bluuebunny/crossref_metadata_2025 huggingface dataset:
Myosin heavy‐chain isoforms in human smooth muscle
Authors: SARTORE Saverio , MARZO Nicoletta DE , BORRIONE Anna Carmen , ZANELLATO Anna Maria Cecilia , SAGGIN Leopoldo , FABBRI Leonardo , SCHIAFFINO Stefano
Published: March 2005

The myosin heavy‐chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme‐immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non‐muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy‐chain isoforms, designated MHC‐1 and MHC‐2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy‐chain isoform (MHC‐3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC‐3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody.

The presence of three myosin heavy‐chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.
Molecular differences between rat‐liver and rat‐kidney biliverdin reductase
Authors: CASCONE Osvaldo , FRYDMAN Rosalìa B. , FERRARA Pascual , TOMARO Marìa L. , ROSENFELD Jorge
Published: March 2005

Rat‐liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, While the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form3) with a molecular mass of 68 kDa by a NAD+‐dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B.(1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore saught. Both form 1 biliverdin reductases, isolasted from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non‐denaturing conditions and on SDS/polyacrylamide gels. On HPLC analysis the peptides resulting from the CNBr cleavage were found to be the same for both enzymes, when either the native enzymes or their thioethylpyridine derivatives were compared. When the HPLC fingerprints of the tryptic digests were compared, they were found to be very similar, except for a peptide eluting at 31.60 min in the liver digest and at 23.60 min in the kidney digest. When the enzyme from both origins was alkylated with 4‐dimethylaminoazobenzene‐4′‐iodoacetamide and then digested with trypsin, the HPLC fingerprints of the alkylated cysteine‐carrying peptides were almost identical, except for a peptide with a retention time of 19.03 min in the liver digest and of 18.19 min in the kidney digest. The liver reductase was not amenable to Edman degradation suggesting a block at the NH2‐terminus; in the kidney enzyme, however, it was free and an NH2‐terminal sequence of 12 amino acids could be determined. The liver enzyme was found to be more sensitive toward p‐hydroxymercuriphenyl sulfonate than the kidney enzme.
Specific antibodies reveal ordered and cell‐cycle‐related use of histone‐H4 acetylation sites in mammalian cells
Authors: TURNER Bryan M. , FELLOWS Gillian
Published: March 2005

Antibodies specific for the acetylated forms of histone H4 (H4) were produced in rabbits with a synthetic peptide corresponding to the 18 N‐terminal residues of tetra‐acetylated H4 (i.e. with acetyllysine at positions 5, 8, 12 and 16). Specificity was determined by inhibition assays using four additional peptides, each acetylated at only a single site. Using an antiserum (R6) specific for the acetylation site at Lys‐5 we have estimated the proportion of Lys‐5 sites acetylated in the mono‐, di‐ and tri‐acetylated forms of H4 from randomly growing human HL‐60 cells. The values obtained (7%, 29% and 61% respectively) differ from those expected if acetylation were randoms (i.e. 25%, 50% and 75%) or if site usage followed a cet order for all H4 molecules (i.e. a jump from 0% to 100%). Antibodies from a second animal (R5) bound preferentially to peptides acetylated at Lys‐12 and also bound to mono‐acetylated H4 relatively weakly in several cell types. In contrast, mono‐acetylated H4 from metaphase HeLa cells labelled more strongly with both antisera, indicating significant acetylation at Lys‐5 and Lys‐12. We conclude that (1) the sites at Lys‐5 and Lys‐12 are under‐used in mono‐acetylated H4 from a variety of mammalian cell types and Lys‐8 and/or Lys‐16 are therefore the first to be acetylated, (2) more than one order of site usage is possible and (3) there is a metaphase‐specific shift in site usage. These results suggest that H4 acetylation plays a role in the modulation of chromatin structure in mammalian cells.
A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP synthase
Authors: STAN‐LOTTER Helga , HOCHSTEIN Lawrence I.
Published: March 2005

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F1 moiety from the Escherichia coli ATP synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20–22 mol/100 mol). Peptide mapping of subunits I and II denatured with sodium dodecyl sulfate showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F1 ATPase from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, halobacteria in general, possess an ATPase which is unlike the ubiquitous F0F1 ATP synthase.
A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6‐phosphate and fructose 1‐phosphate
Authors: van SCHAFTINGEN Emile
Published: March 2005

