type enzyme. The changes in the kinetic parameters kcat/KM, Vmax and S0.5 show that the decreased activities are partly due to a reduced affinity for glutathione. The effect is reflected in lowered kcat values, suggesting that the hydroxyl group of Tyr8 is involved in the activation of glutathione. The proposal of such a role for the Tyr residue has support from the 3D structure of a pig lung class Pi GST [Reinemer et al. (1991) EMBO J. 10, 1997-2005]. Thus, Tyr8 appears to be the first active site residue established as participating in the chemical mechanism of a GST.

Memory for perceived and imagined pictures--an event-related potential study.

Event-related potentials (ERPs) and behavioural measures were used to investigate recognition memory and source-monitoring judgements about previously perceived and imagined pictures. At study, word labels of common objects were presented. Half of these were followed by a corresponding picture and the other half by an empty frame, signalling to the participants to mentally visualise an image. At test, participants in a source-monitoring task made a three-way discrimination between new words and words corresponding to previously perceived and imagined pictures. Participants in an old/new-recognition task indicated whether test words were previously presented or not. In both tasks, correctly identified old items elicited more positive-going ERPs than correctly judged new items. This widely distributed old/new effect was found to have an earlier onset and to be of a greater magnitude for imagined than for perceived items. Task (source versus item-memory) affected the old/new effects over prefrontal areas and the reaction times to remembered old items. The present findings are consistent with the view that a greater amount, or a different type, of information is necessary for accurate source-memory judgements than for correct recognition, and moreover, that different types of source-specifying information revive at different rates. In addition, the results add weight to the view that the late widespread ERP-old/new effect is sensitive to the quality or the amount of information retrieved from memory.

Motor imagery activates the cerebellum regionally. A SPECT rCBF study with 99mTc-HMPAO.

Our earlier findings of a cerebellar activation during motor imagery (Brain Res., 535 (1990) 313-317) were made with a technique with low regional resolution. Therefore we could not elucidate the distribution of the cerebellar activation. In the present study the cerebellar regional cerebral blood flow (rCBF) changes during motor imagery (MI) was measured with a single photon emission computed tomography (SPECT) rCBF method (99mTc-HMPAO) with higher regional resolution during (1) silent counting, and (2) MI (which included silent counting) in 17 normal subjects. Comparing the SPECT results from the two tasks revealed the regional activations during MI. We confirmed that the most pronounced regional activations during MI were found in the cerebellum, especially in its infero-lateral parts on both sides.

The cerebellum participates in mental activity: tomographic measurements of regional cerebral blood flow.

Measurements in man of regional cerebral blood flow (rCBF) have demonstrated a number of cortical and subcortical events coupled to sensory stimulation or motor performance. It has also been shown that local activity changes take place in the cortex during 'pure' mental activity such as motor imagery (unaccompanied by sensory input or motor output). Thus, our group has previously shown that imagination of hand movements gives predominantly a frontal cortical rCBF activation while the corresponding hand movement activates the rolandic hand area mainly. In this paper we report tomographic rCBF measurements with a 133-Xenon SPECT technique during imagined tennis movements and silent counting. Both procedures gave rise to a significant cerebellar activation in addition to cortical rCBF changes. Apparently, the cerebellum may participate in pure mental activity. It possibly plays a role for the temporal organization of neuronal events related to cognition.

An evolutionary approach to the design of glutathione-linked enzymes.

Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.

Semantic processing without conscious identification: evidence from event-related potentials.

Three event-related potential (ERP) experiments examined whether semantic content can be accessed from visually presented words that cannot be consciously identified. Category labels were shown to participants, followed by masked, briefly exposed words that were either exemplars of the category or not exemplars. The task was to verify the category, by guessing if necessary, and to identify the word, naming it if possible. Exposure durations were selected to allow identification in approximately half the trials. For identified words, there was a marked difference in the ERP response between in-category and out-of-category words because of an N400 component. For unidentified words, there was a similar although smaller difference. Conscious identification was defined using a variety of approaches: verbal report, 6-alternative forced choice, and binary categorization (in the context of the regression method; A. G. Greenwald, M. R. Klinger, & E. S. Schuh, 1995). By any definition, ERPs for unidentified words showed evidence of semantic processing. In addition, there were differences in the neuronal populations recruited to process above-threshold versus below-threshold words, suggesting qualitative differences.

EEG topography of acute ethanol effects in resting and activated normals.

