bluuebunny/crossref_metadata_2025 huggingface dataset:
Kininogen‐Derived Fluorogenic Substrates for Investigating the Vasoactive Properties of Rat Tissue Kallikreins — Identification of a T‐Kinin‐Releasing Rat Kallikrein
Authors: Moujahed Abderrahman Ei , Brillard‐Bourdet Michéle , Juliano Maria A. , Moreau Thierry , Chagas Jair R. , Gutman Ninette , Prado Eline S. , Gauthier Francis
Published: July 2004

Peptide substrates with intramolecularly quenched fluorescence that reproduce the rat kininogen sequences at both ends of the bradykinin moiety were synthesized and used to investigate the kinin‐releasing properties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Substrates derived from rat H‐ and L‐kininogen were cleaved best by rK1, especially that including the N‐terminal insertion site of bradykinin, Abz‐TSVIRRPQ‐EDDnp(Abz = 0‐aminobenzoyl, EDDnp = ethylenediamine 2,4‐dinitro‐phenyl), which was cleaved at the R‐R bond with a Kcat/Km of 12400 mM−1 S−1. Replacement of the P2' residue Pro by Val in Abz‐TSVIRRPQ‐EDDnp gave a far less specific substrate that was rapidly hydrolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl‐N‐methyl coumarylamide substrates, which lack prime residues, also had low specificities. The importance of the P2' residue for rK1 specificity was further demonstrated using a human‐kininogen‐derived substrate that included the N‐terminal insertion site of bradykinin (Abz‐LMKRP‐EDDnp). This was cleaved at the M‐K bond by hK1 (kallidin‐releasing site), but at the K‐R bond (bradykinin‐releasing site) by rK1. Competition experiments with Abz‐TSVIRRPQ‐EDDnp, which is resistant to most kallikreins, and Abz‐TSVIRRVQ‐EDDnp, a general kallikrein substrate, demonstrated that the former competitively inhibited hydrolysis by rK9 and hK1, with Ki, values similar to the Km values for the substrate. Thus Pro in P2′ does not prevent the peptide binding to the enzyme active site, but impairs cleavage of the scissile bond. The T‐kininogen‐derived substrate with the T‐kinin C‐terminal sequence (Abz‐FRLVR‐EDDnp) was cleaved by rK10 (Kcat/Km= 2310 mM−1 s−1) and less rapidly by rK1, rK7 and hK1, at the R‐L bond, while that corresponding to the N‐terminal (Abz‐ALDMMISRP‐EDDnp) of T‐kinin was resistant to all five kallikreins used, suggesting that none has T‐kininogenase activity. But this substrate was hydrolysed by a semi‐purified sample of submandibular gland extract. Another kallikrein, identified as kallikrein rK3, was isolated from this fraction and shown to hydrolyze Abz‐ALDMMISRP‐EDDnp; rK3 also specifically released T‐kinin from purified T1/T2‐kininogen after HPLC fractionation. Injection of purified rK3 and of Abz‐ALDMMISRP‐EDDnp‐cleaving fractions into the circulation of anesthesized rats caused transient falls in blood pressure, as did purified rK1 but none of the other purified rat or human kallikreins. This effect occurred via activation of the kinin system since it was blocked by Hoe l40, a kinin receptor antagonist.
Stability and Kinetics of Unfolding and Refolding of cAMP Receptor Protein from Escherichia Coli
Authors: Małecki Jędrzej , Wasylewski Zygmunt
Published: July 2004

cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped‐flow fluorescence spectroscopy at 20°C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three‐state model: (CRPnative)2⇔ 2 (CRPnative) ⇔ 2 (CRPdenatured). The faster process, characterized by the relaxation time τ2= 80 ± 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time τ1, = 1.9 ± 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is Gdis0= 46.9 ± 2.5 kJ/mol and for monomer unfolding Gunf0, = 30.9 ± 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 = 0.3 °C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21 °C is equal to 30.5 ± 20.4 kJ/mol and the apparent spccific heat change is equal to cp,app= 10.7 ± 0.7 kJ/mol−1 K−1. The predicted values of cold denaturation midpoint is equal to t'G=–18.8 ± 1.5°C and for high‐temperature transition tG, = 63.1 ± 1.5°C. The predicted midpoint of high‐temperature unfolding transition is about the same as determined experimentally.
Inhibition of Human Placenta Glutathione Transferase Pl‐1 by the Antibiotic Calvatic Acid and its Diazocyanide Analogue
Authors: Antonini Giovanni , Pitari Giuseppina , Caccuri Anna Maria , Ricci Giorgio , Boschi Donatella , Fruttero Roberta , Gasco Alberto , Ascenzi Paolo
Published: July 2004

