bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoaffinity Labeling Analysis of the Interaction of Pituitary Adenylate‐Cyclase‐Activating Polypeptide (PACAP) with the PACAP Type I Receptor
Authors: Cao Yong‐Jiang , Kojro Elzbieta , Gimpl Gerald , Jasionowski Marek , Kasprzykowski Franciszek , Lankiewicz Leszek , Fahrenholz Falk
Published: July 2004

To identify residues and domains of the peptide hormone pituitary adenylate‐cyclase‐activating polypeptide (PACAP) that interact with the type I receptor, two photoreactive analogues of PACAP‐(1–27)‐peptide were synthesized using solid‐phase peptide synthesis. Phe6 or Tyr22 within the PACAP sequence were replaced by p‐benzoyl‐L‐phenylalanine (Bz‐Phe) thus creating two PACAP derivatives with a photo‐reactive amino acid in either the disordered N‐terminal or the helical C‐terminal part of the peptide. The ligand‐binding properties and the efficiencies of these peptide analogues as photolabels were tested for pig brain PACAP receptors. [Bz‐Phe6]‐PACAP‐(l‐27)‐peptide (KA 1.3 nM) retained the high binding affinity of PACAP‐(1–27)‐peptide (Kd 0.5 nM), wheras Bz‐Phe substitution of Tyr22 reduced the affinity about tenfold (Kd 4.4 nM) thus demonstrating the importance of Tyr22 for receptor binding. Monoiodination of the photoreactive analogues did not change the binding affinity of the photoreactive analogues. Photoaffinity labeling using pig brain membrane demonstrated that the 125I‐labeled photoreactive analogues specifically label a 66000‐Mr protein band. Photoaffinity labeling of the rat brain PACAP receptor expressed in COS cells resulted in two specifically photolabeled proteins: a major band of Mr 58000 and a minor band of Mr 78000. By treatment of photolabeled membranes with W‐glycosidase F1 both of the polypeptide bands were converted to a single polypeptide band of Mr 54000, which corresponds to the deglycosylated PACAP receptor. Despite its lower receptor affinity, [Bz‐Phe22]‐PACAP‐(l‐27)‐peptide labeled the PACAP type I receptor in pig brain membranes and the rat receptor expressed in COS cells with much higher efficiency (20‐fold for the pig receptor) than [Bz‐Phe6]‐PACAP‐(l–27)‐peptide. These findings suggest that Tyr22 in PACAP‐(1–27)‐peptide is located in or close to the hormone‐binding site of the PACAP type I receptor. The results provide evidence that the α‐helical C‐terminal region of PACAP is directly involved in receptor binding.
Characteristics of Protein‐Kinase‐C‐ and ADP‐Ribosylation‐Factor‐Stimulated Phospholipase D Activities in Human Embryonic Kidney Cells
Authors: Rümenapp Ulrich , Schmidt Martina , Wahn Friederike , Tapp Eva , Grannass Andreas , Jakobs Karl H.
Published: July 2004

Phospholipase D (PLD) activity in human embryonic kidney (HEK) cells is stimulated by phorbolester‐activated protein kinase C (PKC) and by membrane receptors, the latter apparently acting via the GTP‐binding proteins, ADP‐ribosylation factor (ARF) and Rho. In the present study, performed in cellfree preparations, we have characterized and compared the regulation of HEK cell PLD activity by the stable GTP analogue, guanosine 5′‐O‐[γ‐thio]triphosphate (GTP[S]), and the phorbol ester, phorbol 12‐myristate 13‐acetate (PMA). In digitonin‐permeabilized HEK cells, prelabeled with [3H]oleic acid, GTP[S] and PMA caused an approximately threefold concentration‐dependent increase in the formation of [3H]phosphatidylethanol, measured in the presence of ethanol. Neomycin, which is known to complex with the PLD cofactor, phosphatidylinositol 4,5‐bisphosphate, decreased basal and GTP[S]‐ or PMA‐stimulated PLD activities with similar sensitivity. GDP and its analogue, guanosine 5′‐O‐[β‐thio]diphosphate, inhibited the stimulatory effect of GTP[S], whereas the PMA response was prevented by the nonselective PKC inhibitor, staurosporine, but not vice versa PLD stimulation by GTP[S], but not by PMA, was markedly reduced upon cytosol depletion and reconstituted by purified recombinant ARF1. In HEK cell membranes, addition of purified recombinant ARNO, a guanine‐nucleotide‐exchange factor for ARF1, potentiated the GTP[S]‐stimulated PLD activity. PLD stimulation by PMA in HEK cell membranes required MgATP and was largely prevented by the selective PKC inhibitors Goe 6976 and bisindolylmaleimide I. Immunoblot analysis demonstrated that both conventional PKC (α, β, γ) and atypical PKC isozymes (ζ, τ) were present in HEK cell membranes. The results indicate that phorbol ester stimulation of PLD activity in HEK cells apparently occurs by a phosphorylation‐dependent mechanism involving membrane‐associated PKC isozymes but not ARF proteins, the major targets of GTP[S]′ action.
A Possible Role for Cathepsins D, E, and B in the Processing of β‐amyloid Precursor Protein in Alzheimer's Disease
Authors: Mackay Elaine A. , Ehrhard Anne , Moniatte Marc , Guenet Chantal , Tardif Chantal , Tarnus Celine , Sorokine Odile , Heintzelmann Blanche , Nay Carole , Remy Jean‐Marc , Higaki Jeffrey , Van Dorsselaer Alain , Wagner Joseph , Danzin Charles , Mamont Pierre
Published: July 2004

