Familial interstitial pneumonia in an adolescent boy with surfactant protein C gene (Y104H) mutation.

Recent studies have suggested that some cases of familial interstitial pneumonia are associated with mutations in the gene encoding surfactant protein C (SFTPC). We report here a case of familial interstitial pneumonia in an adolescent boy whose paternal grandfather and father suffered from idiopathic interstitial pneumonia (IIP). The patient was asymptomatic but showed an abnormal shadow in the chest at his medical check-up. The surgical biopsy of the patient revealed non-specific interstitial pneumonia and showed pathological findings similar to those in his father's autopsy. Genomic DNA from blood leucocytes of the patient was sequenced for the Thy104His (Y104H) SFTPC mutation. Based on these results, he was diagnosed with SFTPC mutation-associated familial interstitial pneumonia. There has been no clinical, physiologic and radiologic progression for 4 years since the diagnosis. The relation between clinical manifestation and the mutation site of the patient may broaden the spectrum of SFTPC mutation-associated interstitial pneumonia.

Aberrations in the fragile histidine triad (FHIT) gene in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) seems to be closely associated with lung carcinogenesis. To identify the genetic characteristics of precancerous IPF lesions in the peripheral lung, we performed PCR-based microsatellite analysis with DNA extracted from microdissected tissues; fluorescent in situ hybridization (FISH) analysis of the fragile histidine triad (FHIT) gene and immunohistochemical analysis of Fhit protein expression in samples of metaplasias and bronchiolar epithelia obtained from patients with IPF. We used four microsatellite markers of the FHIT gene within or flanking the FHIT gene on chromosome 3p for loss of heterozygosity (LOH) analysis. LOH of the FHIT locus was frequently found among the lesions of metaplasias and bronchiolar epithelia in the patients with IPF [62 (52%) of 119 informative lesions]. Fifty-four (73%) of the 74 lesions of metaplasias and bronchiolar epithelia obtained from the IPF patients with lung carcinoma and 8 (17%) of the 46 samples obtained from the IPF patients without lung carcinoma showed LOH at the FHIT gene (P < 0.0001). We confirmed allelic loss in the metaplasias and bronchiolar epithelia of IPF by FISH analysis of the FHIT gene. Additionally, the level of Fhit protein expression in the metaplastic cells of IPF was frequently reduced. Our findings suggest that allelic loss of the FHIT gene may be involved in carcinogenesis in the peripheral lung of patients with IPF.

Site-specific modification of rabbit muscle creatine kinase with sulfhydryl-specific fluorescence probe by use of hydrostatic pressure.

We investigated the effect of pressure on the reactivity of cysteine residues of rabbit muscle creatine kinase (CK). Performing the fluorescent modification under high pressure, a unique sulfhydryl group (Cys-253) of CK was labeled, in addition to Cys-282, which is known as a single reactive sulfhydryl under ambient conditions. CK is composed of two identical subunits, containing four cysteine residues in each subunit. Cys-282 plays an important role in enzymatic activity. In the pressure range from 0.1 MPa to 300 MPa, only one sulfhydryl group for each subunit of CK reacted with the reagents. However, at 400 MPa 2 sulfhydryl groups were modified. The 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage method revealed that both Cys-282 and Cys-253 were modified at 400 MPa. The chemical modification of Cys-282 induced a loss of enzymatic activity. By taking advantage of the modification under high pressure, selective modification of Cys-253 with 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulfonate (IAEDANS) was performed. A reversible blocking of Cys-282 at atmospheric pressure was followed by the reaction of Cys-253 with the fluorescent probe at 400 MPa. After the decompression, Cys-282 was unblocked, and obtained Cys-253-modified CK retained up to 64% of the catalytic activity of the intact CK. The fluorescent properties of IAEDANS covalently bound at Cys-253 were not significantly different from those of IAEDANS covalently bound at Cys-282.

Structure of pressure-induced denatured state of human serum albumin: a comparison with the intermediate in urea-induced denaturation.

