bluuebunny/crossref_metadata_2025 huggingface dataset:
Removal of N‐acetyl groups from blocked peptides with acylpeptide hydrolase
Authors: FARRIES Timothy C. , HARRIS Alan , AUFFRET Anthony D. , AITKEN Alastair
Published: March 2005

Acylpeptide hydrolase, an enzyme that removes the modified residue from N‐terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross‐linking with dimethyl‐suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N, N‐dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis.

Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non‐acetylated peptides are then adsorbed out with isothiocyanato‐glass. The N‐terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N‐terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.
The proteinase yscB inhibitor (PBI2) gene of yeast and studies on the function of its protein product
Authors: SCHU Peter , SUAREZ RENDUELES Paz , WOLF Dieter H.
Published: March 2005

The gene for proteinase yscB inhibitor I2B (PBI2) from Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library, and was sequenced. The gene codes for a single protein of 75 amino acids. In contrast to the published amino acid sequence [Maier, K., Müller, H., Tesch, R., Trolp, T., Witt, I. & Holzer, H. (1979) J. Biol. Chem. 254, 12555–12561] the DNA sequence revealed a valine instead of a leucine at position 33 (32 of the mature protein). Therefore the primary sequences of the isoinhibitors I2B of S. cerevisiae and I1B of Saccharomyces carlsbergensis differ only at position 34 (glutamic acid/lysine).

The open reading frame of PBI2 was replaced in vitro by the URA3gene and a I2B null mutant of S. cerevisiae was constructed by gene replacement. The mutation resulted in an elevation of the protein degradation rate by 50% when grown under nutritional stress compared to the isogenic wild type. Growth and viability of the cells was not significantly affected by the absence of I2B.
The binding of the ferric uptake regulation protein to a DNA fragment
Authors: SAITO Takeshi , WILLIAMS Robert J. P.
Published: March 2005

Using proton NMR, we have studied the binding of a DNA fragment in double‐stranded form to the ferric uptake regulation protein, Fur. We have also looked at the binding of [Cr(CN)6]3− to Fur with a view to testing whether binding is due to electrostatic interaction between Fur and the negative surface of the DNA. No competition at the DNA binding site was observed. Additionally, we have examined the binding of manganese ions to Fur in the presence of the DNA fragment and go on to discuss the likely way in which the Fur · DNA complex responds to metal‐ion binding to Fur.
Developmental expression of rabbit cytochrome P450 CYP1 A1, CYP1 A2 and CYP3 A6 genes
Authors: PINEAU Thierry , DAUJAT Martine , PICHARD Lydiane , GIRARD Franck , ANGEVAIN Jacques , BONFILS Claude , MAUREL Patrick
Published: March 2005

Developmental expression of CYP1A1, CYP1A2 and CYP3A6 in the rabbit have been studied. Cytochromes P450IA1, P450IA2 and P450IIIA6 exhibited comparable patterns of developmental expression. Present at low level (< 0.05 nmol/mg) in the new born animal up to week 3, these proteins sharply accumulated between weeks 3 and 4 to reach a maximum by week 4 (P450IA1, 0.2 nmol/mg; P450IA2, 0.8 nmol/mg; P450IIIA6, 0.12 nmol/mg) and decreased in the adult (P450IA1, 0.2 nmol/mg; P450IA2, 0.4 nmol/mg; P450IIIA6, 0.09 nmol/mg). Cytochromes P450IA1 and P450IA2 were not expressed in the untreated fetus. Onset of CYP3A6 gene expression occurred at day 30 of gestation and both transcription and mRNA accumulation were transplacentally inducible by rifampicin only shortly before birth, i.e. after treatment of the females between days 28 and 30 of gestation. Both long (1.85 kb) and short (1.7 kb) mRNA transcripts were expressed in untreated or rifampicin‐treated fetuses. CYP3A6 gene expression was also induced by rifampicin in 1‐week‐old and 2‐week‐old animals. Developmental expression of CYP1A1 and CYP1A2 genes was shown to be closely related to the diet change accompanying weaning which occurs at weeks 3–4. In animals subjected to either delayed (week 6) or early (week 2) weaning, sharp accumulation of messages, proteins and related activities were delayed or anticipated accordingly with respect to normal weaning. Artificially scheduled weaning gave similar results when repeated with biological‐grade lucern (grown in the absence of chemical fertilizers, pesticides, etc.), the main constituent of commercial rabbit chow. While CYP3A6 gene expression could be brought forward by early weaning at week 2, both message and protein did not exhibit increased accumulation after delayed weaning at week 6, and remained at the low level of the new born animal. Treatment of 1‐week‐old and 2‐week‐old animals with triiodothyronine or of 3‐week‐old animals with propylthiouracyl, an antithyroid factor, did not modify the normal pattern of developmental expression of genes CYP1A1, CYP1A2 and CYP3A6. It is concluded that (a) the onset of CYP3A6 gene expression in the fetus occurs at day 30 of gestation, (b) expression of this gene may be induced transplacentally by rifampicin, (c) CYP1A1, CYP1A2 and CYP3A6 gene expression is sharply activated at weaning, and (d) thyroid hormones appear not to be responsible for the pattern of developmental expression of these genes in the rabbit.
Kringle‐2 domain of the tissue‐type plasminogen activator
Authors: BYEON In‐Ja L. , KELLEY Robert F. , LLINÁS Miguel
Published: March 2005