At a concentration of 1 mM, fructose 1‐phosphate stimulated about twofold, and glucose 6‐phosphate inhibited by about 30%, the phosphorylation of 5 mM glucose in high‐speed supernatants prepared from rat liver or from isolated hepatocytes, but did not affect, or barely so, the activity of a partially purified preparation of glucokinase. Anion‐exchange chromatography of liver extracts separated glucokinase from a fructose‐6‐phosphate‐sensitive and fructose‐1‐phosphate‐sensitive inhibitor of that enzyme. This inhibitor could be further purified by chromatography on phospho‐Ultrogel. It was destroyed by trypsin and was heat‐labile. It inhibited glucokinase competitively with respect to glucose and its inhibitory effect was greatly reinforced by fructose 6‐phosphate although not by glucose 6‐phosphate. Fructose 1‐phosphate relieved the enzyme of the inhibitory effect of the regulator and antagonised the effect of fructose 6‐phosphate in a competitive manner. It is concluded that the regulator plays a role in the physiological control of the activity of glucokinase, particularly with respect to the stimulatory effect of fructose in isolated hepatocytes (see preceding paper in this journal).
The azurin gene from Pseudomonas aeruginosa
Authors: ARVIDSSON Rolf H. A. , NORDLING Margareta , LUNDBERG Lennart G.
Published: March 2005

We have cloned and sequenced the Pseudomonas aeruginosa azurin structural gene and its flanking regions. The DNA sequence predicts a pre‐protein with a signal peptide of 19 amino acids followed by the 128‐amino‐acid mature azurin protein. Nuclease‐S1 mapping and primer elongation experiments indicated two 5′ termini of the azurin transcript. The major transcript of the azurin gene is initiated around 35 base pairs upstream from the translational start. The minor transcript, with a promoter region sharing homology with a consensus nif promoter of Klebsiella pneumoniae and also with other Pseudomonas genes, is initiated 145 base pairs upstreams of the azurin initiation codon. Downstream from the azurin structural gene a sequence similar to a transcriptional terminator is found. Northern blot analysis indicated two sizes of the azurin mRNA (0.54 kb and 0.65 kb) confirming the S1 mapping and the predictions from the nucleotide sequence.
Purification, characterization and subcellular localization of pig liver α‐L‐iduronidase
Authors: OHSHITA Takeyuki , SAKUDA Hitoshi , NAKASONE Susumu , IWAMASA Teruo
Published: March 2005

α‐L‐Iduronidase was purified about 100 000‐fold from pig liver by employing column chromatography on cellulose phosphate (P11), concanavalin A‐Sepharose 4B, heparin‐Sepharose 4B, Toyopearl HW‐55, Sephadex G‐100 and chelating Sepharose 6B charged with cupric ions. The molecular mass of the purified enzyme was estimated to be 70 kDa by Sephadex G‐100 column chromatography. The purified enzyme gave a single band on disc polyacrylamide gel electrophoresis without using sodium doecyl sulfate. However, two separate components of 70 kDa and 62 kDa appeared when it was analyzed by SDS/polyacrylamide gel electrophoresis. These 70‐kDa and 62‐kDa components were confirmed as α‐L‐iduronidase immunochemically. The isoelectric points of these enzymes were both 9.1 as measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Kmvalues were 3.0–3.5 and 65 μM 4‐methylumbelliferyl‐α‐L‐iduronide, respectively. The purified enzyme was stable in the pH range 3.5–6.0 under conditions with or without 0.5 M NaCl. However, in the presence of 0.5 M NaCl, it was unstable at pH 3.0. Moreover, it was conversely stabilized at pH 7.0 in the presence of 0.5 M NaCl. Immunohistochemically, the enzyme was found in the Kupffer cells and was abundant on their lysosomal membranes. In liver cells, however, the immunohistochemical reaction was weak.
Formation and structure of reaction products of cis‐PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di‐, tri‐ and penta‐nucleotides
Authors: DIJT Fransje J. , CHOTTARD Jean‐Claude , GIRAULT Jean‐Pierre , REEDIJK Jan
Published: March 2005

The reaction products of cis‐PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high‐performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300‐MHz 1H‐NMR (pH dependence of the non‐exchangeable base‐protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments.

