Search PubMed⌕ Search

Biomedical subjects

K Yutani

Publications and source records attributed to K Yutani.

At least 55 records · Page 3Linked to original sources
PubMed1999

Feasibility of fluorodeoxyglucose dual-head gamma camera coincidence imaging in the evaluation of lung cancer: comparison with FDG PET.

UNLABELLED: The purpose of this study was to elucidate the feasibility of fluorodeoxyglucose gamma camera coincidence imaging (FDG GCI) in the evaluation of lung cancer in comparison with FDG PET. METHODS: Twenty-three patients with recently diagnosed lung cancer were examined with both FDG PET and FDG GCI on the same day. Pulmonary lesions were analyzed visually and semiquantitatively using the ratio of lesion-to-background counts (L/B ratio). The L/B ratio of FDG PET without attenuation correction (AC) was also calculated and compared. Nodal stations were only visually analyzed. RESULTS: FDG GCI and FDG PET could detect 22 and 23, respectively, of 23 pulmonary lesions by visual analysis (95.7% versus 100%). The L/B ratio of FDG GCI was 4.26 +/- 2.55, and significantly lower than that of FDG PET (9.29 +/- 4.95; P < 0.01). The L/B ratio of FDG PET was significantly higher with AC than that without AC (9.29 +/- 4.95 vs. 6.66 +/- 4.65; P < 0.01). When the L/B ratio threshold was set at 5.0 for FDG PET and 2.7 for FDG GCI, their sensitivity was 87.0% and 73.9%, respectively. Of the 3 and 6 patients with false-negative results on semiquantitative analysis, the lesions in 3 patients on FDG PET and 4 patients on FDG GCI were less than or equal to 2.0 cm in greatest diameter, respectively. In the assessment of mediastinal involvement, FDG PET was 77.8% sensitive, 78.6% specific and 78.3% accurate, whereas FDG GCI was 77.8% sensitive, 92.9% specific and 87.0% accurate. In the hilar regions, FDG PET was 100% sensitive, 84.2% specific and 87.0% accurate, whereas FDG GCI was 75.0% sensitive, 89.5% specific and 87.0% accurate. CONCLUSION: In this study, FDG GCI yielded results comparable to FDG PET on visual analysis to detect pulmonary lesions and lymph node metastases. However, the lesion-to-background contrasts of pulmonary lesions and nodal involvement were lower in FDG GCI than in FDG PET. Comparison between the L/B ratio of FDG PET with and without AC indicated that, with AC, FDG GCI would be closer to FDG PET in the evaluation of lung cancer.

Adenocarcinoma↗
PubMed1999

Applicability of 99mTc-HL91, a putative hypoxic tracer, to detection of tumor hypoxia.

UNLABELLED: To elucidate the applicability of 99mTc-HL91 (HL91) a putative hypoxic tracer, to the imaging of hypoxia in tumors, a biodistribution study of the tracer was performed. The intratumoral distribution of HL91 was compared with that of 14C-deoxyglucose (DG) and the expression of glucose transporter 1 (GLUT1) in an implanted tumor. METHODS: Biodistribution of HL91 after intravenous injection into Wistar rats with rat mammary tumor (Walker-256) was studied by determining blood and tissue levels of radioactivity from 15 min to 6 h after injection. Dual ex vivo autoradiography was performed on sections of the tumor using HL91 (74 MBq) and DG (185 kBq). The same sections were immunohistologically analyzed with anti-GLUT1 antibody. Tumor tissue was histologically divided into areas of viable cancer cells, necrosis and granulation tissue. The viable cancer cell area was further divided into normoxic and hypoxic areas. Uptake of both tracers in each area was measured quantitatively. The intensity of GLUT1 staining (relative optical density [ROD]) in each area was evaluated by densitometry. RESULTS: The uptake of HL91 in the tumor reached a maximal value (0.897 +/- 0.118% ID [injected dose], mean +/- SD, n = 5) at 120 min after intravenous injection of HL91, then gradually decreased. The tumor-to-muscle ratio continued to increase until 360 min (4.34 at 120 min, 7.01 at 240 min and 10.4 at 360 min). HL91 accumulated to significantly higher levels in the hypoxic area than those in the other tissues (P < 0.0001). Uptake of DG and expression of GLUT1 were significantly higher in the hypoxic area than in the normoxic area (P < 0.0001). In the viable cancer cell area, uptake of HL91 and expression of GLUT1 were strongly correlated (r = 0.624-0.868, mean r = 0.743, P < 0.0001), and DG uptake was moderately correlated with GLUT1 expression (r = 0.328-0.669, mean r = 0.505, P < 0.0001). CONCLUSION: These results indicate that HL91 can be used to detect tumor hypoxia.

