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PubMed1991

Structural features of interstitial lung disease in systemic sclerosis.

Most studies of lung histology in systemic sclerosis have been based on autopsy specimens and consequently emphasize end-stage fibrotic disease. Although occasional pathologic descriptions of open-lung biopsies have recognized the presence of inflammatory cells, suggesting a similarity to "lone" cryptogenic fibrosing alveolitis, the two conditions have never been formally compared. In this study we describe the morphologic features of 49 open-lung biopsies from 34 systemic sclerosis patients with interstitial lung disease, many of whom had their lung disease diagnosed at an early stage. None had pulmonary hypertension. Examination of lung tissue by light microscopy showed the earliest changes to include patchy lymphocyte and plasma cell infiltration of the alveolar walls, interstitial fibrosis, and increased macrophages but only occasional polymorphonuclear cells and lymphocytes in the alveolar spaces. Alveolitis was not observed without fibrosis. Comparison of 22 biopsies with a similar number from patients with lone cryptogenic fibrosing alveolitis, matched for age and sex, revealed no qualitative or quantitative differences, other than a higher prevalence of focal lymphoid hyperplasia (follicular bronchiolitis) in the systemic sclerosis patients than in the lone cryptogenic fibrosing alveolitis controls (23 and 5%, respectively). There was an inverse correlation between the extent of interstitial inflammation and patients' age (p less than 0.05), disease duration (p less than 0.05), and age at onset of systemic sclerosis (p less than 0.01). There was also an inverse correlation between DLCO and interstitial fibrosis (p less than 0.01) and loss of lung architecture (p less than 0.05). Ultrastructural studies of eight systemic sclerosis biopsies showed evidence of endothelial and epithelial injury together with interstitial edema and excess collagen deposition. Occasional mast cells were observed, often in close contact with interstitial fibroblasts, but there were no tubuloreticular structures or evidence of immune complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1998

Structural features, physiological roles, and biotechnological applications of the membrane proteases of the OmpT bacterial endopeptidase family: a micro-review.

The proteins of the OmpT family represent a new class of integral membrane peptidases showing no sequence homology with other known classes of proteases. The prototype of the family, the Escherichia coli K-12 protein OmpT (or omptin), is an outer membrane endopeptidase with unusual specificity: it cleaves the peptide bond between two basic amino acids. A second distinct characteristic of OmpT is its ability to function even under extreme denaturing conditions (e.g. high concentration of urea). There is a growing number of reports that associate the proteases of the OmpT family with pathogenicity of certain gram-negative bacteria such as Yersinia pestis, Shigella flexneri, and pathogenic E. coli strains. This article reviews recent developments in the field of the OmpT proteases, provides a guide for the recognition of residues of the active site, and finally discusses potential uses of these proteins in biotechnology applications.

Bacterial Outer Membrane Proteins↗

PubMed2001

The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent.

BACKGROUND: Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5'-PuGC and 5'-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5'-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3'-side of the alkylated guanine. RESULTS: Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5'-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5'-PuGC or 5'-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency. CONCLUSIONS: The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.

Animals↗

PubMed1999

Structural features of a peptide corresponding to human kappa-casein residues 84-101 by 1H-nuclear magnetic resonance spectroscopy.

The peptide Val-Arg-Arg-Pro-Asn-Leu-His-Pro-Ser-Phe-Ile-Ala-Ile-Pro-Pro- Lys-Lys-Ile, which corresponds to residues 84-101 of human kappa-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a beta-turn involving residues Pro87-His90 of human kappa-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human kappa-casein (which is equivalent to Arg97 in bovine kappa-casein) unavailable for interaction with Asp249 of bovine chymosin, and may partly explain why human kappa-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.

Amino Acid Sequence↗

PubMed1985

Structural features involved in the biological activity of insulin and the insulin-like growth factors: A27 insulin/BIGF-I.