The inhibition mechanism of the dimeric human placenta glutathione transferase (GST) P1‐1 by calvatic acid and the reaction intermediates, i.e. the diazocyanide analogue of calvatic acid, has been investigated at pH 7.0 and 30.0°C. Experiments performed at different molar ratios of inhibitor/GST P1‐1 indicate that 1 mol calvatic acid inactivates 1 mol GST PI‐1, containing two catalytically equivalent active sites. However, 2 rnol of the diazocyanide analogue of calvatic acid inactivate 1 mol GST PI‐1. Two disulfide bridgesldimer, probably between Cys47 and CyslOl, have been formed during the reaction of GST P1‐1 with calvatic acid and its diazocyanide analogue. The apparent second‐order rate constants for GST P1–1 inactivation by calvatic acid and its diazocyanide analogue are 2.4 ± 10.3 M−1s−1and (8.5±0.7)×103M−1s−1, respectively. The reaction of calvatic acid with free L‐cysteine can be described by a simple process with an apparent second‐order rate constant of (5.0±0.4)×101M−1s−1. In contrast, a transient species occurs during the reaction of the diazocyanide analogue of calvatic acid with free L‐cysteine. Kinetics may be described by a second‐order process [the rate constant being (8.0±0.5)×103M−1s−1followed by a first‐order decay [the rate constant corresponding to (1.2±0.1)×10−1s−1]. Calvatic acid represents an enzyme inhibitor acting much slower than its reaction intermediates (i.e. its diazocyanide analogue).
Competitive and Non‐Competitive Inhibition of the Multidrug‐Resistance‐Associated P‐glycoprotein ATPase
Authors: Garrigos Manuel , Mir Lluis M. , Orlowski Stéphane
Published: July 2004

P‐glycoprotein, a plasma membrane protein overexpressed in multidrug‐resistant (MDR) cells, exhibits in vitro an ATPase activity and is responsible for the energy‐dependent efflux of structurally unrelated cytotoxic drugs (like vinblastine) and various MDR‐reversing agents (like verapamil and progesterone) from these MDR cells. To investigate the mechanism of P‐glycoprotein interaction with various compounds, we measured the P‐glycoprotein ATPase activity on membrane vesicles prepared from the MDR cell line DC‐3F/ADX, and we studied the effects of vinblastine, verapamil and progesterone on this ATPase activity. The basal P‐glycoprotein ATPase activity is increased by verapamil and progesterone, with respective half‐maximal activating concentrations of ≈1.5 μM and ≈25 μM, and activation factors of ≈1.7 and ≈2.2. Vinblastine inhibits the activation of P‐glycoprotein ATPase induced by verapamil or progesterone with an inhibition constant ≈0.5 μM in both cases. This demonstrates that vinblastine has a specific modulating site on P‐glycoprotein. The combined modulation of P‐glycoprotein ATPase by vinblastine and verapamil reveals that these two drugs are mutually exclusive. Since these two molecules have different effects both on the basal P‐glycoprotein ATPase activity and on the MgATP concentration dependence of P‐glycoprotein ATPase activity, they could bind P‐glycoprotein either on different and overlapping sites, or on distant but interacting sites. In contrast, the combined modulation of P‐glycoprotein ATPase by vinblastine and progesterone reveals a non‐competitive relationship between these two drugs, and hence shows that they can independently and simultaneously bind P‐glycoprotein on distinct sites. Since verapamil and progesterone are mutual inhibitors of P‐glycoprotein ATPase stimulation in a non‐competitive manner, these two molecules can also bind independently P‐glycoprotein on separated sites. This is confirmed here by the observation of a synergistic effect when mixtures of verapamil and progesterone are tested for the modulation of P‐glycoprotein ATPase. Three MDR‐related molecules, taken as models for interaction with P‐glycoprotein, appear thus to bind on at least two different separated specific sites. These results favor a multisite model rather than a universal site model to describe the broad substrate specificity characterizing P‐glycoprotein function.
Intermediates in the Assembly of Bacteriorhodopsin Investigated by Time‐Resolved Absorption Spectroscopy
Authors: Booth Paula J. , Farooq Amjad
Published: July 2004

The in vitro folding and assembly kinetics of bacteriorhodopsin have been studied by absorption spectroscopy. Folding is initiated by rapid stopped‐flow mixing of denatured apoprotein (bacterioopsin) in SDS micelles and mixed dimyristoylglycerophosphocholine/Chaps micelles containing retinal. The apparent mixing rate of the two types of micelles has been determined by time‐resolving the changes in light scattering by the micelles. Micelle mixing appears to occur in two stages: a fast phase with an apparent rate constant of about 420 s−1, and a second phase with an apparent rate constant of about 10 s−1. A rate constant of similar magnitude to the latter has previously been assigned to a protein‐folding event on the basis of protein fluorescence studies [Booth, P. J., Farooq, A. & Flitsch, S. L. (1996)Biochemistry 35, 5902–5909]. However the results presented here show that this rate constant may be associated with a rearrangement of the mixed detergent/lipid micelles.

When the changes in the retinal absorption band are time‐resolved during assembly of bacteriorhodopsin, a retinal–protein intermediate, with an absorption maximum of about 430 nm, has been identified. This absorption maximum lies between that of unbound retinal (at about 380 nm) and the native chromophore (at about 560 nm). A comparison of fluorescence and absorption data, together with previous evidence [Booth, P. J., Flitsch, S. L., Stern, L. J., Greenhalgh, D. A., Kim, P. S., & Khorana, H. G. (1995) Nat. Struct. Biol. 2, 139–143], suggests that the covalent Schiff‐base link to retinal is not formed in the 430‐nm‐absorbing intermediate.
Molecular Structures and Interactions of Pulmonary Surfactant Components
Authors: Johansson Jan , Curstedt Tore
Published: July 2004