A recombinant 90‐residue polypeptide fragment containing the three‐loop kringle‐2 domain of human tissue‐type plasminogen activator (t‐PA) has been studied by two‐dimensional 1H‐NMR spectroscopy at 500 MHz. Complete sequence‐specific resonance assignments were derived. Overall, the kringle exhibits a compact, folded conformation with more than 50% of the residues in irregular structures. Elements of secondary structure were identified from sequential, medium‐ and long‐range dipolar (Overhauser) interproton interactions. These identifications were corroborated by analysis of spin‐spin scalar 3JαN splittings and identification of backbone amide NH protons exhibiting retarded 1H/2H exchange in 2H2O. Three antiparallel β‐sheets and six tight turns were located. In addition, one short α‐helical region was found in the Ser43‐Ala44 ‐Gln44a‐Ala44b‐Leu44c‐Gly45 segment; this region contains three‐residue insertions unique to the t‐PA and urokinase kringles. Although the secondary structure of the t‐PA kringle 2 in solution is in overall agreement with that observed in the crystallographic structure of the prothrombin kringle 1 [Tulinsky, A., Park, C. H. & Skrzypczak‐Jankun, E. (1988) J. Mol. Biol. 202, 885–901], the α‐helical segment and other details of the secondary structure differ somewhat from the prothrombin homolog.
Possible regulation of hypothalamus and lung histidine decarboxylase activity by cAMP‐dependent protein kinase
Authors: HUSZTI Zsuzsanna , MAGYAR Kálmán , KELETI Judith
Published: March 2005

Activity of crude histidine decarboxylases (HisDC) from the hypothalamus and the lungs, was markedly reduced by incubating with ATP. Mg, cAMP and cAMP‐dependent protein kinase A, whereas activity of the crude glandular stomach enzyme changed only slightly under equal condition. The omission of one of these components failed to reduce HisDC activity by as much as the complete system. Addition of bovine heart (type II) or rat cerebellum protein kinase A (types I and II) inhibitor to the assay prevented enzyme inactivation; moreover, protein kinase A inhibitors permitted moderate activation under phosphorylating and control conditions.

Cytosolic hypothalamus HisDC activity was elevated 2—2.2‐fold by incubating the cytosol for 15 min in the presence of MnCl2, a known stimulator of phosphoprotein phosphatase; this was prevented when 20 mM NaF, a common inhibitor of phosphoprotein phosphatase, was added to the cytosol.

The apparent Km of ATP. Mg‐treated hypothalamus HisDC for histidine was elevated 5—10‐fold compared to controls, whereas the Vmax was approximately the same. Under this condition, the Km was calculated as high as 0.5—2.2 mM (depending on phosphorylating conditions), while controls had a Km of 0.1—0.3 mM (depending on the initial phosphorylating states). Addition of rabbit muscle (type I), bovine heart (type II) or rat cerebellum (types I and II) inhibitor of protein kinase A, to the phosphorylating mixture, abolished the difference in Km between control and ATP. Mg‐treated HisDC. Moreover, rat cerebellum protein kinase A inhibitors increased Vmax to above the control level; while 20 mM NaF (inhibitor of phosphoprotein phosphatase) decreased Vmax to approximately one half of that of the controls.

These data indicate that HisDC activity in the hypothalamus and the lungs, but not in the stomach, is affected in oppositely by protein kinase A and phosphoprotein phosphatases.
Studies on RNase T1 mutants affecting enzyme catalysis
Authors: GRUNERT Hans‐Peter , ZOUNI Athina , BEINEKE Marc , QUAAS Rainer , GEORGALIS Yannis , SAENGER Wolfram , HAHN Ulrich
Published: March 2005

Using an Escherichia coli overproducing strain secreting Aspergillus oryzae RNase T1, we have constructed and characterized mutants where amino acid residues in the catalytic center have been substituted. The mutants are His40 → Thr, Glu58 → Asp, Glu58 → Gln, His92 → Ala and His92 → Phe. His92 → Ala and His92 → Phe mutants are inactive. On the basis of their kcat/Km values, the mutants Glu58 → Asp and Glu58 → Gln show 10% and 7% residual activity, relative to wild‐type RNase T1, whereas the His40 → Thr mutant shows 2% activity. The effect of amino acid substitutions on the enzymatic activity of RNase T1 lends further support for a mechanism where Glu58 (possibly activated by His40 and His92 act as general base and acid respectively; this is discussed in terms of the known three‐dimensional structure of the enzyme.
Structure determination of the glycans of human‐serum α1‐antichymotrypsin using 1H‐NMR spectroscopy and deglycosylation by N‐glycanase
Authors: LAINE Anne , HACHULLA Eric , STRECKER Gérard , MICHALSKI Jean‐Claude , WIERUSZESKI Jean‐Michel
Published: March 2005