A synthetic insulin-like compound consisting of the A-chain of insulin extended at its carboxyl terminus with the hexapeptide "D-domain" of insulin-like Growth Factor II, linked via disulfide bonds to a B-chain corresponding to the "B-domain" of insulin-like Growth Factor I, has been examined for insulin-like metabolic activity and for mitogenic activity. The synthetic material (A27 insulin/BIGF-I) is less potent than insulin in metabolic assays, and less potent than both insulin and IGF-I in mitogenic assays. It is proposed that neither the "D-domain" nor the "B-domain" of the IGFs is a major contributor to mitogenic activity. Their presence in the same molecule does not result in significant growth-promoting activity.

Adipose Tissue↗

PubMed2005

Synthesis, structural features, absorption spectra, redox behaviour and luminescence properties of ruthenium(ii) rack-type dinuclear complexes with ditopic, hydrazone-based ligands.

The isomeric bis(tridentate) hydrazone ligand strands 1 a-c react with [Ru(terpy)Cl3] (terpy=2,2':6',2''-terpyridine) to give dinuclear rack-type compounds 2 a-c, which were characterised by several techniques, including X-ray crystallography and NMR methods. The absorption spectra, redox behaviour and luminescence properties (both in fluid solution at room temperature and in rigid matrix at 77 K) of the ligand strands 1 a-c and of the metal complexes 2 a-c have been studied. Compounds 1 a-c exhibit absorption spectra dominated by intense pi-pi* bands, which, in the case of 1 b and 1 c, extend within the visible region, while the absorption spectra of the rack-type complexes 2 a-c show intense bands both the in the UV region, due to spin-allowed ligand-centred (LC) transitions, and in the visible, due to spin-allowed metal-to-ligand charge-transfer (MLCT) transitions. The energy position of these bands strongly depends on the ligand strand: in the case of 2 a, the lowest energy MLCT band is around 470 nm, while in 2 b and 2 c, it lies beyond 600 nm. Ligands 1 a-c undergo oxidation processes that involve orbitals based mainly on the CH3--N--N== fragments. The complexes 2 a-c undergo reversible metal-centred oxidation, while reductions involve the hydrazone-based ligands: in 2 b and 2 c, the bridging ligand is reduced twice and in 2 a once before reduction of the peripheral terpy ligands takes place. Ligands 1 a-c exhibit luminescence from the lowest-lying 1pi-pi* level. Only for complex 2 a does emission occur; this may be attributed to a 3MLCT state involving the bridging ligand. Taken together, the results clearly indicate that the structural variations introduced translate into interesting differences in the spectroscopic, luminescence and redox properties of the ligand strands as well as of the rack-type metal complexes.

Journal Article↗

PubMed1984

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum.

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.

Base Sequence↗

PubMed2003

Interaction of metal ions with caffeine and theophylline: stability and structural features.

The interactions of caffeine and theophylline with divalent cadmium, mercury, strontium and barium ions were studied in aqueous solution and physiological pH. Fourier transform infrared spectroscopy (FTIR) and absorption spectra were used to determine the cation binding mode and association constants. Spectroscopic results showed that Cd(2+), Hg(2+), Sr(2+) and Ba(2+) bind strongly to caffeine and theophylline. Direct and indirect (through metal hydration shell) interactions were observed for caffeine and theophylline with Cd(2+), Hg(2+), Sr(2+) and Ba(2+) through O6 and N9 (caffeine) and O6, N9 and N7 atoms (theophylline). The overall binding constants are:k(Cd-caffeine) = 1.24 x 10(5) M(-1), k(Hg-caffeine) = 1.74 x 10(5) M(-1), k(Sr- caffeine) = 3.3 x 10(4) M(-1), k(Ba-caffeine) = 1.8 x 10(4) M(-1), k(Cd-theophylline) = 5.75 x 10(5) M(-1), k(Hg-theophylline) = 2.14 x 10(5) M(-1), k(Sr-theophylline) = 4.6 x 10(4) M(-1), k(Ba-theophylline) = 3 x 10(4) M(-1). These k values are evidence for weak and strong cation interactions in these metal complexes.

Caffeine↗

PubMed1977

Structural features of Salmonella typhimurium lipopolysaccharide required for activation of tissue factor in human mononuclear cells.

