bluuebunny/crossref_metadata_2025_split huggingface dataset:
A highly active and oxidation‐resistant subtilisin‐like enzyme produced by a combination of site‐directed mutagenesis and chemical modification
Authors: GRØN Hanne , BECH Lene M. , BRANNER Sven , BREDDAM Klaus 
Published: March 2005

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1–10% of that obtained with the wild‐type enzyme. In contrast, the oxidation‐labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222.It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site‐directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild‐type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me‐S‐Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild‐type enzyme when assayed against the substrate Suc‐Ala‐Ala‐Pro‐Phe‐NH‐Ph‐NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc‐Ala‐Ala‐Pro‐Phe‐NH‐Ph‐NO2. This demonstrates the feasibility of replacing the oxidation‐labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.
Endopeptidase variations among different life‐cycle stages of African trypanosomes
Authors: MBAWA Zeres R. , GUMM Ian D. , FISH Wallace R. , LONSDALE‐ECCLES John D. 
Published: March 2005

Lysates of different life‐cycle stages of Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei were analysed for endopeptidase activity, using reaction conditions which permitted a distinction to be made between lysosomal and non‐lysosomal activity [Lonsdale‐Eccles, J. D. & Grab, D. J. (1987) Eur. J. Biochem. 169, 467–475]. Hydrolysis of Z‐Arg‐Arg‐NHMec (Z = benzyloxycarbonyl, NHMec = 7‐amino‐4‐methylcoumaryl) and Z‐Gly‐Gly‐Arg‐NHMec occurred predominantly at alkaline pH and was observed in lysates of both insect and mammalian infective forms of T. brucei and T. congolense. Compared to their other life‐cycle stages, procyclic forms of T. brucei and epimastigote forms of T. congolense exhibited enhanced hydrolysis of these substrates. Low levels of hydrolysis of Z‐Arg‐Arg‐NHMec were observed in the bloodstream and epimastigote forms of T. vivax. The hydrolysis of Z‐Gly‐Gly‐Arg‐NHMec in each of the life‐cycle stages of T. vivax was generally below detectable levels.In lysates of T. congolense, proteolytic and Z‐Phe‐Arg‐NHMec‐hydrolytic activity in bloodstream forms > metacyclic > epimastigote > procyclic forms. In T. vivax Z‐Phe‐Arg‐NHMec‐hydrolytic activity differed slightly according to the origin of the parasite but, in general, followed the same pattern (i.e. bloodstream forms > epimastigote forms, with metacyclic forms usually intermediate between these two). In T. brucei, Z‐Phe‐Arg‐NHMec‐hydrolytic activity in bloodstream forms > procyclic forms. Upon differentiation of the long, slender bloodstream forms into short, stumpy forms the Z‐Phe‐Arg‐NHMec‐hydrolytic activity was elevated even further. Thus, during their life cycle, each of these African trypanosomes exhibits complex changes of endopeptidase activity, suggestive of an induction of lysosomal activity between the insect and mammalian forms.
Conformational change of turkey‐gizzard caldesmon induced by specific chemical modification with carbodiimide
Authors: MARTIN François , HARRICANE Marie‐Cécile , AUDEMARD Etienne , PONS Françoise , MORNET Dominique 
Published: March 2005

Water soluble 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide was used to internally cross‐link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86 974 Da of chicken‐gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13 873–13 879). Comparative circular dichroism analysis indicated a decrease of the α‐helix content from 43% to 36% resulting from the chemical modification. The 1H‐NMR spectra of the native and modified caldesmon showed that the covalent cross‐linking affected mainly the central and N‐terminal parts of the molecule. The C‐terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90‐kDa proteolytic N‐terminal fragment to give an internally cross‐linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F‐actin bundles.These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F‐actin bundles or weaker interactions between actin filaments.
Regulation of the expression of the sex‐specific isoforms of cytochrome P‐450 in rat liver
Authors: KOBLIAKOV Valerji , POPOVA Natalia , ROSSI Lorenzo 
Published: March 2005