Animals↗
PubMed1999

Comparison of dual-head coincidence gamma camera FDG imaging with FDG PET in detection of breast cancer and axillary lymph node metastasis.

UNLABELLED: Dual-head coincidence gamma camera 18F-fluorodeoxyglucose (FDG) imaging was compared with FDG PET in the detection of breast cancer and axillary lymph node metastasis. METHODS: Both coincidence gamma camera FDG imaging and FDG PET were performed in a cylindrical phantom containing spheres of different sizes and activity ratios (5:1, 10:1 and 15:1) and in 30 women (age range 32-78 y) with suspected breast cancer. Biopsies or mastectomies were performed in all patients. Images were visually assessed, and the count ratio between tumor and normal tissue (T/N ratio) was calculated. RESULTS: In the phantom studies, coincidence gamma camera imaging visualized the smallest sphere (1.0 cm) at a ratio of 15:1 but not at ratios of 5:1 and 10:1. Coincidence gamma camera imaging visualized the other spheres (> or =1.3 cm) at all ratios. PET visualized all spheres at all ratios. In the clinical studies, 22 of 26 breast carcinomas detected by PET were also detected by coincidence gamma camera imaging.. Coincidence gamma camera imaging detected all of the carcinomas > or =2 cm in diameter (n = 10) and 12 of 16 carcinomas <2 cm. In breast carcinomas detected by both PET and coincidence gamma camera imaging, the T/N ratio in non-attenuation-corrected PET (7.12 +/- 7.13) was significantly higher than in coincidence gamma camera imaging (2.90 +/- 1.47, P < 0.005). Four of 8 axillary lymph node metastases detected by PET were detected by coincidence gamma camera imaging. Of 9 axillary lymph node metastases <1.0 cm in diameter, 7 and 3 were detected by PET and coincidence gamma camera imaging, respectively. CONCLUSION: Coincidence gamma camera imaging is useful in detecting breast carcinoma > or =2 cm in diameter but is not reliable for breast carcinoma <2 cm in diameter. Coincidence gamma camera imaging may be useless or even dangerous in the detection of axillary lymph node metastasis.

Adult↗
PubMed1998

Structural and thermodynamic responses of mutations at a Ca2+ binding site engineered into human lysozyme.

Structural determinants of Ca2+ binding sites within proteins typically comprise several acidic residues in appropriate juxtaposition. Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are characteristically mutated to Lys, Asp, and Asp, respectively, in natural Ca2+ binding lysozymes and alpha-lactalbumins. The effects of these mutations on the stability and Ca2+ binding properties of human lysozyme were investigated using calorimetry and were interpreted with crystal structures. The double mutant, in which Glu-86 and Ala-92 were replaced with Asp, clearly showed Ca2+ binding affinity, whereas neither point mutant showed Ca2+ affinity, indicating that both residues are essential. The further mutation of Ala-83 --> Lys did not affect the Ca2+ binding of the double mutant. The point mutations Ala-83 --> Lys and Glu-86 --> Asp did not affect the stability, whereas the mutation Ala-92 --> Asp was about 1.3 kcal/mol less stable. Structural analyses showed that both Asp-86 and Lys-83 were exposed to solvent. Side chains of Asp-86 and Asp-91 were rotated in opposite directions about chi1 angle, as if to reduce the electrostatic repulsion. The charged amino acids at the Ca2+ binding site did not significantly affect stability of the protein, possibly because of the local conformational change of the side chains.

Amino Acid Sequence↗
PubMed1998

The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding.

To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate.

Circular Dichroism↗
PubMed1998

Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus.

The structure of methionine aminopeptidase from hyperthermophile Pyrococcus furiosus (PfMAP) with an optimal growth temperature of 100 degreesC was determined by the multiple isomorphous replacement method and refined in three different crystal forms, one monoclinic and two hexagonal, at resolutions of 2.8, 2.9, and 3.5 A. The resolution of the monoclinic crystal form was extended to 1.75 A by water-mediated transformation to a low-humidity form, and the obtained diffraction data used for high-resolution structure refinement. This is the first description of a eukaryotic type methionine aminopeptidase structure. The PfMAP molecule is composed of two domains, a catalytic domain and an insertion domain, connected via two antiparallel beta-strands. The catalytic domain, which possesses an internal 2-fold symmetry and contains two cobalt ions in the active site, resembles the structure of a prokaryotic type MAP from Escherichia coli (EcMAP), while the structure of the insertion domain containing three helices has a novel fold and accounts for a major difference between the eukaryotic and prokaryotic types of methionine aminopeptidase. Analysis of the PfMAP structure in comparison with EcMAP and other mesophile proteins reveals several factors which may contribute to the hyperthermostability of PfMAP: (1) a significantly high number of hydrogen bonds and ion-pairs between side-chains of oppositely charged residues involved in the stabilization of helices; (2) an increased number of hydrogen bonds between the positively charged side-chain and neutral oxygen; (3) a larger number of buried water molecules involved in crosslinking the backbone atoms of sequentially separate segments; (4) stabilization of two antiparallel beta-strands connecting the two domains of the molecule by proline residues; (5) shortening of N and C-terminal tails and stabilization of the loop c3E by deletion of three residues.