Acute effects of ethanol on spectral characteristics of the EEG were studied using 18 recording sites and topographic mapping. The EEG was recorded both at rest and during a mental arithmetic task. Healthy young male volunteers were randomly assigned to an ethanol (n = 22) or a placebo (n = 15) group. The ethanol group received a total dose of 1.0 g/kg, divided into two equal doses given 75 minutes apart. and measurement sessions took place at baseline and after each dose. The placebo group underwent a similar schedule. Power in the theta, alpha and beta bands all increased in the ethanol group, but only the theta and beta bands clearly separated ethanol from placebo. Alpha increases were seen in the placebo group as well. The ethanol-induced changes were greater in the left hemisphere than in the right, having the effect of attenuating the right-over-left asymmetry seen at baseline. Differences between ethanol and placebo were more marked in the mentally activated condition, since the changes seen at rest were inhibited by the activation in the placebo group, but not in the ethanol group. The results indicate (1) that ethanol induces a less differentiated pattern of activity within the brain at rest, and (2) that it impairs the capacity to activate the brain under the challenge of a mental task.

Acute effects of alcohol on regional cerebral blood flow in man.

Acute effects of alcohol in a low (0.7 g/kg) and a high dose (1.5 g/kg) on regional cerebral blood flow (rCBF) were measured with 133Xe inhalation technique at resting conditions in 13 normals. Mean hemisphere CBF increased globally by 12% at the lower dose and 16% at the higher dose. A normal hyperfrontal flow pattern was seen in both alcohol conditions. There were, however, significant regional differences in response to alcohol. The largest rCBF increase was observed in prefrontal regions at the lower dose, and in temporal regions at the higher. Expressed in relative values (% of the whole brain CBF), the temporal rCBF increased linearly with increasing alcohol dosage, while the prefrontal rCBF showed a increase at the lower dose followed by a decrease at the higher dose. It is concluded that alcohol has two types of acute effects on rCBF, a global vasodilatory effect and some regional effects, most clearly seen in prefrontal and temporal regions. The prefrontal flow augmentation following acute alcohol intake may be related to a transient arousal reaction, which has been reported by others. The temporal flow increase may be related to effects of alcohol on emotions and mood.

Effects of nicotine in a bimodal attention task.

Fifteen male users of oral snuff participated in an experiment where we used an auditory-visual vigilance task to study nicotine effects on P300 and response parameters. Quantitative EEG was also studied. Fifteen male nonusers served as controls. We found some decrease of response times and a tendency towards improved signal detection. P300 parameters were not affected in this study. Quantitative EEG analysis indicated an expected increase of arousal, as the activity within the alpha band shifted towards higher frequencies.

Heterologous expression of recombinant human glutathione transferase A1-1 from a hepatoma cell line.

A cDNA clone, lambda GTHA1, encoding human glutathione transferase A1-1 has been isolated from a hepatoma HepG2 cDNA library. At the nucleotide level, the new clone showed minor differences from cDNA deriving from normal liver, but the deduced amino acid sequence was identical to the structure previously described. The protein was expressed from a plasmid, pKHA1, and isolated by a single-step affinity purification on an S-hexylglutathione Sepharose matrix. The yield of the recombinant protein was 165 mg from a 3-liter culture of bacteria.

Restitution of neurophysiological functions, performance, and subjective symptoms after moderate insulin-induced hypoglycaemia in non-diabetic men.

The restoration of cognitive function was studied in 10 healthy men aged 26 years (25.5 +/- 1.2 years; mean +/- SD) after insulin-induced hypoglycaemia (arterialized blood glucose 2.5 +/- 0.4 mmol l-1) for 62 +/- 8 min. Another group of six men participated in a single blind sham study for comparison. The hypoglycaemic event caused a significant increase (p = 0.006) in serum adrenaline levels. Ratings of adrenergically mediated symptoms increased during hypoglycaemia (p = 0.006), as did neuroglycopenic symptoms (p = 0.002), although neuroglycopenia ratings increased in both studies. During hypoglycaemia, P300 amplitudes in a relatively demanding visual search task decreased (p = 0.02), whereas easier tasks were unaffected. The amplitudes were restored after 40 min of normoglycaemia. Reaction time deteriorated after restoration of normoglycaemia, suggesting an effect of hypoglycaemia on learning. Thus, hypoglycaemia at a blood glucose level that is common among patients treated with insulin causes clear cognitive dysfunction, although restoration of the cognitive dysfunction to normal was fast.

Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10).