The hepatic metabolism of steroid hormones and of xenobiotics frequently depends on the expression of the sex‐specific isoforms of cytochrome P‐450 and on differences in sex hormones.Following biochemical, immunological and molecular biological investigations, it was shown that in adult rat liver there exist at least four male‐specific and one female‐specific isoforms of cytochrome P‐450. The designation of these sex‐specific genes is IIC11, IIIA2, IIC13 and IIA2 in males, and IIC12 in females. The irreversible programming of the expression of these isoforms of cytochrome P‐450 in adulthood occurs during the perinatal period of life, and is named enzyme imprinting. One of the main factors that regulates the expression of the sex‐specific isoforms of cytochrome P‐450 is the level of androgens in the blood. Castration of adult rats decreased the level of the male isoforms of cytochrome P‐450 and the activity of the monooxygenase enzyme system that remained higher than in intact females.The mechanism of enzyme imprinting can be explained as follows: neonatal androgens program the secretion of hypothalamic hormones, somatostatin and growth‐hormone‐releasing factor. These factors determine the type of growth hormone secretion in adult rats, and this controls the type of sex‐specific isoforms of cytochrome P‐450 expressed in adulthood.Metabolic regulation similar to that outlined above was shown to occur for several metabolism‐dependent chemical carcinogens. Such a pathway may explain the different sensitivity displayed by male and female rats to treatment with these carcinogenic agents. One possible way of modulating the expression of some isoforms of cytochrome P‐450 in adult rats is by treating neonates with specific xenobiotics that change the constitutive expression of neonatal androgens. It appears that this enzyme imprinting plays an important role in determining the individual sensitivity to the carcinogenic effects of chemicals.
Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol—cytochrome‐c reductase complex
Authors: COCCO Tiziana , LORUSSO Michele , SARDANELLI Anna Maria , MINUTO Michele , RONCHI Severino , TEDESCHI Gabriella , PAPA Sergio 
Published: March 2005

Structural and functional characteristics of subunits of bovine heart cytochrome‐c reductase have been investigated by controlled digestion of soluble and membrane‐reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits.The results obtained show that the N‐terminal segments of core protein II and the 14‐kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe‐S protein, located at the outer C‐periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N‐terminal region. Proteolytic cleavage of 7–11 residues from the N‐terminal segment of the 14‐kDa protein is apparently associated with decoupling of redox‐linked proton pumping. Partial digestion of core protein II, the 6.4‐kDa protein, and the C‐terminal region of the 9.2‐kDa protein, is without effect on the redox and proton‐motive activity of the complex.
Structure/function relationship of proteins belonging to the family of receptors coupled to GTP‐binding proteins
Authors: STROSBERG Arthur Donny 
Published: March 2005

The structural properties of a number of proteins belonging to the family of receptors coupled to GTP binding proteins are discussed in relation to their function.The structure of the ligand binding site and of the regions involved in coupling to the G proteins are analyzed mainly for the adrenergic and muscarinic cholinergic receptors, for which site‐directed mutagenesis and chimaeric constructions have been studied. The structure of the genes are compared and the presence of various regulatory elements is discussed in relation to control of expression.Mechanism of desensitization and internalization, while mostly studied for the β2‐adrenergic receptor, are proposed to be generally applicable to all G‐protein‐coupled receptors.
Core region of Citrobacter O23 lipoplysaccharide
Authors: KATZENELLENBOGEN Ewa , GAMIAN Andrzej , ROMANOWSKA Elzbieta , DABROWSKI Ursula , DABROWSKI Janusz 
Published: March 2005

A novel enterobacterial core region in Citrobacter O23 lipopolysaccharide is described. Its structure was determined by methylation analysis/mass spectrometry, chemical degradation and one‐ and two‐dimensional 1H‐NMR spectroscopy:

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where PPEtN stands for diphosphorylethanolamine, and dOclA for 3‐deoxy‐D‐manno‐octulosonic acid.
Hydrolysis of intact and Cys‐Phe‐cleaved human atrial natriuretic peptide in vitro by human tissue kallikrein
Authors: VANNESTE Yves , MICHEL Alain , DESCHODT‐LANCKMAN Monique 
Published: March 2005