The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15%cis‐Pt(NH3)2[d(ApG)N1(1), N7(2)] and 78%cis‐Pt(NH3)2‐[d(ApG)N7(1), N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81%anti, anti and 19%anti,syn product is present. With d(GpA) about equal amounts of N1.N7 and N7,N7 products were found; for both products the anti, anti and anti,syn conformations were found, respectively. Upon reaction of cis‐PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH > 1.5 these products were present in anti, anti conformation. However, for the d(pApG)‐platinum chelate at ‐ 20°C a small amount (<5%) of a second product could be observed in NMR. For the d(pGpA)‐platinum chelate a second N7,N7‐coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5′‐phosphate group is protonated).

With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra‐molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1‐coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.
Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii
Authors: McFARLAN Sara C. , HOGENKAMP Harry P. C. , ECCLESTON Eric D. , HOWARD James B. , FUCHS James A.
Published: March 2005

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174–9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA−) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114–12119]. In this paper we present the purification and characterization of one of these thioredoxins.

The new thioredoxin from C. nephridii, designated thioredoxin C‐2, is a heat‐stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C‐2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde‐3‐phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide ‐Cys‐Ala‐Pro‐Cys‐ at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence ‐Cys‐Gly‐Pro‐Cys‐. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.
Evaluation of the metal‐ion‐coordinating differences between the 2′‐, 3′‐ and 5′‐monophosphates of adenosine
Authors: MASSOUD Salah S. , SIGEL Helmut
Published: March 2005

The stability constants of the 1:1 complexes formed between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+ and 2′AMP2‐, 3′AMP2‐ or 5′AMP2‐ were determined by potentiometric pH titration in aqueous solution (I= 0.1 M, NaNO3; 25°C). The experimental conditions were carefully selected such that self‐association of the nucleotides and their complexes is negligibly small; i.e. it was made certain that the properties of the monomeric divalent‐metal‐ion–AMP [M(AMP)] complexes were studied. Based on recent measurements with simple phosphate monoesters, R‐MP2‐ where R is a non‐coordinating residue [Massoud, S. S. & Sigel, H. (1988) Inorg. Chem. 27, 1447–1453], it is shown that all the M(AMP) complexes of the alkaline earth ions, with the possible exception of Mg(5′AMP), have exactly the stability expected for a sole‐phosphate coordination of the metal ion. The same property is revealed for the complexes with Mn2+, Co2+, Zn2+ or Cd2+ and 3′AMP2‐; in case of Ni(3′AMP) and Cu(3′AMP) a slight stability increase just at the edge of the experimental‐error limits is indicated. This slight stability increase is attributed to the formation of a macrochelate (possibly with N‐3); in fact, additional information confirms macrochelation for Cu(3′AMP). About 45% of Cu(2′AMP) exists in aqueous solution as a macrochelate (probably involving N‐3); the other M(2′AMP) complexes (M2+= Mn2+, Co2+, Ni2+, Zn2+, Cd2+) form (if at all) only traces of a base‐backbound species. Most pronounced is macrochelate formation with 5′AMP2‐: all mentioned 3d ions and Zn2+ or Cd2+ form to some extent macrochelates via N‐7 (the structures of these closed species are indicated). In case of M(5′AMP) the base‐binding site is certain: replacement of N‐7 by a CH unit (tubercidin 5′‐monophosphate) eliminates any increased complex stability, whereas formation of the 1,N6‐etheno bridge to form 1, N6‐ethenoadenosine 5′‐monophosphate results in the phenanthroline‐like N‐6, N‐7 site which facilitates macrochelation significantly.
CF0, the proton channel of chloroplast ATP synthase
Authors: LILL Holger , JUNGE Wolfgang
Published: March 2005

The discharge of the flash‐induced transmembrane voltage through the exposed proton channel, CF0, of the chloroplast ATP synthase, CF0CF1 was investigated. EDTA treatment of thylakoid membranes exposed approximately 50% of total CF0 by removal of the CF1 counterparts. This greatly accelerated the decay of the transmembrane voltage, as was apparent from electrochromic‐absorption changes of intrinsic pigments and by pH‐indicating‐absorption changes of added dyes. Two decay processes were discernible, one rapid with a typical half‐decay time of 2 ms, and a slower one with a half‐decay time variable between 20 – 100 ms. Both were sensitive to CF0 inhibitors, but only the rapid decay process was also inhibited by added CF1. CF1 was effective in surprisingly small amounts, which were significantly lower than those previously removed by EDTA treatment. This finding corroborated our previous conclusion that the rapid decay of the transmembrane voltage was attributable to only a few high‐conductance channels among many CF0 molecules, typically in the order of one channel CF1‐depleted EDTA vesicle.