α1‐Antichymotrypsin purified from normal human serum was separated by affinity chromatography into three microheterogeneous forms on a concanavalin‐A – Sepharose column: a pass‐through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine‐linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re‐N‐acetylation and further separated by affinity chromatography on a concanavalin‐A – Sepharose column. The complete primary structure of the glycans was determined by high‐resolution 1H‐NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N‐glycanase was used for the deglycosylation of the unfractionated α1‐antichymotrypsin; the successive removal of the N‐linked complex‐type oligosaccharide side chains of α1‐antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that α1‐antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.
Complete nucleotide sequence of the gene for human C1 inhibitor with an unusually high density of Alu elements
Authors: CARTER Philip E. , DUPONCHEL Christiane , TOSI Mario , FOTHERGILL John E.
Published: March 2005

The complete (17159 bp) nucleotide sequence of the gene for the human C1 inhibitor has been determined. The transcription initiation site was examined by primer extension using human liver mRNA, and the messenger 5′‐end sequence was determined on clones obtained by the anchored polymerase chain reaction. The gene of this serpin molecule is split by seven introns, with junctions of phases zero and one. An outstanding feature of the intron sequences is the occurrence of 17 AluI repeats of all four ancestral subgroups, indicating that the gene has been invaded during consecutive waves of Alu amplification, including a recent one. These Alu repeats form the sites of deletion and insertion in several known lesions in the C1‐inhibitor gene. There is no obvious promoter site of the TATA‐box type at the 5′ end of the gene, but instead it contains a region of potential H‐DNA structure similar to that found upstream of the human c‐myc gene.
Zinc ions promote the binding of factor XII/factor XIIA to acidic phospholipids but have no effect on the binding of high‐Mr kininogen
Authors: SCHOUSBOE Inger , HALKIER Torben
Published: March 2005

Binding of high‐Mr kininogen and factor XII/factor XIIa to phospholipids coated on to polystyrene microtiter plates was investigated by ELISA. Both high‐Mr kininogen and factor XII/factor XIIa bound specifically to the phospholipid surface. Binding was observed to negatively charged phospholipids only. The binding of high‐Mr kininogen was not affected by the presence of zinc ions. At a surface concentration of 20% phosphatidylinositol phosphate in phosphatidylcholine a dissociation constant (kD) of 10 nM for the binding of high‐Mr kininogen was calculated. The amount of bound purified α‐factor XIIa could be increased 4–5‐fold in the presence of zinc ions. The lowest zinc ion concentration giving maximal binding was 0.1 mM. The binding of α‐factor XIIa was inhibited by high‐Mr kininogen. Independent of the presence of zinc ions or high‐Mr kininogen, a kD of 7.9 nM was calculated for α‐factor XIIa binding. The binding of prekallikrein was dependent upon the presence and the concentration of high‐Mr kininogen. In plasma containing aprotinin, the binding of high‐Mr kininogen was apparently inhibited in the presence of zinc ions, which was a prerequisite for the binding of factor XII. This apparently inhibitory effect of zinc ions on the binding of high‐Mr kininogen was probably due to the increased binding of factor XII, which displaced high‐Mr kininogen.
Comparison of the action of lipoprotein lipase on triacylglycerols and phospholipids when presented in mixed liposomes or in emulsion droplets
Authors: ROJAS Camilo , OLIVECRONA Thomas , BENGTSSON‐OLIVECRONA Gunilla
Published: March 2005

We have compared the action of lipoprotein lipase on liposomes of egg yolk phosphatidylcholine containing less than saturating amounts of trioleoylglycerol (< 3%) and emulsion droplets of the same lipids. The amounts of the two types of lipid particles (expressed in terms of phosphatidylcholine) needed to reach substrate saturation of the enzyme were similar, indicating similar binding of the lipase to these two lipid/water interfaces. With liposomes, as opposed to emulsion droplets, albumin was not necessary for continued hydrolysis of triacylglycerols, presumably because product fatty acids could be accommodated in the phospholipid bilayer. The maximal rate of trioleoylglycerol hydrolysis was more than 10‐fold higher, and the ratio of trioleoylglycerol/phosphatidylcholine hydrolysis was more than 50‐fold higher with the emulsion droplets. Qualitatively similar results were obtained with hepatic lipase, and a lipase from Pseudomonas fluorescence. The data suggest that the lipases remained at the interface for several catalytic cycles, and that a continued supply of substrate molecules to the active site favored triacylglycerol entry from the core of the lipid particle, rather than sliding in from the side through lateral diffusion in the surface layer.
Isolation and characterisation of milk growth factor, a transforming‐growth‐factor‐β2‐related polypeptide, from bovine milk
Authors: COX David A. , BÜRK Robert R.
Published: March 2005