Amino Acid Sequence↗
PubMed1998

A general rule for the relationship between hydrophobic effect and conformational stability of a protein: stability and structure of a series of hydrophobic mutants of human lysozyme.

To get a general rule for the relationship between hydrophobic effect and conformational stability, five Ile to Val and nine Val to Ala mutants (3SS mutants) from 3SS (C77A/C95A) human lysozyme were constructed. As known from previous studies, the 3SS protein lacking a disulfide bond between Cys77 and Cys95 is destabilized by enthalpic factors, as revealed by a decrease of about 20 kJ/mol in the denaturation Gibbs energy change (DeltaG) value, as compared to the wild-type protein, which has four disulfide bonds. In this study, the stabilities and structures of the 3SS mutants were determined by differential scanning calorimetry and X-ray crystal analysis, respectively, and compared with those of the mutants (4SS mutants) from the wild-type (4SS) protein published previously. The stabilities of all the 3SS mutants, except for V110A-3SS were decreased as compared with that of the 3SS protein, coinciding with the results for the 4SS mutants. The change in the denaturation Gibbs energy change (DeltaDeltaG) values of the 3SS mutants relative to the 3SS protein at the denaturation temperature (49.2 degreesC) of the 3SS protein at pH 2.7 were similar to those of the equivalent 4SS mutants relative to the wild-type at 64.9 degreesC. The Delta DeltaG values of the 3SS mutants correlated with the changes in hydrophobic surface area exposed upon denaturation (Delta DeltaASAHP) for all of the hydrophobic residues when the effects of the secondary structure propensity were considered. This correlation is identical with that previously found for the 4SS mutants. The linear relation between Delta DeltaG and Delta DeltaASAHP for all of the hydrophobic residues with the same slope was found also for the mutants of T4 lysozyme already reported, indicating that this is a general relationship between changes in conformational stability and changes in ASA values of hydrophobic residues due to mutations.

Calorimetry↗
PubMed1998

Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six tyrosine --> phenylalanine mutants.

The contribution of hydrogen bonds to the conformational stability of human lysozyme was investigated by the combination of calorimetric and X-ray analyses of six Tyr --> Phe mutants. Unfolding Delta G and unfolding Delta H values of the Tyr --> Phe mutant proteins were changed by from +0.3 to -4.0 kJ/mol and from 0 to -16 kJ/mol, respectively, compared to those of the wild-type protein. The net contribution of a hydrogen bond at a specific site to stability (Delta Gwild/HB), considering factors affected by substitutions, was evaluated on the basis of X-ray structures of the mutant proteins. In the present study, one of six mutant proteins was suitable for evaluating the strength of the hydrogen bond. Delta Gwild/HB for the intramolecular hydrogen bond at Tyr124 was evaluated to be 7.5 kJ/mol. Results of the analysis of other mutants also suggest that hydrogen bonds of the hydroxyl group of Tyr, including the hydrogen bond with a water molecule, contribute to the stabilization of the human lysozyme.

Calorimetry, Differential Scanning↗
PubMed1998

Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus.

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

Aminopeptidases↗
PubMed1998

Characterization of soluble artificial proteins with random sequences.

The structural and catalytic properties of two soluble random proteins, RP3-42 and RP3-45, of 141 amino acid residues were investigated. Although no marked secondary structure was detected by CD spectrum, sedimentation equilibrium and small-angle X-ray scattering studies showed that they form an oligomeric structure and are as compact as the molten globule. The random proteins have low but distinct esterase activity; the values of the second-order rate constant for the hydrolysis of p-nitrophenol were 0.78 and 1.39 M(-1) s(-1) for RP3-42 and RP3-45, respectively. The differences in the properties of the random and the native proteins are discussed from the evolutionary point of view.

Amino Acid Sequence↗
PubMed1998

High-resolution crystals of methionine aminopeptidase from Pyrococcus furiosus obtained by water-mediated transformation.