The structure of human serum albumin (HSA) in the pressure-induced denatured state was investigated by fluorescence spectroscopy. HSA undergoes a conformational change in the pressure range from 0.1 MPa to 400 MPa, at 25 degrees C. Several ligands bind to specific sites in HSA, and the fluorescence spectra of these ligands were used to study the conformational state of this protein. The warfarin-binding site (site I) and the dansylsarcosine-binding site (site II), are located in subdomains II and III, respectively. The fluorescence spectra of these probes reflected the structural changes in each of these subdomains. Dansylsarcosine completely dissociated from its binding site in domain III above 300 MPa, but substantial affinity of warfarin remained in this pressure range. Similar results were obtained for the urea-induced denaturation of HSA; although dansylsarcosine completely dissociated at urea concentration above 6 M, warfarin remained bound to site I in domain II at these concentrations. These results suggest that the structure of domain III is unfolded both in the initial stages of both pressure- and urea-induced denaturation of HSA. HSA possesses a single tryptophan residue (Trp-214) in domain II, and fluorescence from this residue reflects structural changes in this domain. In the urea-induced denatured state of HSA, a red-shift in the wavelength of maximum fluorescence occurred over urea concentrations ranging from 4 M to 6 M. This shift indicated that a structural change in domain II occurred simultaneously with the unfolding of domain III in this concentration range. On other hand, the shift in the wavelength of maximum fluorescence of Trp-214 was comparatively small in the pressure range from 0.1 MPa to 400 MPa indicating that the environment of Trp-214 was not affected. These results indicate that preferential unfolding of domain III occurs in the pressure-induced denatured state of HSA.

Pressure dependence of trypsin-catalyzed hydrolyses of specific substrates.

The effects of pressure on the trypsin-catalyzed hydrolyzed hydrolyses of three specific substrates, N-benzoyl-L-arginine ethyl ester (BzArgOEt), amide (BzArgNH2) and p-nitroanilide (BzArgNA), have been examined. The volume of the activation (delta V++) for kcat was -2.4 ml/mol for BzArgOEt and +3 - +6 ml/mol for BzArgNH2. Because of different rate-determining steps in the steady-state kinetics, the delta V++ value for BzArgOEt would indicate the activation volume of the deacylation step, whereas that for BzArgNH2 the delta V++ for the acylation step. The activation volumes were accounted for in terms of the difference in the mechanisms on the formation and decomposition of the tetrahedral-like intermediates during the acylation and deacylation steps. The delta V values for the formation of BzArgNH2- and thionine-trypsin complexes were several ml/mol, consistent with the fact that the main driving force of the substrate binding to this enzyme is electrostatic interaction, and in contrast to the delta V values of alpha-chymotrypsin complex formation with indole (approximately 0 ml/mol) or 2-furylacryloyl-D-tryptophan methyl ester (approximately 0 ml/mol), for which the hydrophobic interaction is the dominant force of the substrate binding. For the hydrolysis of BzArgNA, which showed a distinct substrate activation at high substrate concentrations, the pressure dependence of the four parameters, ks, Ks, (the catalytic rate and dissociation constant of the normal enzyme-substrate complex, respectively), Kss and Kss (those of the complex activated by the binding of the second substrate molecule), were measured at 1 atm and 1000 atm (25 degrees C). All of the four parameters increased with increase in pressure.

In vitro increases in plasmid DNA supercoiling by hydrostatic pressure.

By conducting topoisomerase I-mediating supercoiling assays, effects of elevated pressure on DNA supercoiling were investigated for the first time. It was found that pressure elevations induced a progressive increase in plasmid DNA linking numbers, winding the DNA duplex by a magnitude of 1.1-1.6x10(-3) angular degree/base/MPa. Implications for the findings were discussed in terms of disturbance of the tertiary structure of DNA by elevated pressure.

Differences in pressure and temperature transitions of proteins and polymer gels.

Pressure-driven and temperature-driven transitions of two thermoresponsive polymers, poly(N-isopropylacrylamide) (pNIPAM) and poly(N-vinylisobutyramide) (pNVIBA)), in both a soluble linear polymer form and a cross-linked hydro-gel form, were examined by a dynamic light-scattering method and direct microscopic observation, respectively. Their behavior was compared with that of protein systems. Changes in some characteristic parameters in the time-intensity correlation functions of dynamic light-scattering measurement of aqueous solutions of pNIPAM at various pressures and temperatures showed no essential differences during temperature and pressure scanning and, as a whole, the motions of polymers in aqueous solutions were similar in two types of transitions until chain shrinkage occurred. The gels (cross-linked polymer gels) prepared from the thermoresponsive polymers also showed similar volume transitions responding to the pressure and temperature increase. In temperature transitions, however, gels showed drastic volume shrinkage with loss of transparency, while pressure-induced transition showed a slow recovery of transparency while keeping the size, after first transient drastic volume shrinkage with loss of transparency. At a temperature slightly higher than the transition under atmospheric temperature, so-called reentry of the volume change and recovery of the transparency were observed during the pressure-increasing process, which implies much smaller aggregation or non-aggregated collapsed polymer chains in the gel at higher pressures, indicating a certain mechanistic difference of the dehydration processes induced by temperature and pressure.