Activation of mononuclear cell tissue factor was examined utilizing lipopolysaccharides obtained from wild-type and both Rc and Re mutants of Salmonella typhimurium. Wild-type (smooth) lipopolysaccharide, galactose-deficient (Rc) lipopolysaccharide, heptose-deficient (Re) lipopolysaccharide, and lipid A preparations were all active in their ability to generate tissue factor activity in human mononuclear cells grown in tissue culture. Polymyxin B has been reported to prevent some of the lethal effects of endotoxin in vivo, and the drug reportedly binds to the 2-keto-3-deoxyoctulosonate-lipid A region of the lipopolysaccharide molecule. Polymyxin B was effective in inhibiting the tissue factor generating activity of wild-type lipopolysaccharide, Re lipopolysaccharide, and lipid A in a dose-dependent fashion. Treatment of lipid A preparations with mild alkali abolished the ability of these preparations to activate tissue factor in cells. Analogous to many of the other biologic properties of lipopolysaccharide, tissue factor activation in human mononuclear cells appears to depend upon the integrity of the lipid A portion of the molecule.

Cells, Cultured↗

PubMed1995

Structural features and functions of principal cells of the intermediate zone of the epididymis of adult rats.

BACKGROUND: In the present study, principal cells of the intermediate zone of the epididymis, an area situated between the initial segment and proximal caput, were observed to present morphological features distinct from those of principal cells of other regions. METHODS: The epididymides of adult rats were fixed by perfusion with glutaraldehyde and embedded in Epon. Administration of fluid phase tracers was performed in the case of several animals. Localization of anti-SGP-2 and anti-immobilin antibodies in conjunction with light (LM) and electron (EM) microscope immunocytochemistry was also performed. RESULTS: In the LM and EM, the most distinctive feature of many principal cells of this zone was the presence of apically located vacuoles referred to as giant endosomes due to their large size and because they readily incorporated tracers introduced into the lumen of the epididymal duct and were acid phosphatase-negative. Giant endosomes, containing electron-dense granular patches, appeared to form by the progressive fusion of small, medium, and large endosomes. In the supranuclear region, multivesicular bodies (MVBs) and lysosomes were present. Although smaller in size than the giant endosomes, MVBs and lysosomes contained the electron-dense patches. It is suggested from morphological images that giant endosomes fragment into smaller units corresponding to MVBs which gradually transform into lysosomes. Experiments using anti-SGP-2 and anti-immobilin antibodies revealed gold particles over the Golgi apparatus and secretory vesicles (150-300 nm) of principal cells of this zone as well as the luminal contents indicative of secretion of these proteins. Interestingly, giant endosomes were also immunolabeled with both antibodies as were stereocilia, coated pits and vesicles, and endosomes of various sizes; lysosomes were minimally labeled. These results suggest that principal cells of the intermediate zone endocytose as well as secrete SGP-2 and immobilin. The internalized SGP-2 and immobilin may correspond to that secreted further upstream and that, possibly due to their short half-life and terminated function, are removed from the lumen of the duct. Principal cells of this zone secrete these proteins possibly to replenish that lost by endocytosis. CONCLUSIONS: Principal cells of the intermediate zone contain giant endosomes. The presence of such large structures suggests that the early events in endocytosis is a slower process in principal cells of this zone as compared to other regions. The fact that these cells both secrete and endocytose SGP-2 and immobilin adds to the complexity of our understanding of how principal cells function along the length of the epididymis.

Animals↗

PubMed2001

Structural features of arabinogalactan-proteins from the fruit of Lycium chinense Mill.

In addition to the other arabinogalactan-proteins (AGPs) (Cp-1-C and -D) already reported, two kinds of AGP (Cp-2-B and Hp-2-C) were obtained from the fruit of Lycium chinense Mill. The ratio of arabinose to galactose was approximately 1:1 in both samples, and the carbohydrate was linked O-glycosidically to serine in Cp-2-B, and to both serine and threonine residues of the protein in Hp-2-C. The weight-average molecular weight was 71,000 for Cp-2-B and 120,000 for Hp-2-C. Both samples also contained non-reducing terminal 3-O- and 4-O-substituted galacturonic acids. The ratio of 6-O-substituted galactose (linear part) and 3,6-di-O-substituted galactose (branching point) was almost unity in both samples, being obviously different from the case of Cp-1-C (predominant in the branching domain) and Cp-1-D (predominant in the linear domain). These results offer fresh insight into the grouping of the AGPs, based on the ratio of 6-O- and 3,6-di-O-substituted galactosyl residues.