The dominating functional property of pulmonary surfactant is to reduce the surface tension at the alveolar air/liquid interface, and thereby prevent the lungs from collapsing at the end of expiration. In addition, the system exhibits host‐defense properties. Insufficient amounts of pulmonary surfactant in premature infants causes respiratory distress syndrome, a serious threat which nowadays can be effectively treated by airway instillation of surfactant preparations. Surfactant is a mixture of many molecular species, mainly phospholipids and specific proteins, surfactant protein A (SP‐A), SP‐B, SP‐C and SP‐D. SP‐A and SP‐D are water‐soluble and belong to the collectins, a family of large multimeric proteins which structurally exhibit collagenous/lectin hybrid properties and functionally are Ca2+‐dependent carbohydrate binding proteins involved in innate host‐defence functions. SP‐A and SP‐D also bind lipids and SP‐A is involved in organization of alveolar surfactant phospholipids. SP‐B belongs to another family of proteins, which includes also lipid‐interacting polypeptides with antibacterial and lytic properties. SP‐B is a 17.4‐kDa homodimer and each subunit contains three intrachain disulphides and has been proposed to contain four amphipathic helices oriented pairwise in an antiparallel fashion. SP‐A, SP‐B and SP‐D all have been detected also in the gastrointestinal tract. SP‐C, in contrast, appears to be a unique protein with extreme structural and stability properties and to exist exclusively in the lungs. SP‐C is a lipopeptide containing covalently linked palmitoyl chains and is folded into a 3.7‐nm α‐helix with a central 2.3‐nm all‐aliphatic part, making it perfectly suited to interact in a transmembranous way with a fluid bilayer composed of dipalmitoylglycerophosphocholine, the main component of surfactant. Homozygous genetic deficiency of proSP‐B causes lethal respiratory distress soon after birth and is associated with aberrant processing of the precursor of SP‐C. This review focuses on the chemical composition, structures and interactions of the pulmonary surfactant, in particular the associated proteins.
Prothrombin, Albumin and Immunoglobulin A form Covalent Complexes with α1‐Microglobulin in Human Plasma
Authors: Berggård Tord , Thelin Nils , Falkenberg Cecilia , Enghild Jan J. , ÅkerströM Bo
Published: July 2004

Molecules containing the 33‐kDa plasma protein α1‐microglobulin were isolated from human plasma by anti‐(α1‐microglobulin) affinity chromatography. Five major bands could be seen after electrophoretic separation of the α1‐microglobulin‐containing proteins under native conditions. Immunoblotting demonstrated α1‐microglobulin in all five bands. Two of these have been described previously: free α1‐microglobulin and α1‐microglobulin complexed with IgA (IgA α1‐microglobulin). The other three bands were identified as prothrombin α1‐microglobulin, albumin α1‐microglobulin and dimeric α1‐microglobulin. Prothrombin α1‐microglobulin were 1:2 and 1:1 complexes which carried approximately 1% of total α1‐microglobulin, had molecular masses of about 145 kDa and 110 kDa upon SDS/PAGE and dissociated completely to free α1‐microglobulin and prothrombin (72 kDa) when reducing agents were added, suggesting that the complexes were stabilized by disulfide bonds. The α1‐microglobulin molecules did not inhibit cleavage of prothrombin by factor Xa and were bound to the peptides which were released upon activation of prothrombin. Albumin α1‐microglobulin, corresponding to 7% of total plasma α1‐microglobulin, was a mixture between 1:1 and 1:2 complexes, with masses upon SDS/PAGE of approximately 100 kDa and 135 kDa, respectively. Both these complexes dissociated only partially to free α1‐microglobulin and albumin when reducing agents were added. The albumin α1‐microglobulin complexes carried a yellow‐brown chromophore similar to free α1‐microglobulin. The complex‐binding to α1‐microglobulin did not block the fatty‐acid‐binding ability of albumin. The plasma concentrations of albumin α1‐microglobulin and prothrombin α1‐microglobulin were estimated to 5.2 mg/l and 1.1 mg/l, respectively.
Isolation of the Tetrathionate Hydrolase from Thiobacillus Acidophilus
Authors: De Jong Govardus A. H. , Hazeu Wim , Bos Piet , Kuenen J Gijs
Published: July 2004

An enzyme capable of hydrolysing tetrathionate was purified from cell‐free extracts of Thiobacillus acidophilus. The purified enzyme converts tetrathionate into thiosulfate, sulfur and sulfate. In addition, pentathionate could also be converted by the same enzyme. Measurement of the enzyme activity during purification is based on the absorbance of the initial intermediates formed from tetrathionate in the ultraviolet region, which have not been identified. Enzyme activity could also be measured by the scattering of insoluble sulfur in the visible region. The purified enzyme has a pH optimum of 2.5 and a temperature optimum of 65°C. Enzyme activity is strongly stimulated by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of 48kDa. The ultraviolet–visible absorption spectra and denaturation experiments indicate the presence of an organic cofactor.
The Binding of Azide to Copper‐Containing and Cobalt‐Containing Forms of Hemocyanin from the Mediterranean Crab Carcinus Aestuarii
Authors: Alzueti Gloria , Bubacco Luigi , Casellai Luigi , Rocco Gian Paolo , Salvato Benedetto , Beltramini Mariano
Published: July 2004