Observation of the pre-steady state process in thermolysin catalysis with a fluorescent displacement probe at low pH.

The pre-steady state process in the thermolysin-catalyzed hydrolysis of Cbz-Gly-Phe-Ala was observed at pH 4.5 by fluorescence stopped-flow method using Dns-Phe as a displacement probe. After the confirmation of the pre-equilibrium hypothesis for the binary interaction, the nonlinear substrate concentration dependence of the apparent kinetic constant for the pre-steady state process was analyzed and an existence of multi-intermediates was proposed.

Single-point amino acid substitutions at the 119th residue of thermolysin and their pressure-induced activation.

The effect of amino acid substitution at the 119th site of thermolysin (TLN) on the pressure activation behavior of this enzyme was studied for four mutants at pressures < 300 MPa. For Q119Q, Q119N and Q119R, the highest activation was observed to be over 30 times that at atmospheric pressure and the activation volumes (deltaV++) were about -75 ml/mol. However, we obtained only 10 times higher activation for Q119E and Q119D (deltaV++ approximately -60 ml/mol). The intrinsic fluorescence of TLN changed at pressures > 300 MPa, and the latter two mutants showed a smaller deltaGapp and deltaVapp of transition than the wild type. These results are discussed with respect to the hydration change in the enzyme protein around the substituted region.

Kinetic study on the acylation step of alpha-chymotrypsin-catalyzed hydrolysis of acylimidazole. A model reaction of specific peptide substrate activated by binding to the enzyme.

As a model reaction of the enzyme-bound activated peptide substrates in proteinase-catalyzed hydrolysis, the alpha-chymotrypsin-catalyzed hydrolysis of three acylimidazoles (trans-cinnamoylimidazole, indoleacryloylimidazole, and furylacryloylimidazole) was studied, especially as regards the acylation process. The complicated pH-dependences of the reactions, due mainly to the existence of the protonated forms of these acylimidazoles, were analyzed based on a reaction scheme considering the so-called inactive monomeric enzyme and the dimeric enzyme. The intrinsic reaction parameters for individual species were evaluated. The acylation through the mono-protonated enzyme-substrate complex (ESH) reflects the rate of attach by Ser-195 O gamma on the carbonyl carbon of acylimidazolium, where the formation of the possible tetrahedral intermediate is rate-determining. The acylation via the non-protonated enzyme-substrate complex (ES) is intermediate between those of esters (as regards the single bond character of the cleaved linkage) and of amides (with respect to the proton transfer as a key process of the acylation). This was confirmed by the observation of acceleration of the acylation rate upon addition of external proton donors. Based on these results, the mechanism of acylation in peptide substrate hydrolysis is discussed.

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities.

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).

Kinetic study of carboxypeptidase P-catalyzed reaction. Pressure and temperature dependence of kinetic parameters.

Detailed kinetic analyses of carboxypeptidase P-catalyzed reactions were carried out spectrophotometrically using 3-(2-furyl)acryloyl-acylated peptide substrates. The maximum kcat/Km was observed at around pH 3.5 for the synthetic peptide substrates. The kcat/Km value decreased with increasing pH, with an apparent pKa value of 4.43. However, the maximum kcat was observed at neutral pH (pH congruent to 6) and the pKa was 4.49. These apparently different pH profiles for kcat/Km and kcat of this enzyme were due to the decreasing Km value in the acid pH region. The pressure and temperature dependences of these kinetic parameters were also measured. N-Benzoylglycyl-L-phenyllactate (Bz-Gly-OPhLac) gave dependences similar to those of the peptide substrate, suggesting that there is no distinct difference in the catalytic mechanism between the peptide and the ester hydrolyses.

pH dependence of the formation of dimeric alpha-chymotrypsin and its catalytic activity.