Amino Acids↗

PubMed1996

Different CD40-mediated signaling events require distinct CD40 structural features.

Signals delivered to B cells through CD40 are critical to the development of humoral immune responses. In this study we characterize regions of the 62-amino acid cytoplasmic domain of human CD40 (hCD40) that are essential for signal transduction and examine the functional consequences of mutations in these regions. A panel of mutant hCD40 molecules was stably expressed in mouse B cell lines and tested for its ability to stimulate Ab secretion, homotypic adhesion, and increased surface expression of B7, Fas, CD23, LFA-1, and intracellular adhesion molecule-1. Our results indicate that CD40 contains at least two major signaling determinants in the cytoplasmic domain: one disrupted by a truncation of 22 amino acids, and a second disrupted by the removal of 10 additional amino acids. The second determinant includes threonine 234, a residue previously shown to be important in CD40 signal transduction. Our functional analysis of alanine and serine substitutions at position 234 indicates that phosphorylation of this residue may be important for full CD40 signaling activity.

Amino Acid Sequence↗

PubMed1992

Structural features of substituted purine derivatives compatible with depletion of human O6-alkylguanine-DNA alkyltransferase.

A series of O6- and S6-substituted purine derivatives were tested for their ability to deplete the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) in cell-free extracts from HT29 colon tumor cells and intact HT29 cells. The order of potency was O6-(p-Y-benzyl)-guanine (Y = H, F, Cl, and CH3) > O6-benzyl-2'-deoxyguanosine > O6-(p-Y-benzyl)guanosine (Y = H, Cl, and CH3) > or = a series of 9-substituted O6-benzylguanine derivatives > or = O6-allylguanine > O6-benzylhypoxanthine > O6-methylguanine. A series of 7-substituted O6-benzylguanine derivatives, 2-amino-6-(p-Y-benzylthio)purine (Y = H, CH3), 2-amino-6-[(p-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and 7-benzylguanine were inactive. It is concluded that for efficient AGT depletion, an allyl or benzyl group attached through exocyclic oxygen at position 6 of a 2-aminopurine derivative is required. Activity is preserved with a variety of substituent groups attached to position 9 while substitution at position 7 leads to a complete loss of activity.

DNA Repair↗

PubMed1982

Oligonucleotide structural features involved in binding to and activation of the 2-5A-dependent endoribonuclease of L cells.

Several analogues of 2-5A were prepared and evaluated for their ability to bind to and/or activate the 2-5A dependent endonuclease from murine L cells. Replacement of one of the two 2',5'-phosphodiester bonds of ppp5'A2'p5'A2'p5'A with 3',5'-phosphodiester bonds gave analogues 40-50 X less active than 2-5A in binding to or activation of the 2-5A-activated endonuclease whereas replacement of both 2',5'-linkages with 3',5'-linkage resulted in a compound (3-5A) which was 10,000 X less effective in binding to the endonuclease and devoid of activity as an inhibitor of protein synthesis. The sequence of periodate oxidation/Schiff base formation/borohydride reduction gave a derivative of ppp5'A2'p5'A2'p5'A2'p5'A in which the 2'-terminal ribose was replaced with a N-hexyl morpholine ring. This material was 10 X more active than 2-5A an a inhibitor of translation. One possible explanation for this increased activity is that the 2'-terminally modified oligomer is resistant to degradation by the 2',5'-phosphodiesterase responsible for the degradation of 2-5A in cell extracts.

Adenine Nucleotides↗

PubMed1985

Structural features of the binding site of cholera toxin inferred from fluorescence measurements.