The monoclinic crystal form of methionine amino-peptidase from Pyrococcus furiosus (MAP-Pfu) has been crystallized from four different conditions. Native crystals belong to space group P2(1) with typical unit-cell dimensions a = 53.4, b = 85.1, c = 72.7 A, beta = 107.7 degrees and diffract to 2.9-4.5 A resolution. However, there is a problem of nonisomorphism among the crystals. Water-mediated transformation to low-humidity form occurs by reduction of the relative humidity of crystal environment to 79%. The unit-cell dimensions of transformed crystals are a = 51.9, b = 83.3, c = 70.3 A, beta = 105.9 degrees, and the calculated solvent content is 3.9% less than in original crystals. Transformation to low-humidity form is accompanied by 1.7 times reduction of overall temperature factors, extension of diffraction resolution up to 1.75 A, without change or reduction of crystal mosaicity, and improvement in stability to X-ray radiation. The water-mediated transformation also appears to relieve the problem of nonisomorphism among the original MAP-Pfu crystals.

Aminopeptidases↗
PubMed1998

Assessment of reperfused myocardium using a new ischaemia-avid imaging agent, technetium-99m HL91: comparison with myocardial glucose uptake.

We evaluated the efficacy of a new ischae-mia-avid imaging agent, technetium-99m labelled 4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime (99mTc- HL91) as a marker of myocardial viability in ischaemic and reperfused myocardium. The left coronary artery of rats was ligated for 15 or 60 min and released. The myocardium was reperfused for 60 min [stunned myocardium, or acute myocardial infarction (MI)] or 7 days (subacute MI). Thereafter, 99mTc-HL91 and carbon-14 2-deoxyglucose (DG) were co-injected 30 min before sacrifice. We evaluated the myocardial accumulation of 99mTc-HL91 and DG by dual-tracer ex vivo autoradiography. The uptake of each tracer in the myocardial region was normalized by that in the septum (control), and expressed as percent uptake (%HL or %DG, respectively). Individual hearts were also histopathologically examined. The %HL in the stunned myocardium (n = 8) and in the septum were identical (101%+/-15%, mean+/-SD, P = ns), whereas the %DG was significantly increased (149%+/-27%, P<0.05) compared with that in the septum. These results suggest that 99mTc-HL91 cannot visualize stunned myocardium, whereas DG can. In acute MI (n = 7), the %HL (423%+/-96%, P<0.005) and the %DG (318%+/-91%, P<0.001) in the non-infarcted area at risk were significantly augmented compared with those in the septum. The %DG (181%+/-17%) in the infarcted area was also augmented (P<0.001), whereas the %HL (106%+/-25%) in the infarcted area was identical to that in the septum (P = ns). These results indicate that 99mTc-HL91 detected viable myocardium in the area at risk. In subacute MI (n = 8), the%HL in the infarcted area (101%+/-45%) and in the septum was identical, whereas %DG (292%+/-57%) was significantly higher than that in the non-infarcted risk area or the septum (P<0.0001). These findings suggest that DG detected viable myocardium in the area at risk, but that 99mTc-HL91 was not retained. We conclude that 99mTc-HL91 is a potent marker of myocardial viability when used during the early acute phase after reperfusion.

Animals↗
PubMed1997

Calorimetric observation of a GroEL-protein binding reaction with little contribution of hydrophobic interaction.

Binding of Escherichia coli chaperonin, GroEL, to substrate proteins with non-native structure, reduced alpha-lactalbumin (rLA) and denatured pepsin, were analyzed by isothermal titration calorimetry at various temperatures in the presence of salt (0.2 M KCl). Both proteins bound to GroEL with 1:1 stoichiometry and micromolar affinity at all temperatures tested. However, thermodynamic properties of their binding to GroEL are remarkably different from each other. While heat capacity changes (DeltaCp) of rLA-GroEL binding showed large negative values, -4.19 kJ mol-1 K-1, that of denatured pepsin-GroEL binding was only -0.2 kJ mol-1 K-1. These values strongly indicate that the hydrophobic interaction is a major force of rLA-GroEL binding but not so for denatured pepsin-GroEL binding. When salt was omitted from the solution, the affinity and DeltaCp of the rLA-GroEL binding reaction were not significantly changed whereas denatured pepsin lost affinity to GroEL. Thus, in the non-native protein-GroEL binding reaction, thermodynamic properties, as well as the effect of salt, differ from protein to protein and hydrophobic interaction may not always be a major driving force.

Calorimetry↗
PubMed1997

Contribution of water molecules in the interior of a protein to the conformational stability.