The dimerization of alpha-chymotrypsin (alpha-CT) has been known to involve a specific interaction between the Tyr-146 alpha-carboxyl of one molecule and the His-57 imidazole, which is a member of the catalytic triad in the active site, of the other. This interaction determines the pH dependences of the dimerization constant and of the catalytic activities of the monomeric and dimeric enzymes. We compared the pKa values for catalytic activities with known pKa values for the dimerization constant in order to assign pKa values to residues of the enzyme. In the monomeric enzyme, the catalytic triad has a pKa value of 3.6 at the site between the Asp-102 carboxyl and His-57 N delta 1, and the Tyr-146 alpha-carboxyl has a pKa value of 4.6. In the dimeric enzyme, the site between the Asp-102 carboxyl and His-57 N delta 1 has a pKa value of around 5.5 and the site between His-57 N epsilon 2 and the Tyr-146 alpha-carboxyl has a pKa value around 2.4. Protonation at the site between the Asp-102 carboxyl and His-57 N delta 1 reduced the catalytic activity of the dimeric enzyme for p-nitrophenyl propionate, indicating that the Asp-102 carboxyl plays an important role in the monomeric alpha-CT-catalyzed reactions.

Specific and non-specific effects of inert anions and cations on the dimerization of alpha-chymotrypsin and the catalytic activities of monomeric and dimeric alpha-chymotrypsins.

The effect of salts on the dimerization and the catalytic activity of dimeric alpha-chymotrypsin (alpha-CT) was investigated. The observed effect was mainly related to the anionic constituent of the salt, and its order depended on the salt concentration. The order of effect of anions on several parameters at [salt] less than 0.02 M was SO4 2-, ClO4-, NO3-, Br-, Cl-, which reflected the electrostatic interaction between the anion and the positively charged surface of alpha-CT. On the other hand, the anion dependence at moderate salt concentrations (0.1-0.2 M) followed the Hofmeister series, SO4 2-, Cl-, Br-, NO3-, ClO4-, which was related to the direct and indirect interactions between the anion and non-charged groups of the protein. The anion order for the dimerization constant of this enzyme, however, was the complete reverse of that generally observed in the aggregation of proteins at high salt concentration (greater than 1 M). This result was accountable for in terms of a specific interaction in the formation of the dimeric enzyme (probably that between the active site of one monomer and Tyr-146 of the other).

Interaction of specific amino acid derivatives with dimeric alpha-chymotrypsin.

Binding and catalytic activities of dimeric alpha-chymotrypsin from specific amino acid derivatives were investigated with special reference to the equilibrium between active and inactive monomeric forms of this enzyme occurring in a low pH range. The low catalytic activity of the dimeric enzyme towards these specific substrates was revealed to be due to the difficulty in binding of the dimer. However the free tryptophanates and N-acetyl-L-tryptophan, which are slightly smaller (in molecular size) than the above substrates and comparable to the nonspecific phenyl acetate substrates (towards which the dimeric alpha-chymotrypsin showed an distinct catalytic activity in our previous study [J. Biochem. 87, 871-880 (1980)]), were bound to the dimer more strongly than to the monomeric enzyme. Hence they enhanced dimer formation when added at low concentrations.

Catalytic activity of dimeric alpha-chymotrypsin. Acylation kinetics at low pH's.

Dimeric alpha-chymotrypsin (D) formed over a range of low pH (3.7-5.2) showed a different specificity from monomeric alpha-chymotrypsin (M) for the acylation step, though the catalytic system functions as well in the dimeric enzyme as in the monomeric enzyme. For aliphatic substituted phenyl esters a bulky substituent at the ortho-position of the phenyl ring reduced the acylation rate of the dimeric enzyme compared with the monomeric enzyme. Especially for aliphatic p-nitrophenyl esters, the dissociation constants of the DS complexes were always smaller than those of the MS complexes, independent of the aliphatic chain length of the substrate. The intrinsic acylation rate constant of the dimeric enzyme increased with the chain length of the substrate. These catalytic properties are discussed with reference to the structures of the catalytic and binding sites of the dimeric enzyme.