To establish the competence of the active site of hemocyanin to acquire diverse coordination geometries, the binding of azide to three forms of a crab hemocyanin, the dinuclear cupric or met‐hemocyanin, the mononuclear cupric or met‐apo‐hemocyanin, and the mononuclear Co(II)‐substituted derivative has been studied by near‐ultraviolet circular dichroism and EPR spectroscopies. The near‐ultraviolet circular dichroism spectra of the various derivatives present qualitatively similar features, namely a negative peak around 335 nm in the case of the two copper‐containing derivatives and a three‐component pattern with the Co(II) derivative. Upon decreasing the pH from 7.0 to 5.5 a decrease of optical activity is observed with all protein samples. The characteristic CD features, attributable to N(imidazole)‐to‐metal and to OH‐to‐metal charge‐transfer transitions, are strongly affected by azide binding. In particular, the intensity of the negative band exhibited by the two copper‐containing protein forms decreases with the onset of a new negative feature with maximum around 400 nm diagnostic for azide‐to‐Cu(II) charge‐transfer transitions. The visible region is affected as well, indicating that changes in the coordination sphere of copper take place. The affinity for azide of the different protein forms is higher at low pH. EPR measurements on the paramagnetic met‐apohemocyanin derivative as a function of pH demonstrate heterogeneity in the coordination environment at low pH. In the presence of azide an increase of rhombic distortion of the EPR spectra is observed and on the basis of the identified sets of copper hyperfine features in the course of azide titration experiments two different azide bound forms of met‐apo‐hemocyanin can be detected. The CD and EPR data at the different pH values are consistent with a reaction scheme in which azide replaces a fourth ligand in the metal‐coordination sphere, identified as a water or hydroxide molecule.
Substrate Specificity of Trypanothione Reductase
Authors: Marsh Ian R. , Bradley Mark
Published: July 2004

Trypanothione redutase, one of the family of flavin‐dependent disulfide oxidoreductases, catalyses the reduction of trypanothione disulfide [N1,N8‐bis(glutathionyl)spermidine] and related glutathionyl‐polyamine disulfides. A series of subtly different, designed substrate analogues based on trypanothione were prepared by means of a solid‐phase approach and used to study the catalytic efficiency of the parasitic enzyme. Kinetic analysis showed that the size of the polyamine bridge was relatively unimportant, for catalysis, while replacement of the ammonium bridge was much more dramatic. The highly charged glutathionylspermidine disulfide had the largest Km of all the substrates tested. N1‐acetylcysteinylglycinyl‐N8‐glutathionyl spermidine and N1‐glutathionyl‐N8‐acetylcysteinylglycinylspermidine both showed a tenfold reduction in catalytic efficiency compared with trypanothione.
Production and Structure Characterization of Recombinant Chromogranin A N‐Terminal Fragments (Vasostatins)
Authors: Corti Angelo , Sanchez Lincidio Perez , Gasparri Anna , Curnis Flavio , Longhi Renato , Brandazza Anna , Siccardi Antonio G. , Sidoli Alessandro
Published: July 2004

Vasostatins (VS) are vasoinhibitory peptides derived from the N‐terminal domain of chromogranin A, a secretory protein present in the electron‐dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1–78 (VS‐I), and 1–115 (VS‐2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/PAGE and mass spectrometry analysis. However, their hydrodynamic behaviours in size‐exclusion chromatography corresponded to that of proteins with a larger size. SDSPACE analysis of VS‐1 and VS‐2 after cross‐linking with disuccinimidyl suberate indicated that both polypeptides form dimers. VS‐2 was almost entirely dimeric at > 4 μM, but rapidly converted to monomer after dilution to 70 nM. The rapid dimer‐monomer transition of VS‐2 after dilution could be part of a mechanism for regulating its activity and localising its action. Immunological studies of VS‐1 have shown that residues 37–70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides. The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments.
Kinetic Analysis of two Closely Related Receptor‐Like Protein‐Tyrosine‐Phosphatases, PTPα and PTPε
Authors: Lim Kah Leong , Lai Delia S. Y. , Kalousek Markus B. , Wang Yue , Pallen Catherine J.
Published: July 2004

Among transmembrane protein‐tyrosine‐phosphatases, the membrane distal catalytic domain (D2) of protein‐tyrosine‐phosphatase α (PTPα) is unusual in having low but detectable activity in the absence of the membrane proximal catalytic domain (D1). To investigate the catalytic properties of PTPα D2 in association with D1, kinetic parameters of activity were established for PTPα D1D2 proteins containing an inactivating point mutation in D1 and/or D2. In this context, D2 activity was unchanged by the presence (N‐terminal or C‐terminal) or absence of inactive D1, and the presence or absence of inactive D2 affected the velocity but not the Km of D1 catalysis. While D1 appears to be the major catalytic contributor to PTPα activity, D2 possesses a significantly higher substrate‐specific activity relative to wild‐type D1D2 than the D2 domains of other protein‐tyrosine‐phosphatases. Also, PTPα D2 is an active phosphatase with comparable or better efficiency, on the basis of kcat/km criteria, to some of the dual specificity phosphatases. Kinetic parameters of a closely related receptor‐like protein‐tyrosine‐phosphatase, PTPε, were determined. PTPε D1 is the major, if not the only, catalytic moiety of PTPε, and has much higher turnover numbers than D1 of PTPα. The PTPα D2 activity is insignificant compared to that of PTPε‐D1D2, with lower turnover numbers than PTPα D2. Thus, the intrinsic activity of PTPα D2 is high compared to other D2 domains and, more outstandingly, its activity relative to D1 appears unique. These are also apparent upon in vitro assay of full‐length PTPα catalytic mutants expressed in mammalian cells. Together, these results suggest potential catalytic and regulatory roles for PTPα D2, and that PTPα may be an optimal model transmembsane protein‐tyrosine‐phosphatase for investigating the former within the cell.
Developmentally Controlled Cleavage of the Calliphora Arylphorin Receptor and Posttranslational Action of the Steroid Hormone 20‐Hydroxyecysone
Authors: Burmester Thorsten , Scheller Klaus
Published: July 2004