A strategy was developed for the purification of a biologically active polypeptide growth and migration factor from skimmed bovine milk. This 25‐kDa dimeric molecule, termed milk growth factor (MGF), was isolated by a method consisting of a combination of strong cation‐exchange chromatography, low‐pressure hydrophobic‐interaction chromatography, hydrophobic‐interaction HPLC and size‐exclusion HPLC steps, which separated the protein according to its properties of charge, hydrophobicity and size, respectively. On average, a total purification of 106—107‐fold and a yield of approximately 115 ± 78 ng/MGF/milk was obtained using the method described. All purification steps were performed with novel combinations of ethanol and volatile acidic salt (ammonium acetate) solutions in order to retain biological activity of the protein. These conditions, together with the easy removal of salt by lyophilisation, facilitated the detection of biological activity in fractions collected at each step of the purification by means of a sensitive in vitro fibroblast‐migration assay in which the half‐maximal activity was obtained at a concentration of approximately 17 ± 4 pg/ml (i.e. approximately 1 pM) of the pure protein. Biological activity of the dimeric protein was unaffected by heat treatment or exposure to acid (pH 2.0), but was lost upon reduction to its monomeric form. Amino acid composition and sequence analyses demonstrate that MGF is related to transforming growth factor type β2.
Complete sequence and model for the A2 subunit of the carotenoid pigment complex, crustacyanin
Authors: KEEN Jeffrey N. , CACERES Isabel , ELIOPOULOS Elias E. , ZAGALSKY Peter F. , FINDLAY John B.C.
Published: March 2005

The complete sequence has been determined for the A2 subunit of crustacyanin, an astaxanthin‐binding protein from the carapace of the lobster Homarus gammarus. The polypeptide chain is 174 residues long and is similar to proteins of the retinol‐binding protein superfamily. Some regions of the sequence are most similar to the retinol‐binding protein, β‐lactoglobulin subgroup, while the disulphide bonding pattern is more akin to that seen in the porphyrin binding proteins insecticyanin and bilin‐binding protein. It is beginning to appear as though this superfamily of proteins, characterized by a similar gross structural framework, may be further subdivided into interrelated subclasses. Model building based on the coordinates of the known structure of human plasma retinol‐binding protein and on empirical prediction algorithms has allowed the putative identification of side‐chains which line the binding cavity. This pocket is larger than in retinol binding protein and β‐lactoglobulin but does not allow the carotenoid to adopt a folded conformation. The amino acid composition of the pocket does not support a ‘charge‐shift’‐type hypothesis to support the bathochromic shift phenomenon which takes place on interaction of the chromophore with the protein. Instead aromatic side‐chains may play a prominent role.
Microheterogeneity of rat submaxillary gland kallikrein k10, a member of the kallikrein family
Authors: GUTMAN Ninette , ELMOUJAHED Abdel , BRILLARD Michèle , DU SORBIER Bertrand MONEGIER , GAUTHIER Francis
Published: March 2005

A tissue‐kallikrein‐related proteinase present in rat submaxillary glands, which was previously called endopeptidase k, has been further characterized and compared with other members of the kallikrein family. The partial primary structure of this proteinase, now called kallikrein k10, is very similar to that of proteinase B [Kato, H., Nakanishi, E., Enjyoji, K., Hayashi, I., Oh‐Ishi, S. & Iwanaga, S. (1987) J. Biochem. (Tokyo) 102, 1389–1404] and T‐kininogenase [Xiong, W., Chen, L. M. & Chao, J. (1990) J. Biol. Chem. 265, 2822–2827], but no corresponding gene or mRNA has so far been found. Kallikrein k10 is microheterogeneous due to variable glycosylation of its N‐terminal light chain and to variable processing at its kallikrein loop, as shown by endo‐β‐N‐acetylglucosaminidase F treatment, amino acid sequence analysis and mass spectrometry. The enzymatic properties of the two molecular varieties of kallikrein k10 towards synthetic fluorogenic substrates are not significantly different. Both cleave specifically after Arg residues, but, in contrast to true tissue kallikrein, may accomodate either polar or nonpolar residues at position P2. Kallikrein k10 also differs from tissue kallikrein by its sensitivity to soyabean trypsin inhibitor. Its biological function may therefore differ from that of tissue kallikrein, especially as it does not induce a transient decrease in blood pressure when injected in vivo.
Binding of long‐chain fatty acids to serum albumin in healthy humans Relationship to obesity
Authors: BRODERSEN Rolf , VORUM Henrik , KRUKOW Niels , PEDERSEN Anders Overgaard
Published: March 2005

Equilibria of the binding of palmitate to serum albumin in adults are studied by dialysis‐exchange‐rate determinations. The results are used for a description of binding equilibria of fatty acids in general, as follows.

1. The reserve albumin concentration, p, for binding of palmitate is used as an approximate measure of p*, the reserve albumin concentration for binding of mixed fatty acids present in serum.

2. The total availability of fatty acids is defined as C*/p*, where C* is the total concentration of non‐esterified fatty acid.

3. The fatty‐acid‐binding property of albumin is described by L*=p*/P—αC*/P is the albumin concentration. The numerical value of α is −0.05.