Inhibition of photophosphorylation in control thylakoids was measured as function of the concentration of CF0 inhibitors. It was compared with the inhibition of proton conduction through exposed CF0 in EDTA vesicles. Photophosphorylation and proton conduction by the high‐conductance form of CF0 were inhibited by the same low inhibitor concentrations. This suggested that the high‐conducting form of CF0 with a time‐averaged single‐channel conductance of 1 pS [Lill, H., Althoff, G. & Junge, W. (1987) J. Membrane Biol. 98, 69–78] represented the proton channel in the integral enzyme, which acted as a low‐impedance access from the thylakoid lumen to the coupling site in CF0CF1.

The slow decay process was attributed to a majority of low‐conductance CF0 channels, i. e. about 50 molecules/vesicle. The conductance of these channels was more than 100‐fold lower and they did not compete with the very few highly conducting channels for rebinding of added CF1. The low proton conduction of the majority of exposed CF0 molecules, possibly due to a structural rearrangement, may be protecting the thylakoid membrane against rapid energy dissipation caused by accidental loss of CF1. It may also explain the low single‐channel conductance of bacterial F0 reported in the literature.
Proton translocation coupled to the oxidation of carbon monoxide to CO2 and H2 in Methanosarcina barkeri
Authors: BOTT Michael , THAUER Rudolf K.
Published: March 2005

Cell suspensions of acetate‐grown Methanosarcina barkeri mediate the conversion of CO and H2O to CO2 and H2. The reaction is coupled with the phosphorylation of ADP. Evidence is presented that CO oxidation by the cells is associated with the transient acidification of the suspension medium. Up to 2 mol vectorial protons were measured/mol CO oxidized when the transmembrane electrical gradient was kept low by the addition of valinomycin (20 nmol/mg protein) and KCl (200 mM) or of KSCN (50 mM). No transient acidification was observed in the presence of the protonophore tetrachlorosalicylanilide which stimulated rather than inhibited CO oxidation. Proton extrusion remained unaltered when the proton‐translocating ATPase was specifically inhibited by dicyclohexylcarbodiimide. The latter finding indicates that proton translocation is associated with CO conversion to CO2 and H2 rather than with ATP hydrolysis in the cells. The data substantiate that the coupling of CO oxidation with ADP phosphorylation in M. barkeri occurs via a chemiosmotic mechanism.
Preparation, characterization and application of interleukin‐1β mutant proteins with surface‐accessible cysteine residues
Authors: WINGFIELD Paul , GRABER Pierre , SHAW Alan R. , GRONENBORN Angela M. , CLORE G. Marius , MacDONALD H. Robson
Published: March 2005

Two mutants of interleukin‐1β (K27C and K138C) were produced using site‐specific mutagenesis in which lysine residues at positions 27 and 138 of the mature protein sequence were substituted by cysteine residues. The conformations of the mutant proteins were studied by 1H‐NMR spectroscopy and shown to be similar to the wild‐type protein. The receptor‐binding affinity and biological activity of K27C and K138C were also similar to wild‐type protein. The substituted cysteines in both mutant proteins were shown to be solvent‐accessible as judged by their reactivity towards sulfhydryl reagents. As the wild‐type protein contains two cysteines, which are both solvent‐inaccessible in the native state, the mutants offer the opportunity to introduce probes in a sequence‐specific manner via reaction with sulfhydryl groups. Examples of this are described in which the K138C was disulfide‐linked to phycobiliproteins. The highly fluorescent conjugates had similar receptor‐binding affinities to that of the wild‐type unconjugated protein and were found suitable for flow‐cytometric analysis.
Comparative characterization of membrane‐associated and cytosolic Tyr‐protein kinases in human erythrocytes
Authors: CLARI Giulio , MORET Vittorio
Published: March 2005

In recent years, two protein‐tyrosine kinase activities, phosphorylating tyrosine residues on the transmembrane band‐3 protein, have been isolated from human erythrocyte membranes and partially characterized by different laboratories, i. e. one extracted by non‐ionic detergent (Triton X‐100 or Nonidet P‐40), the other solubilized by 0.25 M NaCl from the detergent‐insoluble residue.