In response to a rise in ecdysteroid titre, fat body cells of insect larvae take up storage proteins from the haemolymph by receptor‐mediated endocytosis. Here we show that the receptor responsible for incorporation of the major haemolymph protein arylphorin of the blowfly, Calliphora vicina, is subject to an unusual posttranslational processing that involves three distinct cleavage steps. After the removal of a 17‐amino‐acid signal peptide, a receptor precursor of 141 kDa is released. Before reaching the cell surface, the precursor is cleaved a second time, giving rise to the active 92‐kDa arylphorin receptor, plus a 48‐kDa peptide. The function of this 48‐kDa peptide may be the prevention of premature ligand‐ receptor interaction in the endoplasmic reticulum. 20‐Hydroxyecdysone initiates a third cleavage step of the arylphorin receptor, which results in a 62‐kDa arylphorin binding protein and a 30‐kDa peptide. Contrary to the standard model of steroid hormone action, the process which give rise to receptor cleavage can be induced by 20‐hydroxyecdysone in vivo and in vitro even in absence of protein biosynthesis.
Cloning, Expression, Purification and Characterization Of Triosephosphate Isomerase from Trypanosoma Cruzi
Authors: Ostoa‐Saloma Pedro , Garza‐Ramos Georgina , Ramírez Jorge , Becker Ingeborg , Berzunza Myriam , Landa Abraham , Gomez‐Puyou Armando , De Gómez‐Puyou Marietta Tuena , Pérez‐Montfort Ruy
Published: July 2004

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3‐phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2‐SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.
Structural Studies of the Cell‐Envelope Oligosaccharide from the Lipopolysaccharide of Haemophilus Influenzae Strain RM.118‐28
Authors: Risberg Anna , Schweda Elke K. H. , Jansson Per‐Erik
Published: July 2004

The structure of the oligosaccharide part of the Haemophilus influenzae RM.118‐28 lipopolysaccharide (LPS) has been investigated. The oligosaccharide was obtained from the LPS by mild acid hydrolysis followed by gel‐permeation chromatography, and was studied by methylation analysis, NMR spectroscopy and mass spectrometry. The structure of the major compound, which is a hexasaccharide, is proposed as follows.
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In the structure, Kdo is 3–deoxy‐d‐manno‐octulosonic acid, PEtn is phosphoethanolamine, PCho is phosphocholine and l,d‐Hep is l‐glycero‐d‐manno‐heptose. Electrospray‐ionization mass spectrometry on O‐deacylated LPS obtained after treatment with anhydrous hydrazine gave evidence for the presence of two minor compounds, which show additional substitution of the main structure with phosphate and PEtn, respectively. These substitutions have not been localized.
Dimerization of Cdc25p, the Guanine‐Nucleotide Exchange Factor for Ras fromSaccharomyces Cerevisiae, and its Interaction with Sdc25p
Authors: Camusi Christelle , Geymonat Marco , Garreau Hervé , Baudet‐Nessler Sylvie , Jacqueti Michel
Published: July 2004

The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches. The two‐hybrid system showed that the C‐terminal part of Cdc25p (Cdc25‐Ct) can interact with itself but also with Sdc25p‐Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast. The homotropic interaction of Cdc25p‐Ct has been confirmed in yeast using immunoprecipitation experiments with epitope‐tagged and P‐galactosidase‐fused polypeptides. No other component was required for this interaction, since dimerization was shown to occur with material synthesizedin vitro.The size of Cdc25‐Ct produced inEscherichia colihas been directly measured on gel filtration columns and corresponds to a dimer. The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization. The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex cornbinatorial regulation of ras in yeast.
26‐Hydroxylation of Ecdysteroids is Catalyzed by a Typical Cytochrome P‐450‐Dependent Oxidase and Related to Ecdysteroid Resistance in an Insect Cell Line
Authors: Kayser Hartmut , Wtnkler Tammo , Spindler‐Barth Margarethe
Published: July 2004

The epithelial cell line from the dipteran Chironomus tentans responds to the insect steroid hormone 20‐hydroxyecdysone and the non‐steroidal analogue tebufenozide by undergoing a morphogenetic and biochemical differentiation program. Long‐term culture in the presence of 20‐hydroxyecdysone has resulted in the selection of subclones that are resistant to the steroid but respond normally to the non‐steroidal analogue. In the present study, several subclones that were resistant to the steroid hormone have been compared with steroid‐sensitive subclones with respect to their capability to metabolize 20‐ hydroxyecdysone. Homogenates of both types of cells, when incubated with 3H‐labelled steroid in the presence of NADPH, produced 20,26‐dihydroxyecdysone, which was further metabolized to two compounds, which behaved less polar than 20‐hydroxyecdysone on reverse‐phase HPLC. Ecdysone, a less‐ active hormone precursor, provided 26‐hydroxyecdysone as the only product. The metabolites were identified by mass spectrometry coupled to HPLC, chromatography with authentic samples, and formation of acetonides. The structure of 20,26‐dihydroxyecydsone was confirmed by 1H‐NMR.