The above parameters are measured in sera from four healthy volunteers, in whom large variations of serum fatty acid concentration occurred. A group of 64 healthy students showed considerable variation of L* from one individual to another. It is found that L* decreases significantly with increasing body mass index (body mass divided by the square of the body length). In 42 patients with diabetes type I, L* was independent of body mass index. These findings are consistent with a previously formulated hypothesis of the mechanism of obesity.
Reactive blue 2 is a potent inhibitor of a thylakoid protein kinase
Authors: COUGHLAN Sean J. , DAVENPORT James W. , HIND Geoffrey
Published: March 2005

The anthraquinone dye reactive blue 2 was found to be a potent inhibitor of a protein kinase isolated and purified from thylakoids. This enzyme was also inhibited in situ, with corresponding inhibition of ATP‐dependent quenching of the chlorophyll fluorescence. The mode of inhibition was noncompetitive, with a Ki of 8 μM for the membrane‐bound kinase, and 6 μM for the purified kinase. The inhibitor did not modify the substrate preference of the endogenous kinase and could be removed from the membrane by washing. Unlike reactive blue 2, the enzyme did not partition into detergent micelles and is therefore presumably not a hydrophobic, intrinsic membrane protein.
Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor β
Authors: WRANA Jeffrey L. , OVERALL Christopher M. , SODEK Jaro
Published: March 2005

Transforming growth factor β (TGF‐β) and secreted protein acidic rich cysteine (SPARC) have been associated with the rapid remodeling of connective tissues that occurs in wound healing and developmental processes. To study the temporal and mechanistic aspects of TGF‐β‐regulated extracellular‐protein gene expression in human fibroblasts, confluent cells were pulse labeled for 30 min with [35S]methionine at various times following the single addition of 1.0 ng/ml TGF‐β. After a 4‐h chase period, specific radiolabeled media proteins were isolated by either immunoprecipitation or affinity chromatography and quantitated. Stimulation of SPARC synthesis was first apparent 5 h after addition of TGF‐β, reached a maximum (3.5‐fold increase) at 24 h and persisted for at least 96 h. A similar temporal response to TGF‐β was observed for the extracellular matrix proteins collagen and fibronectin. In contrast, TGF‐β induced a strong (> sixfold increase at 9 h after addition of TGF‐β), but transient stimulation of the synthesis of endothelial‐type plasminogen activator inhibitor. Northern blot analysis showed that SPARC mRNA levels were increased by TGF‐β in parallel with increase in SPARC synthesis; a maximum 3.9‐fold increase in SPARC mRNA being reached at 24 h. Similarly, the levels of both collagen and fibronectin mRNA were increased by TGF‐β treatment. In each case the stimulation of mRNA was blocked by the presence of the translation inhibitor, cycloheximide. Stability of SPARC mRNA (half‐life of approximately 50 h) was not significantly altered by TGF‐β. In contrast, the stability of collagen and fibronectin mRNA were both increased in the presence of TGF‐β; the increased stability being pronounced in less dense cells. In addition to effects on stability, transcription of the collagen and fibronectin genes was increased 7 h after TGF‐β addition, but returned to control levels by 24 h. However, transcription of the SPARC gene was unaffected by TGF‐β at both time points and, together with the stability data, indicates that TGF‐β regulates SPARC expression via a nuclear post‐transcriptional mechanism. Differential regulation of gene expression by TGF‐β in a precise temporal pattern via transcriptional and post‐transcriptional pathways may be an important aspect of the response of fibroblast cells in a wound environment.
Complex formation between chlorophyll a and cytochrome c: surface properties at the air‐water interface
Authors: LAMARCHE François , PICARD Gilles , TÉCHY Françoise , AGHION Jacques , LEBLANC Roger M.
Published: March 2005

The binding of cytochrome c to an insoluble monolayer of chlorophyll a was studied. Surface pressure (∏), surface potential (ΔV) and [14C]cytochrome c surface‐concentration (Γ) isotherms were measured versus molecular area (σ) in mixed films. Compared to the successive‐addition method, this procedure allows the formation of homogeneous mixed films. The cytochrome c is incorporated into a chlorophyll a monolayer, compressed at a surface pressure of 20 mN · m−1. On expansion, the quantity of protein incorporated into the monolayer gradually increases. Subsequent compression‐expansion cycles result in similar isotherms, distinct from that measured during the first expansion. All surface properties measured, but more specifically the surface radioactivity of [14C]cytochrome c, indicate the irreversibility of protein incorporation into the chlorophyll a monolayer. In fact, surface properties of the binary film are completely different from the properties of either of the pure components. As a result, calculated values of surface potentials for mixed films using the additivity law deviate from experimentally measured potentials. The absorption and fluorescence spectra of mixed films transferred onto a solid substrate by the Langmuir‐Blodgett technique, indicate a dilution effect of chlorophyll a by cytochrome c. However, the dilution effect cannot be detected by the fluorescence lifetimes of pure chlorophyll a and mixed chlorophyll a‐cytochrome c films, both shorter than 0.2 ns. This provides support for the existence of an energy‐transfer mechanism between chlorophyll a monomer and chlorophyll a aggregates which could serve as an energy trap. The role of the protein could be related to that of the matrix.
Differences in 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐inducible CYP1A1 expression in human breast carcinoma cell lines involve altered trans‐acting factors
Authors: THOMSEN Jane S. , NISSEN Lena , STACEY Simon N. , HINES Ronald N. , AUTRUP Herman
Published: March 2005