Atrial natriuretic peptide (ANP) is a 28‐amino‐acid hormone involved in the regulation of fluid balance. In circulation, the proteolytic inactivation of ANP has been demonstrated to involve both membrane metallo‐endopeptidase and an aprotonin‐sensitive activity, probably corresponding to kallikrein [Vanneste, Y., Pauwels, S., Lambotte, L., Michel, A., Dimaline, R. & Deschodt‐Lanckman, M. (1990) Biochem. J. 269, 801–806]. In the present study, we focused on the aprotinin‐sensitive pathway of ANP metabolism. In order to identify the cleavage sites recognized by kallikrein within the sequence of the hormone, tissue kallikrein was purified to homogeneity from human urine and the degradation of human ANP by the enzyme preparation was studied.Our results demonstrate that both intact and Cys7‐Phe8‐cleaved ANP, the initial metabolite produced in circulation by the metallo‐endopeptidase, are substrates in vitro for purified tissue kallikrein. However, the Cys‐Phe‐cleaved peptide was degraded approximately fourfold faster than the intact hormone by the purified enzyme. The first degradation step of ANP by tissue kallikrein involves two cleavages occurring at the bonds Arg3‐Arg4 and Gly16‐Ala17, generating an inactive, open‐ring metabolite. Incubation of ANP for a longer period with the enzyme led to the generation of several additional degradation fragments. Ten peaks were separated by HPLC and characterized by amino acid analysis. The results allowed the identification of a total of eight peptide bonds susceptible to hydrolysis by tissue kallikrein in the sequence of ANP: Arg3‐Arg4, Ser5‐Ser6, Cys7‐Phe8, Arg11‐Met12, Gly16‐Ala17, Gly20‐Leu21, Ser25‐Phe26 and Arg27‐Tyr28.These results indicate that the aprotinin‐sensitive activity involved in the metabolism of ANP in circulation could correspond to tissue kallikrein. However, clear identification of ANP as a novel physiological substrate of the enzyme will need further investigation.
Visible, EPR and electron nuclear double‐resonance spectroscopic studies on the two metal‐binding sites of oxovanadium (IV)‐substituted D‐xylose isomerase
Authors: BOGUMIL Ralf , HÜTTERMANN Jürgen , KAPPL Reinhard , STABLER Rainer , SUDFELDT Christoph , WITZEL Herbert 
Published: March 2005

The two metal‐binding sites of the D‐xylose isomerase from Streptomyces rubiginosus were studied using VO2+ as a sensor for the ligand environment. Titration of the tetrameric enzyme with VO2+, followed by EPR spectroscopy and inhibition studies, show that the first four VO2+ equivalents occupy, in analogy to Co2, Cd2+ and Pb2+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high‐affinity B site. The low‐affinity A site could be studied selectively by blocking the B site with visible and EPR‐silent Cd2+. The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high‐affinity site could be demonstrated by electron nuclear double‐resonance (ENDOR) studies of the 4VO2+ enzyme, and was assigned to a histidine ligand. The 14N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 1H‐ENDOR coupling of 1.7 MHz, exchangeable in D2O, has been assigned to the N–H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low‐affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 1H‐ENDOR couplings observed in this sample were assigned to H2O ligands in the vicinity of VO2+. The results closely relate to what is known from X‐ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations.In mixed metal samples with four VO2+ and four Co2+ equivalents, the VO2+ is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+.
Conformational fluctuations and protein reactivity
Authors: LAVALETTE Daniel , TETREAU Catherine , BROCHON Jean‐Claude , LIVESEY Alastair 
Published: March 2005