Effect of pressure on the deuterium exchange reaction of alpha-lactalbumin and beta-lactoglobulin.

The effect of pressure on the deuterium exchange reaction of alpha-lactalbumin (LA) and beta-lactoglobulin (LG) was investigated to determine the structural change in these proteins induced by elevated pressure. LG, one of the main components of milk whey, has been degraded selectively from other milk proteins including LA by protease treatment under high pressure (Hayashi, R., Kawamura, Y. and Kunugi, S. J. Food Sci. 1987; 52: 1107-1108). This was considered to occur because LG lost its native structure under high pressure more remarkably than LA. In the present study, the H/D exchange reaction was carried out under high pressure and the resulting structures were analysed by Fourier-transform infra-red (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, after the release of elevated pressure. The wavenumber of amide I bands in the FTIR spectrum assigned to alpha-helix and beta-sheet structures of the proteins, shifted to lower regions as the H/D exchange of protons proceeded. The integral band area of the amide proton signal in low-field regions of the NMR spectrum is related to the H/D exchange of less stable protons in the protein. H/D exchanges for LA at 200 MPa and LG at 50 MPa were detectable by NMR as a decrease in the amide proton signals, but they were detected less unambiguously by FTIR. This apparent difference may be explained by reference to an intermediary unfolding stage of the protein that is generated under moderately high pressure.

Chemical modification of proteins under high pressure. Preparation of ferrocene-attached bovine serum albumin and glucose oxidase.

High hydraulic pressure was used for denaturing proteins during chemical modifications. Bovine serum albumin and glucose oxidase were selected as the first targets of this unique technique and the ferrocene group was introduced into them, to obtain a macromolecular electron mediator and a self-electron-mediating oxidase, respectively. The result was compared with those obtained under non-denaturing conditions and under urea-denatured conditions. As for the number of ferrocene group linked to the protein, the pressure denaturation is superior to chemical denaturants, where ferrocenecarboxyaldehyde was used as the modifier. In both proteins the ferrocene group seemed to be introduced mainly inside the molecules with the pressure method, as the native conformation of the protein was restored when the high pressure was removed.

Pressure-induced change in proteins studied through chemical modifications.

Pressure-induced change of two bovine proteins, alpha-lactalbumin (LA) and beta-lactoglobulin (LG), was investigated at neutral pH by means of fluorescence and CD spectroscopy. The rate and the extent of modification was considerably increased by applying high pressure during the dansylation reaction of LG, while those for LA were only moderately affected. This difference was accounted for by the structural deformation of these proteins under high pressure. The fluorescence spectrum of these proteins measured under elevated pressure, as well as their fluorescence and CD spectra after the pressure release, indicated different responses towards pressure. The structural change of LA was practically reversible up to 400 MPa, whereas that of LG lost reversibility at 150 MPa or lower. Fluorescent measurement of dansylated (prepared at atmospheric pressure) proteins, especially the energy transfer from the intrinsic Trp residue to the dansyl group, showed that the protein structure was deformed by pressure and that the energy transfer facility of the two proteins was differently affected by high pressure, probably reflecting the degree of compactness of their pressure-perturbed structures.

Modification of the single unpaired sulfhydryl group of beta-lactoglobulin under high pressure and the role of intermolecular S-S exchange in the pressure denaturation [single SH of beta-lactoglobulin and pressure denaturation].

Chemical modification reactions of the unpaired sulfhydryl group of beta-lactoglobulin (LG) under high pressure and the role of this group in the pressure-induced denaturation were investigated. When LG was incubated at 400 MPa (pH 6.8) for 1 h, dimerization through intermolecular reaction of SH was observed. The generation of the covalently linked dimers were prevented by the presence of N-ethylmaleimide (NEM), an agent for SH-specific modification. The reactivity of the SH group of LG, which is buried inside in its native state, was increased by high pressure, as a result of its exposure to the protein surface accompanied by the pressure denaturation. The effect of NEM was also observed in the fluorescence change caused by high pressure, in both the intrinsic fluorescence of LG and the retinol fluorescence of the LG-retinol complex. The control showed an irreversible change at neutral pH, but it became mostly reversible in the presence of NEM. Compatible results were obtained by CD spectroscopy. Inter- and intramolecular reactions of the SH group are suggested to be main causes for the pressure-induced irreversible denaturation of LG.