Differences in expression of the CYP1A1 gene have previously been observed in human breast carcinoma cell lines exposed to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). Using an expression vector containing the functional 5′‐regulatory region of human CYP1A1 (up to – 1140) fused to the reporter gene CAT (for chloramphenicol acetyltransferase), the breast carcinoma cell lines, MCF‐7, T47‐D and ZR‐75‐1, classified as highly responsive to TCDD, were highly responsive to TCDD in the chloramphenicol acetyltransferase assay as well. Gel mobility shift assays have shown that these cell lines express a nuclear protein that binds the aryl hydrocarbon (Ah) receptor responsive element. The low or non‐responsive cell lines, AL‐1, BT‐20 and CAMA‐1, were low or non‐responsive to TCDD in the chloramphenicol acetyltransferase assay, suggesting that the low‐responsive phenotype is caused by altered trans‐acting factors. However, the mechanism appears to differ among the cell lines. Whereas no induction was observed in AL‐1, a fivefold induction in activity was observed in BT‐20 and CAMA‐1. The TCDD concentration giving half‐maximum induction differed greatly between CAMA‐1 and BT‐20. The gel mobility shift assay showed the presence of a protein that bound specifically to the Ah responsive element in the non‐responsive cell line AL‐1, as well as the low‐responsive cell lines, BT‐20 and CAMA‐1. The high basal activity but low induction observed in CAMA‐1 may be due to an Ah receptor constitutively bound to the Ah responsive element.
Structure determination of [d(ATATATAUAT)]2 via two‐dimensional NOE spectroscopy and molecular dynamics calculations
Authors: KERWOOD Deborah J. , ZON Gerald , JAMES Thomas L.
Published: March 2005

Proton homonuclear two‐dimensional (2D) NOE spectra were obtained for the decamer [d(ATATATAUAT)]2 as a function of mixing time, and proton resonance assignments were made. Quantitative assessment of the 2D NOE cross‐peak intensities was used in conjunction with the program MARDIGRAS, which entails a complete relaxation matrix analysis of the 2D NOE peak intensities, to obtain a set of upper and lower bound interproton distance constraints. The analysis with MARDIGRAS was carried out using three initial models: A‐DNA, B‐DNA and Z‐DNA. The distance constraints determined were essentially the same regardless of initial structure. These experimental structural constraints were used with restrained molecular dynamics calculations to determine the solution structure of the decamer. The molecular dynamics program AMBER was run using A‐DNA or B‐DNA as starting model. The root‐mean‐square (rms) difference between these two starting models is 0.504 nm. The two starting models were subjected to 22.5 ps of restrained molecular dynamics calculations. The coordinates of the last 10.5 ps of the molecular dynamics runs were averaged to give two final structures, MDA and MDB. The rms difference between these two structures is 0.09 nm, implying convergence of the two molecular dynamics runs. The 2D NOE spectral intensities calculated for the derived structures are in good agreement with experimental spectra, based on sixth‐root residual index analysis of intensities. A detailed examination of the structural features suggests that while the decamer is in the B‐family of DNA structures, many torsion angle and helical parameters alternate from purine to pyrimidine. with kinks occuring at the U‐A steps.
1H‐NMR studies of the interaction between a self‐complementary deoxyoligonucleotide duplex and indolo[2,3‐b]quinoxaline derivatives active against herpes virus
Authors: PATEL Naina , BERGMAN Jan , GRÄSLUND Astrid
Published: March 2005

1H NMR has been used to study the interactions of ellipticine and the ellipticine analogues 2,3‐dimethyl‐6‐(2‐dimethylaminoethyl)6H‐indolo‐[2,3‐b]quinoxaline and 6‐(2‐dimethylaminoethyl)6H‐indolo‐[2,3‐b]quinoxaline with the self‐complementary decadeoxyribonucleotide d(CGCGATCGCG)2. The Watson‐Crick H‐bonded imino proton resonances were studied. The drugs were shown to bind to the duplex by intercalation involving slow exchange kinetics for the imino proton resonances on the NMR time scale (500 MHz). Ellipticine and the 2,3‐dimethyl analogue were found not to show strong base preferences, while the other analogue was found to have a preferred primary binding site between the A · T base pairs with a probable minor secondary binding site between the A · T and adjacent G · C base pairs. The new drug‐shifted imino proton resonances were assigned through saturation transfer experiments. The base‐specific interactions were accompanied by drug‐induced non‐uniform broadening of the resonances (due to intermediate chemical exchange kinetics), in the spectral region of the non‐exchangeable aromatic and sugar H1′ proton resonances of the oligonucleotide at 25°C.
Proton‐NMR studies of the effects of ionic strength and pH on the hyperfine‐shifted resonances and phenylalanine‐82 environment of three species of mitochondrial ferricytochrome c
Authors: MOENCH Susan J. , SHI Ting‐Mei , SATTERLEE James D.
Published: March 2005