When a protein's active site happens to be strongly coupled with the protein structure, the rate constant of the reaction may eventually be modulated by the conformational fluctuations. Evidence for this effect has long been provided by extensive flash photolysis investigations of liganded hemoproteins and more recently of the nonheme respiratory protein hemerythrin in hydro‐organic solvents. Within a given protein conformational substate, an elementary reaction step is characterized by one single free energy barrier and by a first‐order rate constant, k, which changes with temperature according to an Arrhenius law. At physiological temperature and low viscosity, ultrafast conformational relaxation causes efficient averaging of the reaction rates and the protein displays exponential kinetics with and average rate constant 〈k〉. Under sufficiently general conditions, it can be shown that 〈k〉 also follows a simple Arrhenius law with ‘effective’ values of the pre‐exponential factor Aeff and activation enthalpy Heff. It is found that Aeff strongly depends on the overall shape of the rate constant distribution and that Heff actually corresponds to the lower limit of the enthalpy of activation, i.e. the value associated with the highest possible reaction rate.The underlying distribution of rate constants can be reconstructed from a set of experiments in which the kinetics depart from an exponential, i.e. at low temperature and high viscosity. The most probable distribution of exponentials consistent with the observed kinetics of the geminate recombinations of oxygen with photodissociated hemerythrin has been determined by using a new approach, known as the maximum entropy method. The results are consistent with a single pre‐exponential value and a distributed enthalpy spectrum. As expected, Heff does not coincide either with the most probable nor with the average value of the enthalpy. The most salient findings are that the probability for any protein molecule to have an enthalpy of activation equal to the effective value Heff vanishes and that Aeff differs by nearly three orders of magnitude from the true value Ao.Biochemical reaction rates are actually average values, since protein reactions are measured under physiological conditions, where conformational relaxation is always fast. Our understanding of the significance of Aeff and Heff is therefore entirely dependent on the knowledge of the distribution function of the rate constants. In particular, enthalpy and entropy terms of similar reactions performed by different proteins cannot be compared as long as the distribution of the rate constants remains unknown.
Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban
Authors: MACDOUGALL Lindsay K. , JONES Larry R. , COHEN Philip 
Published: March 2005

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)‐associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70–80% of the SR‐associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser‐16 (the residue labelled by cAMP‐dependent protein kinase, PK‐A) or Thr‐17 (the residue phosphorylated by an SR‐associated Ca2+/calmodulindependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity.A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160kDa, responsible for targetting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK‐A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser‐16 or Thr‐17) phosphatase activity in the material sedimented by centrifugation at 45000xg, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X‐100, but not with 0.5 M NaCl or digestion with α‐amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK‐A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR‐bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser‐16 and Thr‐17) in vivo.Hearts left in situ post mortem lose 85–95% of their PP1 activity within 20–30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognised previously.
Binding of α‐ and βγ‐subunits of G0 to β1‐adrenoceptor in sealed unilamellar lipid vesicles
Authors: KURSTJENS Nicol P. , FRÖHLICH Margareta , DEES Christian , CANTRILL Richard C. , HEKMAN Mirko , HELMREICH Ernst J. M. 
Published: March 2005

First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid‐rich vesicles with average diameters ranging over 150–200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30–70 nm (fraction II). When the lipid mixture containing dimyristoylglycero‐phosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoyglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with α‐ and γ‐subunits of G0‐proteins purified to homogeneity from bovine brain, the lipid‐rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a β1‐adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside‐out. On co‐reconstitution of either αo or alone with β1‐adrenoceptors, some of these subunits appear together with β1‐adrenoceptors in the small vesicle fraction II, but much more αo was bound to the receptor in the presence of βγ‐subunits.The observations reported are novel and surprising in several respects: firstly, they suggest that βγ‐subunits can bind to the non‐activated β1‐receptor where they may serve as an anchor for α‐subunits. Secondly, the binding of αo‐ and βγ‐subunits to the β1‐adrenoceptors enhances the basal GTPase activity of αo. Thirdly, since the binding domains of the β1‐adrenoceptor for G‐proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G‐proteins with the β‐receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213–215 (1987)] for the transducin‐rhodopsin interactions. Finally, the binding of Go‐subunits from bovine brain to a β1‐adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.