Water molecules frequently occur in the interior of globular proteins. To elucidate the contribution of buried water molecules to the conformational stability of a protein, we examined the crystal structures and the thermodynamic parameters of denaturation of six Ile to Ala/Gly mutant human lysozymes, in which a cavity is created at each mutation site by the substitution of a smaller side-chain for a larger one. One or two ordered water molecules were found in the cavities created in some mutants (I106A, I59A and I59G). The cavity volumes for these three mutants were bigger than those that remained empty in the other mutants. The stability of the mutant proteins with the newly introduced water molecules was about 8 kJ/mol higher than that expected from the change in hydrophobic surface area (DeltaDeltaASAHP) exposed upon denaturation. It was concluded that a water molecule in a cavity created in the interior of a protein contributes favorably to the stability.

Amino Acid Substitution↗
PubMed1997

Crystallization and preliminary X-ray analysis of methionine aminopeptidase from the hyperthermophilic bacterium Pyrococcus furiosus.

Methionine aminopeptidase (MAP) from Pyrococcus furiosus (Pfu) has been crystallized in four different forms (A, B, C and D). Form A crystals belong to space group P2(1) with unit-cell dimensions a = 54.18, b = 85.72, c = 72.84 A, beta = 108.34 degrees. Forms B, C and D belong to space group P6(2(4)) with unit-cell dimensions a = 139.1, c = 63.7 A for form B, a = 198.6, c = 243.8 A for form C, and a = 111.0, c = 125.0 A for form D. Forms A and D diffract to 2.9 A, form B diffracts to 3.5 A, and form C crystals diffract to 4.5 A. Form A contains two molecules of MAP-Pfu per asymmetric unit. The binuclear metal center positions and a non-crystallographic twofold symmetry matrix has been determined for the form A crystals.

Journal Article↗
PubMed1997

Thermal conversion from low- to high-activity forms of catalase I from Bacillus stearothermophilus.

Catalase I from Bacillus stearothermophilus has the interesting property of increasing its enzyme activity on heating. It was confirmed that after heating at 70 degrees C for 10 min or 65 degrees C for 20 min, almost all the enzyme molecules were converted irreversibly to the activated form. The increase in kcat from 1400 to 3930 s-1 and the decrease in Km for H2O2 from 4.4 to 2.7 mM by heat activation indicate changes in the kinetic property of the enzyme molecule. Therefore, it follows that catalase I has two active forms, a high-activity form and a low-activity form. The heat activation process followed the first-order kinetics with an activation enthalpy (DeltaH*) of 191 kJ/mol while the heat denaturation process had a DeltaH* of 545 kJ/mol. The CD spectra of the two enzyme forms had small but marked differences. The conversion of the low-activity form to the high-activity form was an endothermic process with a Tm of 56 degrees C, which is much lower than that of the heat denaturation (Tm = 76 degrees C), and the enthalpy change for the transition was only 5% of that for the denaturation. It has to be noted that the high-activity form of the enzyme was converted back to a low-activity form through the process of denaturation, refolding, and reconstitution with heme. In addition, the newly obtained low-activity form was brought to a high-activity form by heating. These results suggest that the native state of catalase I has two active conformations that are roughly the same but not identical and are separated by a high energy barrier.

Calorimetry, Differential Scanning↗
PubMed1997

Roles of hydrogen bonding residues in the interaction between the alpha and beta subunits in the tryptophan synthase complex. Asn-104 of the alpha subunit is especially important.

The interaction of the alpha subunit with the beta2 subunit of tryptophan synthase is known to be necessary for the activation of each subunit and for the catalytic efficiency of the alpha2beta2 complex. To elucidate the roles of hydrogen bonds in the interaction site between the alpha and beta subunits for subunit association, eight mutant alpha subunits at five hydrogen bonding residues (N104D, N104A, N108D, N108A, E134A, E135A, N157D, and N157A) were constructed, and the thermodynamic parameters of association with the beta subunit were obtained using a titration calorimeter. The N104D and N104A mutations remarkably decreased the stimulation activities, the association constants, and the association enthalpies. Although the association constant and the stimulation activities of E134A were reduced in the absence of salt, the change in the association enthalpy was relatively small, and the addition of salt could repair its defects. The substitutions at positions 135 and 157 did not affect the stimulation activity and decreased the Gibbs energy of association corresponding to the defect in 1 mol of hydrogen bond. The present results suggest that the alpha subunit which has a mutation at position 104 cannot fold into an intact conformation upon complex formation, resulting in reduced stimulation activities. The hydrogen bond with Asn-104, which is a conserved residue among 16 microorganisms, was especially important for alpha/beta interaction and mutual activation.

Escherichia coli↗