The enzyme responsible for the synthesis of 20,26‐dihydroxyecdysone in the Chironomus cell preparations has been characterized as a typical cytochrome P‐450‐dependent monooxygenase. It was a strictly microsomal enzyme, sensitive to inhibition by carbon monoxide and imidazole/triazole‐based fungicides, and required NADPH for maximal activity. NADH could partly replace NADPH. The Michaelis constant (Km) for 20‐hydroxyecdysone was 0.96 μM, and the maximal enzyme velocity (Vmax) was 50 pmol substrate metabolized mg protein−1. min−1. 26‐Hydroxylation of 20‐hydroxyecdysone was inhibited by ecdysone, an alternative substrate, and by inokosterone, a product analogue, to 50% at 1.4 μM and 0.73 μM, respectively. When various subclones were compared with respect to their in vitro rate of 20‐hydroxyecdysone metabolization, those clones known to be resistant to the steroid were ‘high metabolizers’ (>70% relative rate), whereas the sensitive clones were ‘poor metabolizers’ (<30% relative rate). Hence, it is tempting to conclude that ecdysteroid resistance of the Chironomus cell clones is due to metabolic inactivation of the steroid hormone.
Structural Determination of the O‐Linked Sialyl Oligosaccharides Liberated from Fetuin with Endo‐α‐N‐Acetylgalactosaminidase‐S by HPLC Analysis and 600‐MHz 1H‐NMR Spectroscopy
Authors: Ishii‐Karakasa Ikuko , Iwase Hitoo , Hotta Kyoko
Published: July 2004

The endo‐α‐N‐acetylgalactosaminidase from the culture medium of Streptomyces sp. OH‐I1242 (endo‐GalNAc‐ase‐S) hydrolyzed the O‐glycosidic linkage between GalNAc and Ser (Thr) in fetuin, liberating oligosaccharides. The O‐linked oligosaccharides liberated from the fetuin with endo‐GalNAc‐ase‐S were pyridylaminated following fractionation on a Bio‐Gel P‐4 column. The structure of the pyridylaminated O‐linked oligosaccharides from fetuin has been determined by reverse‐phase HPLC and 600‐MHz 1H‐NMR spectroscopy. The chemical shifts and the coupling constants of pyridylaminated (PA) NeuAcα2‐3Galβ1‐3GalNAc were refined by computer simulation of the spectrum. The structures of NeuAcα2‐3Galβ1‐3(NeuAca2‐6) GalNAc‐PA and NeuAcα2‐3Galβ1 ‐3(NeuAcα2‐3Galβ1‐4GlcN Acβ1‐6)GalNAc‐PA were determined by their structural reporter groups.
Conservation of Hexamerin Endocytosis in Diptera
Authors: Burmester Thorsten , Scheller Klaus
Published: July 2004

In cyclorrhaphan Diptera at least two different types of haemolymph proteins exist which belong to the class of hexamerins. In the last larval instar of Calliphora vicina, the highly aromatic hexamerin, arylphorin, and the second hexamerin, PII, make up about 90% of haemolymph proteins. Both of these proteins are selectively taken up by the fat body cells at the end of larval life and share a common membrane‐bound receptor. In addition, hexamerins and possible hexamerin receptors of Calliphora vicina, Calliphora vomitoria, Drosophila melanogaster, Ceratitis capitata, Sarcophaga bullata, Musca domestica and Protophormia terraenovae were investigated. Uptake of arylphorin by the larval fat bodies of Calliphora vicina as well asarylphorin‐receptor binding can be competed in vitro by haemolymph from other Diptera. Therefore, hexamerin‐receptor binding must be conserved among related cyclorrhaphan Diptera and between different types of hexamerins within a species. As the degree of competition is in good agreement with the presumed phylogenetic distances between these species, the method described here provides a simple tool to estimate evolutionary distances.
Deletion of the Heptosyltransferase Genes rfaC and rfaF in Escherichia Coli K‐12 Results in an Re‐Type Lipopolysaccharide with a High Degree of 2‐Aminoethanol Phosphate Substitution
Authors: Brabetz Werner , Muller‐Loennies Sven , Holst Otto , Brade Helmut
Published: July 2004

The chromosomal genes rfaC and rfaF of Escherichia coli W3110 were inactivated by allelic‐replacement mutagenesis to generate a defined strain lacking both heptosyltransferases which catalyze in lipopolysaccharide (LPS) biosynthesis the transfer of the first two L‐glycero‐D‐manno‐heptose (Hep) residues to 3‐deoxy‐D‐mannm‐2‐octulosonic acid (Kdo). The LPS of the mutant was isolated and its chemical structure was investigated by compositional analysis and nuclear magnetic resonance spectroscopy of isolated, deacylated oligosaccharide phosphates. The basic structure was a tetrasaccharide α‐Kdo‐(2 → 4)‐a‐Kdo‐(2 → 6)‐β‐D‐GlcN4P‐(1 → 6)‐α‐D‐GlcNlP which in LPS was substituted at position 07 of Kdo II by 2‐aminoethanol phosphate in non‐stoichiometric amounts. 2‐Aminoethanol was cleaved during deacylation of the LPS by successive hydrazinolysis and KOH treatment and, in addition, phosphate migration from 07 to 08 of Kdo II occurred. Thus, the oligosaccharides u‐Kdo7P‐(2→4)‐α‐Kdo‐(2→6)‐β‐D‐GlcN4P‐(1→6)‐α‐D‐GlcNlP and α‐KdO8P‐(2→4)‐α‐Kdo‐(2→6)‐β‐D‐GICN4P‐(1→6)‐α‐D‐GlcN1P could be isolated. KOH treatment of the two trisphosphates and authentic methyl 3‐deoxy‐D‐manno‐octulopyra‐noside 7‐(2‐acetamidoethyl phosphate) proved that phosphate migration only took place when the phosphate group was substituted with 2‐aminoethanol. Complementation studies with plasmid‐encoded rjaC and rfaF genes revealed that the mutant strain can be used in combination with LPS‐specific antibodies for the cloning and characterization of heptosytransferases which glycosylate Kdo residues of the inner core region of LPS.
Palytoxin‐Induced Channel Formation within the Na+/K+‐ATPase does not Require a Catalytically Active Enzyme
Authors: Scheiner‐Bobis Georgios , Schneider Heike
Published: July 2004