The present paper shows that these two membrane‐associated Tyr‐protein kinases purified, in the presence of bovine serum albumin, by phosphocellulose chromatography followed by heparin‐Sepharose chromatography, have the same apparent molecular mass (36 kDa) determined by Ultrogel Ac44 filtration. Moreover, both Tyrprotein kinases exhibit several identical properties, including Km values for band 3, the random acidic copolymer poly(Glu, Tyr)4:1 and angiotensin II, pH dependence, response to Mn2+ and Mg2+, response to NaCl and 2,3‐bisphosphoglycerate.

All these properties are identical or very similar to those exhibited by the Tyr‐protein kinase previously isolated by us from human erythrocyte cytosol.

These results suggest that the two membrane‐associated and the cytosolic Tyr‐protein kinase activities are mediated by the same enzyme, distributed between the cytosol and the membrane structures.
Permeability to cefsulodin of the outer membrane of Pseudomonas aeruginosa and discrimination between β‐lactamase‐mediated trapping and hydrolysis as mechanisms of resistance
Authors: HEWINSON R. Glyn , CARTWRIGHT Steven J. , SLACK Mary P. E. , WHIPP Roger D. , WOODWARD Martin J. , NICHOLS Wright W.
Published: March 2005

A pair of strains of Pseudomonas aeruginosa (3‐Pre: cefsulodin‐sensitive, inducible β‐lactamase; and 3‐Post: cefsulodin‐resistant, elevated β‐lactamase, derived from 3‐Pre by subculture in the presence of cefsulodin) were taken as representative of the class of bacteria resistant to third‐generation cephalosporins due to elevated synthesis of the normally inducible, chromosomally encoded β‐lactamase. These two strains were used to differentiate between ‘trapping’ and ‘hydrolytic’ mechanisms of cefsulodin resistance by (a) measuring the outer‐membrane permeabilities‐to cefsulodin, (b) measuring the kinetics of cefsulodin hydrolysis and the stoichiometry of cefsulodin trapping by the periplasmic β‐lactamase, and (c) comparing the predictions of the trapping and hydrolysis hypotheses with the minimum inhibitory concentrations (MIC) of cefsulodin. The MIC of cefsulodin for strains 3‐Pre and 3‐Post were 2.35 μM (1.25 μg ml−1) and 37.6 μM (20.0 μg ml−1) respectively. The permeability parameter for cefsulodin of the outer membrane of the resistant strain was 0.0034 cm3 min−1 mg dry mass−1, so the flux of cefsulodin across its outer membrane at the MIC was calculated to be 0.120 nmol min−1 mg dry mass−1. Hydrolysis of cefsulodin by the β‐lactamase in the periplasm occurred at a rate of 0.118 nmol min−1 mg dry mass−1 which can thus account for resistance by matching the above rate of inflow. Trapping by the β‐lactamase, even with a 1:1 stoichiometry, would require the enzyme to be synthesized at 5.0 μg protein min−1 mg dry mass−1 or about 40% of the dry mass/generation. We conclude that hydrolysis, but not trapping, adequately explains the resistance to cefsulodin in P. aeruginosa 3‐Post. A similar calculation for latamoxef resistance, using data taken from the literature, led to the same conclusion.
Modulation of the GTPase activity of the chloroplast F1‐ATPase by ATP binding at noncatalytic sites
Authors: XUE Zhixiong , BOYER Paul D.
Published: March 2005

Although the binding of nucleotides at the noncatalytic sites of F1‐ATPase has been regarded as probably having some type of regulatory function, only limited observations have been reported that support such a role. We present here results showing that the presence of ATP at noncatalytic sites can give a fivefold enhancement of the rate of GTP hydrolysis by the chloroplast F1‐ATPase. Heat‐activation of the chloroplast F1‐ATPase in the presence of ATP, followed by column separation from the medium nucleotides gives an enzyme with two of the three noncatalytic sites filled with ATP. In contrast, heat‐activation in the presence of ADP gives an enzyme with only one noncatalytic site filled with ADP. Such an enzyme with two noncatalytic sites empty catalyzes MgGTP hydrolysis only very slowly. The filling of a second noncatalytic site with ATP by exposure of the enzyme to ATP without Mg2+ present, followed by column separation, markedly increases the rate of GTP hydrolysis. A further increase occurs when a third noncatalytic site is filled by exposure to Mg2+ and ATP. The rate of MgATP hydrolysis is the same for the enzyme heat‐activated in the presence of ATP or ADP, probably because MgATP, unlike MgGTP, rapidly binds to both catalytic and noncatalytic sites.
The high‐affinity binding of laminin to cells
Authors: NURCOMBE Victor , AUMAILLEY Monique , TIMPL Rupert , EDGAR David
Published: March 2005