The dependence on pH of the fluorescence of cholera toxin and its A and B subunits has been studied at 25 degrees C. The fluorescence intensity of cholera toxin is highly pH-dependent. In the pH range 7-9.5 it reaches a maximum corresponding to a quantum yield of 0.076. In the pH range 4-7 a strong increase in fluorescence intensity is observed (delta Q/Qmax = 0.64). Evaluation of the pH sensitivity of the fluorescence intensity of the A and B subunits reveals that the B subunit is mainly responsible for the observed pH effect (delta Q/Qmax for B subunit = 0.64). The intensity changes are paralleled by similar although less pronounced changes in the average fluorescence excited state life-time tau (delta tau/tau max = 0.33 for cholera toxin). Fluorimetric titration of the B subunit, which is related to the indole fluorescence of the lone Trp-88, reveals that the fluorescence intensity changes in the pH range 4-7 are due to reaction of two types of ionizable quencher displaying apparent pKa values of 4.4 and 6.2, respectively. It is suggested that the increase in fluorescence intensity with a midpoint at pH 6.2 is the result of deionization of the imidazolium side-chain of one or two out of the four histidine residues present in each beta-polypeptide chain, whereas a deionized carboxyl group is responsible for the quenching with midpoint at pH 4.4. Complex formation of cholera toxin or B subunit with the monosialoganglioside GM1 or the oligosaccharide moiety of GM1 (oligo-GM1) completely prevents the quenching by both quenchers. Addition of 6 M urea also eliminates the pH effect. The quenching is not the result of the dissociation of the B subunit into its constituent monomers. Upon fluorimetric titration of cholera toxin or B subunit above pH 9, a progressive drop in both fluorescence intensity and tau occurs. This decrease could be due to energy transfer from the indole moiety of Trp-88 to ionized tyrosines or by quenching through an unprotonated epsilon-amino group of lysine. Fluorimetric titration of the A subunit indicates that the tryptophan fluorescence is only moderately altered by ionizable groups displaying a pKa in the range 4 to 9. Activation of A subunit does not affect this lack of pH sensitivity. Above pH 9, however, a much more significant drop in the fluorescence intensity of activated A subunit occurs. The structural implications of the results are discussed.

Animals↗

PubMed2006

Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition.

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K(d) value (2.9 microM) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K(d) values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIalpha led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.

Amino Acid Sequence↗

PubMed1985

Structural features of imidazole derivatives that enhance styrene oxide hydrolase activity in rat hepatic microsomes.

The present study is an investigation of the in vitro effects of several model imidazole compounds and three antifungal drugs on styrene oxide hydrolase activity in hepatic microsomes from control, PB-induced, and 3MC-induced rats. We first determined the influence of the position of substitution of a phenyl group in the imidazole or imidazoline ring on the ability of 10(-3) M concentration of the imidazole derivative to enhance microsomal epoxide hydrolase activity. This study showed that 1-phenylimidazole enhanced epoxide hydrolase activity to the greatest extent and that, for any one imidazole derivative, the extent of enhancement was similar for microsomes from untreated, PB-induced, or 3MC-induced rats. We next studied the potency and maximum effectiveness of several N-1-substituted imidazole derivatives with differing lipophilicities and pKa values. Hansch analysis showed that potency for activating epoxide hydrolase correlated with both lipophilicity and electron-withdrawing effects of imidazole ring substituents. The most potent compounds had aryl or aralkyl substitutents at the 1-position of imidazole and pKa in the range 7-9 and were clotrimazole, ketoconazole, miconazole, 1-benzylimidazole, and 1-(4-hydroxyphenyl)imidazole. These compounds enhanced styrene oxide hydrolase by 100% at concentrations less than 2 X 10(-4)M. 1-Benzylimidazole increased both Km and Vmax and abolished the inhibition observed at high concentrations of styrene oxide.

Animals↗

PubMed1999

Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I.

PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M. laminosus. The soluble PsaD could assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7x10(6) M-1, and the enthalpy change of -93.6 kJ mol-1. This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped. The Cys66, Lys21, Arg118 and Arg119 residues were exposed on the surface of soluble PsaD whereas the Lys129 and Lys131 residues were not exposed on the surface. Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of alpha-helical conformation.

Amino Acid Sequence↗