It has been demonstrated that palytoxin binds to and forms a channel within the Na+/K+‐ATPase. To investigate whether palytoxin‐induced channel formation within the sodium pump can occur independently of ATP hydrolysis and phosphorylation of the enzyme, an Asp369→Ala mutant of the α1 subunit of the sheep sodium pump was produced and coexpressed with β subunits in the yeast Saccharomyces cerevisiae. This aspartic acid residue, which during ion transport becomes phosphorylated from ATP, is essential for the function of the sodium pump. Therefore, as expected, microsomes isolated from yeast expressing the mutant sodium pump do not exhibit any ouabain sensitive ATPase activity, whereas in microsomes from yeast expressing the wild‐type sodium pump, 60% of the total ATPase activity is ouabain‐sensitive. Ouabain binds to yeast membranes containing either wild‐type or mutant sodium pumps with similar Bmax (1.45±0.05 versus 1.37±0.02 pmol/mg) and Kd values (27.7±0.91 versus 29.57±0.93 nM), thus indicating that the mutant sodium pumps are expressed in the yeast and that the mutation does not considerably affect the conformation of the enzyme. In the presence of phosphate ouabain binds to microsomes containing the wild‐type sodium pump with a Kd of 3.62±0.34 nM, showing that, although not necessary, phosphoenzyme formation enhances binding of the steroid. Phosphate or ATP, however, inhibit binding of ouabain to microsomes containing the mutant sodium pump with IC50 values of 78±3 μM and 3.0±0.4 μM, respectively.

Despite these radical changes in the interactions of the mutant enzyme with ouabain, the interactions with palytoxin are not affected by the mutation. Palytoxin causes K+ efflux from yeast cells expressing the wild‐type or mutant sodium pumps with EC50 values of 3.5±0.4 nM and 6.2±0.9 nM, respectively. Palytoxin‐induced efflux from cells expressing wild‐type or mutant sodium pumps occurs with similar t1/2 values of 20.3±2.1 min and 22.2±3.1 min, respectively. Ouabain inhibits K+ efflux from both cell types with IC50 values of 28±2 μM and 210±15 μM, respectively. Cells expressing the Asp369→Ala mutants have an IC50 7.5‐fold higher than that obtained with cells expressing the wild‐type sodium pumps, possibly because ATP or phosphate present in the cytosol of the yeast cells influence and decrease ouabain binding to the mutant sodium pump. Thus, while ouabain binding and the associated inhibition of ion fluxes is promoted by phosphorylation of the wild‐type enzyme by phosphate or ATP, palytoxin‐induced channel formation is independent of phosphorylation and can be separated from the ATPase function of the sodium pump. Since ion fluxes through the sodium pump protein do not depend on ATP hydrolysis, the results suggest that the ionophores of pumps and ion channels might share common structural features.
Rat Liver Golgi Apparatus Contains a Protein Kinase Similar to the Casein Kinase of Lactating Mammary Gland
Authors: Lasa Marina , Marin Oriano , Pinna Lorenzo A.
Published: July 2004

By using a β‐casein‐derived specific peptide substrate for mammary gland Golgi‐enriched‐fraction casein kinase, phosphorylating activity has been detected in the Golgi apparatus of rat liver, spleen and to a lesser extent, kidney and brain, while the other post‐nuclear cytoplasmic fractions are totally devoid of such a casein kinase activity. In contrast ubiquitous protein kinases CK1 and CK2 (casein kinases 1 and 2), tested with their specific peptide substrates, display different subcellular distribution and are almost undetectable in the Golgi fraction. The absence of CK2 in the Golgi fraction has been also confirmed using specific antibodies. The relatedness between the liver Golgi apparatus casein kinase (G‐CK) and the bona fide mammary gland Golgi‐enriched‐fraction casein kinase (GEF‐CK) is supported by a variety of observations, notably: (a) identical peptide substrate specificity, consistent with an S‐X‐E‐X consensus sequence; (b) preference for Mn2+, and, to a lesser extent, Co2+, over Mg2+, as activating cation; (c) superimposable elution profiles from DEAE‐Sepharose, heparin‐Sepharose, and Superdex 200, this latter consistent with a molecular mass around 500kDa; (d) insensitivity to staurosporine and heparin (a potent inhibitor of CK2) and inability to use GTP as phosphate donor (by contrast to CK2). These data provide the evidence for the existence of a third class of ubiquitous casein kinases here termed G‐CK, distinct from CK1 and CK2, specifically located to the Golgi apparatus and related to the bona fide casein kinase(s) responsible for the phosphorylation of casein secreted from lactating mammary gland. The possible involvement of G‐CK in the phosphorylation of secretory pathways proteins at S‐X‐E motifs is discussed.
The N‐Domain of the Signal Recognition Particle 54‐kDa Subunit Promotes Efficient Signal Sequence Binding
Authors: Newitt John A. , Bernstein Harris D.
Published: July 2004