The laminin proteolytic fragments 1 (derived from the intersection of the short arms of the cruciform laminin molecule) and 8 (derived from the laminin long arm) bind to distinct receptors on HT‐1080 human fibrosarcoma cells; both fragments are shown here to inhibit the high‐affinity binding of laminin to these cells. Inhibition of binding between fragment 8 and laminin was competitive, whereas that between fragment 1 and laminin was noncompetitive. This indicates that laminin and fragment 8 most probably share the same cellular receptors, whereas laminin and fragment 1 bind to distinct receptors, inhibition being due to steric hindrance. Surprisingly, fragment 1–4 (corresponding to the complete short arms of laminin) neither bound to HT‐1080 cells nor inhibited the binding of laminin or fragment 1. After treatment of fragment 1–4 with pepsin, however, the smaller subfragment 1 was liberated, which could then bind to the cells, and so was shown to block the binding of laminin and fragment 1. We conclude that native laminin bound to HT‐1080 cells via the fragment‐8‐binding site near the end of its long arm. Although these cells also have distinct receptors for the short arm fragment 1, this receptor‐binding site was not used as it appeared to be latent within the native laminin molecule.
The primary structure of the Chloroflexus aurantiacus reaction‐center polypeptides
Authors: SHIOZAWA Judith A. , LOTTSPEICH Friedrich , OESTERHELT Dieter , FEICK Reiner
Published: March 2005

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction‐center genes has been obtained.

The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N‐chlorosuccinimide peptide fragments isolated from the purified reaction‐center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2‐propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.
Structural studies on the fucosamine‐containing O‐specific polysaccharide of Proteus vulgaris O19
Authors: VINOGRADOV Evgeny V. , KACA Wieslaw , KNIREL Yuriy A. , RÓZLSKI Antoni , KOCHETKOV Nikolay K.
Published: March 2005

The polysaccharide chain of Proteus vulgaris O19 lipoppolysaccharide contains D‐galactose, N‐acetyl‐D‐glucosamine, N‐acetyl‐D‐galactosamine and N‐acetyl‐L‐fucosamine in the ratio 1:1:1:1. The structure of the polysaccharide was established by full acid hydrolysis and methylation analysis, as well as by non‐destructive methods. i.e. the computer‐assisted evaluation of the 13C‐NMR spectrum and computer‐assisted evaluation of the specific optical rotation by Klyne's rule. The polysaccharide is regular and built up of tetrasaccharide repeating units of the following structure: →3)‐α‐L‐FucNAcp‐(1→3)‐β‐D‐GlcNAcp‐(1→3)‐α‐D‐Galp‐(1→4)‐α‐D‐GalNAcp‐(1→

The O19‐antiserum cross‐reacts with lipopolysaccharide from P. vulgaris O42, the structure of which is still unknown. No cross‐reactions were observed with O‐polysaccharides Pseudomonas aeruginosa O7 and Salmonella arizonae O59 in spite of some structural similarities.
Analysis of N‐acetyl‐4‐O‐acetylneuraminic‐acid‐containing. N‐linked carbohydrate chains released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F
Authors: DAMM Jan B. L. , VOSHOL Hans , HÅRD Karl , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

The carbohydrate chains of equine fibrinogen were enzymatically released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl) asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500‐MHz 1H‐NMR spectroscopy. Four monosialo and four disialo diantennary N‐acetyllactosamine type of carbohydrate chains occur:

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Fe(II)‐substituted horse liver alcohol dehydrogenase, a model for non‐heme iron enzymes
Authors: BILL Eckhard , HAAS Claus , DING Xiao‐Qi , MARET Wolfgang , WINKLER Heiner , TRAUTWEIN Alfred X. , ZEPPEZAUER Michael
Published: March 2005