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3‐CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso‐1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the ß‐CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3‐CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870–7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3‐CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 β‐CH2 group and the heme 3‐CH3 substituent. In addition, studies of the effect of pH on the 1H‐NMR spectrum of yeast iso‐1 ferricytochrome c show that the heme 3‐CH3 proton resonance exhibits a pH‐dependent shift with an apparent pK in the range of 6.0 –7.0. The chemical shift change of the yeast iso‐1 ferricytochrome c heme 3‐CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934–4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C‐terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3‐CH3 group in yeast iso‐1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8‐CH3 and the 7α‐CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of ∼ 7.0. The titrating group responsible for these spectral changes is proposed to be His39.
Two‐dimensional 1H, 15N‐NMR investigation of uniformly 15N‐labeled ribonuclease T1
Authors: SCHMIDT Jürgen M. , THÜRING Harald , WERNER Angela , RÜTERJANS Heinz , QUAAS Rainer , HAHN Ulrich
Published: March 2005

Uniformly 15N‐enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear 1H, 15N‐shift correlation spectra were recorded utilizing proton detection. Direct 1H, 15N connectivities were established applying the heteronuclear multiple‐quantum coherence technique. Additional 1H, 1H‐TOCSY or 1H, 1H‐NOESY transfer steps allowed for sequential assignments. Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple‐bond correlation experiment. Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple‐quantum filtering methods. 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended. Preliminary 1H, 15N NMR investigations were performed on the RNase‐T1–3′GMP inhibitor complex. Results were interpreted with respect to nucleotide binding.
A study on the inactivation of wheat carboxypeptidase
Authors: KUNUGI Shigeru , TANABE Kazuo , KONDOH Toshihiro , YOKOYAMA Masahito , ITO Nobuya , NOMURA Akihiko , FUKUDA Mitsuhiro
Published: March 2005

The mechanism of inactivation of carboxypeptidase from wheat at high pH was studied kinetically and spectrophotometrically.

Inactivation of wheat carboxypeptidase is characterized by initial, transient high activity, soon followed by loss of activity, accompanied by an increase in both fluorescence intensity and anisotropy and a decrease a decrease in circular dichroism.

A scheme was proposed in which the enzyme undergoes dissociation into monomers, the total activity of which becomes twice that of the normal dimer on a mass basis, and soon further denatures and aggregates.

Treatment of the enzyme with a bifunctional reagent partly prevented denaturation of the monomer and hence increased the peptide synthetic activity.
Bacillus subtilis expresses two kinds of haem‐A‐containing terminal oxidases
Authors: LAURAEUS Marko , HALTIA Tuomas , SARASTE Matti , WIKSTRÖM Mårten
Published: March 2005

The expression of two different aa3‐type cytochrome oxidases is demonstrated in Bacillus subtilis. One of them (denoted caa3‐605), was predicted by DNA‐sequencing of Bacillus cytochrome oxidase genes, but has not been found previously. It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3. The other oxidase (denoted aa3‐600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al. [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97–103]. It shows no immunological cross‐reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem‐A chromophores. It catalyses oxidation of quinols, which is proposed to be its physiological function.
Molecular modelling of bacterial deep rough mutant lipopolysaccharide of Escherichia coli
Authors: KASTOWSKY Manfred , SABISCH Andreas , GUTBERLET Thomas , BRADACZEK Hans
Published: March 2005

Molecular modelling techniques have been applied to compute the conformations accessible to bacterial deep rough lipopolysaccharide of Escherichia coli (Re‐LPS). Analyses of the results showed that the models typically exhibit a tilt of the diglucosamine backbone with respect to the membrane normal of 53 ± 7° while both the glucosamine ring planes are orientated approximately parallel to the membrane normal. Different models were found to show compact and elongated types of acyl chain arrangements, both producing anisotropic lateral dimensions of the models of 1.0–1.1 nm and 1.7–2.0 nm for the shorter and the longer side, respectively. The conformationally allowed range of the isolated dOclA(α‐2‐4)dOclA disaccharide (dOclA = 3‐deoxy‐d‐manno‐octulosonic acid) was found to be extremely limited. It appeared that the dOclA disaccharide (dOclA)2 is centred at the top of the Re‐LPS molecule preferring two orientations stabilized by hydrogen bonds involving only one phosphate group of the lipid A moiety at a time.

The effect of charges on the Re‐LPS conformations has been studied in separate calculations. From these calculations it was obvious that charges have no significant effects on the conformations of the isolated lipid A and (dOclA)2 moieties. However, it was found that the orientation of (dOclA)2 with respect to the lipid A part is highly sensitive to charges, i.e. in the charged models the proximity of phosphate and carboxyl groups is prevented by strong electrostatic repulsion between these negatively charged groups.

In order to rationalize the acyl chain packing of the models, a simple geometrical model which correlates the tilt of the diglucosamine backbone with the energetically favoured close packing of the acyl chains is proposed. Furthermore, the possibility of a chelate‐like complexation of divalent cations and its contribution to head group mobility is discussed.
Analysis of the promoter region of napin genes from Brassica napus demonstrates binding of nuclear protein in vitro to a conserved sequence motif
Authors: ERICSON Mats L. , MURÉN Eva , GUSTAVSSON Hans‐Olof , JOSEFSSON Lars‐Göran , RASK Lars
Published: March 2005