The signal recognition particle 54‐kDa subunit (SRP54) binds to the signal sequences of nascent presecretory and transmembrane proteins. Previous studies have shown that signal sequences bind to the C‐terminal methionine‐rich domain of the protein (M‐domain), but have raised the possibility that either the N‐terminal domain (N‐domain) or the central guanosine triphosphatase module (GTPase‐domain) also contribute to signal‐sequence‐binding activity. We have generated a series of N‐domain and GTPase‐domain mutants to investigate this issue further. Mutations in a conserved N‐domain motif (ALLEADV) produced significant defects in signal sequence binding that correlate with the severity of the mutation. The magnitude of the defect was independent of the preprotein substrate, which suggested that the mutations do not alter the specificity of signal sequence recognition. The N‐domain mutants also showed defects in promoting the translocation of presecretory proteins across the membrane of microsomal vesicles, but these defects appeared to be a direct consequence of the reduction in signal‐sequence‐binding activity and not separate effects of the mutations. By contrast, mutations in the guanosine triphosphatase consensus sequence had no effect on signal sequence binding, but instead severely impaired protein translocation activity. These results indicate that a principal function of the SRP54 N‐domain is to promote efficient signal sequence binding. These data also suggest that the SRP54 GTPase regulates the cycle of signal sequence binding and release, perhaps by modulating the relative orientation of the N‐ and M‐domains.
Use of Paramagnetic NMR Probes for Structural Analysis in Cytochrome c3 from Desulfovibrio Vulgaris
Authors: Salgueiro Carlos A. , Turner David L. , Xavier António V.
Published: July 2004

The dipolar field generated by each of the four haems in the tetrahaem ferricytochrome c3 from Desulfovibrio vulgaris (Hildenborough) (c3DvH) is determined by means of a novel procedure. In this method the 13C chemical shifts of the nuclei directly bound to the haems are used to determine the in‐plane orientations of the rhombic perturbation in each of the four haems with respect to a model of molecular orbitals of eg symmetry which are subject to a rhombic perturbation [Turner, D. L., Salgueiro, C. A., Schenkels, P., LeGall, J. & Xavier, A. V. (1995) Biochim. Biophys. Acta 1246, 24–28]. These orientations, together with the components of the magnetic susceptibility tensors obtained from the EPR g values and the crystal structure of c3DvH, can be used to calculate the dipolar shifts induced by each haem throughout the protein. Thus the observed 13C paramagnetic shifts of the c3DvH haem substituents were fitted considering both the pseudocontact and contact shifts of each haem simultaneously. The dipolar shifts calculated by this method were tested against the observed dipolar shifts for some amino acid residues strategically placed in the protein and also for the haem propionate groups. The effect of considering the calculated dipolar extrinsic shifts on the behaviour of the chemical shifts of the haem methyl groups in the intermediate stages of oxidation at different pH values was also analysed. The several tests applied to the calculated dipolar shifts have shown that the method is extremely useful for predicting chemical shifts as an aid to complete proton assignment, and to add further constraints in the refinement of solution structures of paramagnetic proteins and hence to probe subtle structural rearrangements around the haem pocket.
Isolation and Characterization of a cDNA from the Rat Brain that Encodes Hemoprotein Heme Oxygenase‐3
Authors: Mccoubrey William K. , Huang T. J. , Maines Mahin D.
Published: July 2004

Two isozymes of heme oxygenase (HO), HO‐1 or HSP32 and the constitutive form HO‐2, have been characterized to date. We report the discovery of a third protein species and refer to it as HO‐3. HO‐3 is the product of a single transcript of ≈2.4 kb and can encode a protein of ≈33 kDa. The HO‐3 transcript is found in the spleen, liver, thymus, prostate, heart, kidney, brain and testis and is the product of a single‐copy gene. The predicted amino acid structure of HO‐3 differs from both HO‐1 (HSP32) and HO‐2 but is closely related to HO‐2 (≈90%). Escherichia coli expressed and purified HO‐3 protein does not cross react with polyclonal antibodies to either rat HO‐1 or HO‐2, is a poor heme catalyst, and displays hemoprotein spectral characteristics. The predicted protein has two heme regulatory motifs that may be involved in heme binding. These motifs and the hemoprotein nature of HO‐3 suggest a potential regulatory role for the protein in cellular processes which are heme‐dependent.
Expression and Regulation of Cyclooxygenase‐2 in Rat Microglia
Authors: Bauer Manuel K. A. , Lieb Klaus , Schulze‐Osthoff Klaus , Berger Mathias , Gebicke‐Haerter Peter J. , Bauer Joachim , Fiebich Bernd L.
Published: July 2004

Increased levels of prostanoids have been implicated in various neuropathological diseases, although little is known about their cellular sources inside the brain. In this study, we analyzed the expression of cyclooxygenase‐2 (COX‐2), a key enzyme in arachidonic acid metabolism, in rat microglia. COX‐2 mRNA and protein as well as prostaglandin E2 formation were almost undetectable in unstimulated microglial cultures but were found to be strongly upregulated in response to lipopolysaccharide. However, in contrast to most peripheral cells, proinflammatory cytokines such as tumor necrosis factor α, interleukin‐1 β or interleukin‐6 failed to markedly induce COX‐2 expression. Similar effects were observed by analyzing transcription nuclear factor‐κ B (NF‐κB) which was strongly activated in microglia by lipopolysaccharide but not by incubation with cytokines. Moreover, known inhibitors of NF‐κB activation, such as dexamethasone and the antioxidant pyrrolidine dithiocarbamate, as well as the protein kinase C (PKC) inhibitor Gö6976, strongly reduced lipopolysaccharide‐induced COX‐2 transcription, indicating the involvement of NF‐κB and PKC in COX‐2 expression. Our results suggest that microglia may represent an important source of prostanoids in the brain, thus reinforcing their prominent role in cerebral inflammatory processes.