The catalytic Zn(II) ion of horse liver alcohol dehydrogenase (EE isozyme) was replaced by Fe(II), providing a novel iron protein with the unusual coordination of Fe(II) to two cysteines residues, one histine residue and water. The electronic structure of iron in this system was characterized by Mössbauer spectroscopy at various temperatures as well as applied magnetic fields and analysed in terms of the spin Hamiltonian formalism. The novelty we found is an unusually weak spin coupling (|J| < 0.1 cm−1) of a paramagnet (S= 1) with iron (S= 2). From EPR and biochemical studies we conclude that the corresponding chemical species is triplet oxygen (O2). The quantitative determination of the coupling energy was possible utilizing the competition between Zeemann interaction and spin coupling at weak magnetic fields and low temperature. Oxidation experiments followed by Mössbauer spectroscopy showed that the spin‐coupled system is an outer‐sphere Fe(II) · (O2)aq complex occurring as an intermediate during a Fe(II)‐catalyzed dioxygen activation. We observed two additional Fe(II) species after treatment with O2 and dithionite. The spin Hamiltonian parameters of iron in the coupled system are presented. The results are compared with those of iron in other non‐heme iron proteins.
Zonal distribution of fatty acid synthase in liver parenchyma of male and female rats
Authors: KATZ Norbert , THIELE Jutta , GIFFHORN‐KATZ Susanne
Published: March 2005

1. A sensitive radiochemical assay was established to determine the activity of fatty acid synthase in microdissected liver tissue of less than 1 μ dry mass.

2. In female rats, the enzyme activity in perivenous tissue was twice that in periportal liver tissue while it was homogeneously distributed in livers of male animals. The overall activity was higher in female than in male animals.

3. The absolute activity, as well as the perivenous/periportal ratio, was reduced during starvation and in diabetes. They were greatly increased after refeeding to values above those observed in animals during normal feeding.

4. Overectomy or administration of testosterone to female rats resulted in a significant reduction of the zonal heterogeneity.

5. Castration of administration of estradiol to male animals was followed by an increase in the enzyme activity exclusively in the perivenous tissue, resulting in a zonal heterogeneity as observed in female rats.
Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver
Authors: HASLER Thomas , CURTIUS Hans‐Christoph
Published: March 2005

Salmon liver was chosen for the isolation of 6‐pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500‐fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N‐(7‐dimethylamino‐4‐methylcoumarinyl)‐maleimide. The enzyme is heat‐stable up to 80°C, has an isoelectric point of 6.0–6.3, and a pH optimum at 7.5. The enzyme is Mg2+‐dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 μ. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues.

Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6‐pyruvoyl tetrahydropterin synthase, nor was any cross‐reaction in ELISA tests seen.
Control of mitochondrial transcription by thyroid hormone
Authors: MUTVEI Ann , KUZELA Stefan , NELSON B. Dean
Published: March 2005

Thyroid hormone regulation of rat liver mitochondrial transcription was investigated. Steady‐state levels of mitochondrial transcripts were measured by Northern blot analysis using cloned fragments of rat mtDNA. Thyroid hormone increased the steady‐state concentrations of all mitochondrial mRNAs by 2–8 fold after 1–3 days of hormone treatment, whereas no significant change in the mitochondrial rRNA was observed. Analysis of transcript synthesis in isolated mitochondria shows that part or all of this increase is accounted for by elevated synthesis. Mechanisms by which thyroid hormone regulates transcription of the mitochondrial genome are discussed.
Trypanothione reductase from Trypanosoma cruzi
Authors: JOCKERS‐SCHERÜBL Maria C. , SCHIRMER R. Heiner , KRAUTH‐SIEGEL R. Luise
Published: March 2005

Tryanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate.

Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the kcat/Km ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 × 106 M–1 s–1) and trypanothione disulfide (4.63 × 106 M–1 s–1).

Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH‐reduced forms, were irreversibly modified by the cytostatic agent, 1,3‐bis(2‐chloreethyl)‐1‐nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki= 40μM) than of trypanothione reductase (IC50= 200 μM).

Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374–5378] a nitrofuran derivative, 2‐(5‐nitro‐2‐furanylmethylidene)‐N,N′ ‐ [1,4‐piperazinediylbis (1,3‐propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki= 0.5 μM) than for glutathione reductase (IC50= 10μM). A naphthoquinone derivative, 2,3‐bis[3‐(2‐amidinohydrazono)‐butyl]‐1,4‐naphthoquinone dihydrochloride, turned out to be both an inhibitor (IC50= 1 μM) and an NADPH‐oxidation‐inducing substrate (Km= 14 μM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three‐dimensional structures of glutathione and trypanothione reductases.
Mutant proteins of human interleukin 2
Authors: WEIGEL Ulrich , MEYER Marita , SEBALD Walter
Published: March 2005

Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (I12) were generated by oligonucleotide‐directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein. The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined.

All muteins besides two (K44/T125 and Q110/T125) could be renatured and purified. One mutein (K94/T125) exhibited a more than tenfold‐improved renaturation yield.

One amino exchange (Asp‐20 to Asn) resulted in an about 20‐fold reduction in proliferative activity and high‐affinity receptor binding. The binding to the low‐affinity I12‐binding protein (Tac antigen) was unimpaired.

A second exchange (Arg‐38 to Gln) had no effect on proliferative activity. The binding to both the high‐ and the low‐affinity receptor, however, was reduced about 20‐fold.

Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild‐type interleukin 2 and muteins.

It is suggested that Asp‐20 forms part of the binding site for the large receptor subunit whereas Arg‐38 is involved in the contact site to the small subunit.
Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae
Authors: SCHWELBERGER Hubert Gernot , KOHLWEIN Sepp Dieter , PALTAUF Fritz
Published: March 2005

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6‐bisphosphate aldolase from Saccharomyces cerevisiae. Radio‐labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990‐base‐pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5′ end and the 90 base pairs at the 3′ end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high‐copy‐number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fbal::URA3lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.
Role of a sulfhydryl group in gastric lipases
Authors: GARGOURI Youssef , MOREAU Hervée , PIERONI Gérard , VERGER Robert
Published: March 2005

Native human and rabbit gastric lipases (HGL and RGL, respectively) were inactivated after modification of one sulfhydryl group/enzyme molecule. HGL and RGL were covalently labeled using 5,5′‐dithiobis(2‐nitro‐[14C]benzoic acid) and the interaction of 2‐nitro‐5‐thio‐[14C]benzoic‐acid‐labeled lipases ([14CINbs‐lipases) with monomolecular lipid films was investigated. Our results show that [14C]Nbs‐lipases bind to lipid films as efficiently as native HGL or RGL. The critical surface pressure πc and the maximal surface pressure (Δπmax) of [14C]Nbs‐lipases were enhanced in comparison with those of native RGL and HGL. These change in behavior were probably due to an increase in hydrophobicity brought about, directly or indirectly, by the binding of the Nbs radical. This chemical modification thus blocks the hydrolysis site and reinforces the hydrophobic character of the gastric lipases
The stereospecificity of the ferrous‐ion‐dependent alcohol dehydrogenase from Zymomonas mobilis
Authors: GLASFELD Arthur , BENNER Steven A.
Published: March 2005

Alcohol dehydrogenase from Zymomonas mobilis has been found to transfer the pro‐R hydrogen of NADH to acetaldehyde. This is the first report of the sterospecificity of a dehydrogenase in the mechanistic and structural class of Fe2+‐dependent alcohol dehydrogenases and offers an opportunity to expand mechanistic hypotheses relating stereospecificity, reaction mechanism and reaction thermodynamics in dehydrogenases.
Inhibition of PhoE translocation across Escherichia coli inner‐membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation
Authors: DE VRIJE Truus , BATENBURG A. Maximiliaan , JORDI Wilco , DE KRUIJFF Ben
Published: March 2005

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration‐dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.
A human melanoma‐derived cell line (IGR39) with a very high number of vasoactive‐intestinal‐peptide (VIP) receptors
Authors: MARTIN Jean‐Michel , LUIS José , MARVALDI Jacques , PICHON Jacques , PIC Pierre
Published: March 2005

Vasoactive intestinal peptide (VIP) stimulated in a dose‐dependent manner the accumulation of cAMP in human melanoma‐derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half‐maximum cAMP production was obtained at 0.78 nM VIP. VIP‐related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP ≫ peptide histidine–methioninamide > human growth‐hormone‐releasing factor(1–44) > secretin > glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production.

After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 < 2 min) and homologous.

Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP‐binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37°C with a VIP concentration of 1 μM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half‐maximum decrease in binding of mono[125I]iodinated VIP (125I‐VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP‐binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP‐free medium.

The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.