Napin is a seed storage protein from Brassica napus (rape) that is encoded by a gene family. We have isolated and characterized a novel napin gene, nap B. Comparisons of the 5′‐upstream region of nap B to the promoter regions of previously published napin genes reveal that certain sequence motives are evolutionary conserved and may be implicated in gene regulation. These consensus motives, that overlap with purine/pyrimidine stretches, are TACACAT and CATGCA both of which frequently occur as overlapping, direct repeats. Related or identical sequences are also found in the upstream regions of the homologous genes of Arabidopsis thaliana. One copy of the CATGCA motif occurs in close proximity to the TATA box in all the above genes. In this case it overlaps with an octamer sequence (ATGCAAAT) which is a sequence element common in many eukaryotic promoters and enhancers. The TACACAT sequence, as part of a longer purine/pyrimidine stretch, was found to interact with a protein present in crude nuclear extracts from developing B. napus seeds. Napin genes appear to be methylated to almost equal extents whether present in expressing or non‐expressing tissue.
Nuclear transition protein 1 from ram elongating spermatids
Authors: CHIRAT Frédéric , MARTINAGE Arlette , BRIAND Gilbert , KOUACH Mostafa , VAN DORSSELAER Alain , LOIR Maurice , SAUTIÈRE Pierre
Published: March 2005

The ram transition protein 1 (TP1) is present in spermatid cell nuclei in the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its primary structure was determined by automated Edman degradation of S‐carboxamidomethylated protein and of peptides generated by cleavage with thermolysin and endoproteinase Lys‐C.

The ram TP1 is a small basic protein of 54 residues and structurally very close to other mammalian TP1. The mass spectrometric data obtained from the protein and its fragments reveal that ram TP1 is indeed a mixture (approximately 5:1) of two structural variants (Mr 6346 and 6300). These variants differ only by the nature of the residue at position 27 (Cys in the major variant and Gly in the minor variant). The study of phosphorylation sites has shown that four different serine residues could be phosphorylated in the monophosphorylated TP1, at positions 8, 35, 36 or 39.

From previous physical studies, it has been postulated that the Tyr32 surrounded by two highly conserved basic clusters was responsible for the destabilization of chromatin by intercalation of its phenol ring between the bases of double‐stranded DNA.

The presence of three phosphorylatable serine residues in the very conserved sequence 29–42 is another argument for the involvement of this region in the interaction with DNA.
Identification of Lys116 as the target of N‐ethylmaleimide inactivation of ferredoxin:NADP+ oxidoreductase
Authors: ALIVERTI Alessandro , GADDA Giovanni , RONCHI Severino , ZANETTI Giuliana
Published: March 2005

Oxidized ferredoxin:NADP+ oxidoreductase (FNR) was slowly and irreversibly inactivated by N‐ethyl‐maleimide. Complete protection against inactivation was afforded by saturating concentrations of NADP+. In the presence of NADPH, a rapid inhibition of the enzyme ensued; however, this inhibition was found to be reversible. In the tryptic map of the flavoprotein, modified with N‐ethyl[2, 3‐14C]maleimide in oxidizing conditions, a unique radioactive peptide was found. Its sequence comprised residues 110–117 of the enzyme; Lys116 was shown to be the residue alkylated by N‐ethylmaleimide. It is noteworthy that the same residue of FNR was found to be modified by 5‐dimethylaminoaphthalene‐1‐sulfonyl(dansyl) chloride at the putative NADP(H)‐binding site [Cidaria, D., Biondi, P. A., Zanetti, G. & Ronchi, S. (1985) Eur. J. Biochem. 146, 295–299]. Furthermore, the data reported here demonstrate that the sulfhydryl groups of FNR are not involved in enzyme inactivation by N‐ethylmaleimide.
The monoclonal antibody 2G8 is carbohydrate‐specific and distinguishes between different forms of vertebrate cholinesterases
Authors: LIAO Jian , HEIDER Harald , SUN Man‐Chi , STIEGER Susi , BRODBECK Urs
Published: March 2005

The monoclonal antibody (mAb) 2G8 (subclass IgG2a) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys flesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1.8) from human serum. Binding of mAb 2G8 to the globular G2 form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen‐tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme‐linked immunosorbent assay and Western blot showed that heat‐denatured, diisopropylfluorophosphate‐treated, CNBr‐ and trypsin‐digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N‐glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N‐linked carbohydrates.
Inorganic‐pyrophosphate‐dependent phosphorylation of spinach thylakoid proteins
Authors: PRAMANIK Alauddin M. , BINGSMARK Sophie , LINDAHL Marika , BALTSCHEFFSKY Herrick , BALTSCHEFFSKY Margareta , ANDERSSON Bertil
Published: March 2005

It has been shown for the first time that several photosystem‐II thylakoid proteins and the main chlorophyll‐a/b light‐harvesting complex can be phosphorylated with inorganic pyrophosphate as phosphate donor. With pyrophosphate, as with ATP, the protein‐kinase reaction is dependent on light or a strong reducing agent. The reaction which can be demonstrated in well‐washed spinach thylakoids is dependent on electron transport and is controlled by the redox state of the plastoquinone pool. It is suggested that the pyrophosphate‐dependent thylakoid protein phosphorylation is mediated by the same kinase which is responsible for the ATP‐dependent protein phosphorylation. This pyrophosphate‐dependent kinase activity may be derived from an evolutionary precursor from which ATP‐dependent protein phosphorylation also developed.