Formation of the 4‐kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by β‐ and γ‐secretases. The carboxy‐terminal 99‐amino‐acid peptide which is liberated from APP by β‐secretase was used as a potential native substrate of the γ‐secretase(s). With the addition of an initiator Met and a FLAG sequence at the C‐terminus (β5APP100‐FLAG), it was expressed in Escherichia coli under the control of the T7 promoter. The preferred site(s) of cleavage in the N‐terminal 40‐amino‐acid β‐amyloid peptide and βAPP100‐FLAG by potential γ‐secretase(s) were rapidly identified using matrix‐assisted laser‐desorption/ionization time‐of‐flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since γ‐secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the βAPP100‐FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both β‐amyloid peptides and the βAPP100‐FLAG. As cathepsin D was found to cleave the βAPP100‐FLAG in the vicinity of the C‐terminus of the β‐amyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.
Interactions of a Bicyclic Analog of Colchicine with β‐Tubulin Isoforms αβII, αβIII and αβIV
Authors: Banerjee Asok , Engelborghs Yves , D'hoore An , Fitzgerald Thomas J.
Published: July 2004

Tubulin exists as various isoforms, which differ in their assembly, drug‐binding properties, and the dynamic properties of the microtubules they compose. One of the most striking differences in drug binding among the isoforms is observed with colchicine, which binds much better to the αβII and αβIV isoforms than to the αβIII isoform. Here we have studied the interaction of these isoforms with 2‐methoxy‐5‐(2′,3′,4′‐trimethoxyphenyl) tropone (MTPT), an analog of colchicine that lacks the B‐ring, The kinetics of association and dissociation were studied fluorometrically, and the kinetic parameters for the two‐step binding were determined for different β‐tubulin isoforms. The apparent on‐rate constants for αβII, αβIII and αβIV were 13358, 4558 and 10828 M−1 s−1, the off‐rate constants (k−2) were 0.04, 0.03 and 0.02 s−1, and the affinity constants are 3.33 × 105, 1.56 × 105 and 5.44 × 105 M−1, respectively. The differences in kinetic parameters among different β‐tubulin isoforms are greatly reduced when the B‐ring is removed. Our results indicate that the B‐ring plays a major role in determining the isoform differences, and the results might be of importance for designing tissue‐specific analogs of colchicine for cancer chemotherapy.
The Signal Transducer Gp130 — Bacterial Expression, Refolding and Properties of the Carboxy‐Terminal Domain of the Cytokine‐Binding Module
Authors: Müller‐Newen Gerhard , Pflanz Stefan , Hassiepen Ulrich , Stahl Jürgen , Wollmer Axel , Heinrich Peter C. , Grötzinger Joachim
Published: July 2004

Gp130 is the signal transducing receptor subunit of the so‐called interleukin‐6‐type cytokines. This transmembrane protein is a member of the cytokine‐receptor superfamily predicted to consist of six fibronectin‐type‐III‐like domains in its extracellular part. The second and the third domain constitute the so‐called cytokine‐binding module. Domain 2 is characterized by a set of four conserved Cys residues, domain 3 by a conserved WSXWS motif. As a first approach to a more detailed characterization of the cytokine‐binding domains of human gp130, we have expressed in Escherichia coli two forms of domain 3 differing in length. Both proteins were purified and refolded in a single step applying size‐exclusion chromatography. According to the rotational correlation times deduced from fluorescence anisotropy decay, they do not form aggregates. CD and fluorescence spectroscopy were used to study thermal unfolding and denaturation by guanidinium hydrochloride. It was shown that N‐ and C‐terminal extension by residues of the adjacent hinge regions substantially increase the thermal stability of the domain, which is conceivable from a molecular model. These results are the basis for further structural investigation by NMR spectroscopy.
Modified Helix‐Loop‐Helix Motifs of Calmodulin
Authors: Sharma Yogendra , Chandani Sushil , Sukhaswami Malladi Balasubrahmanyam , Uma Lakshmipathy , Balasubramanian Dorairajan , Fairwell Thomas
Published: July 2004

The four calcium‐binding sites, called the helix‐loop‐helix, or the EF‐hand motifs, of calmodulin differ in their ion‐binding affinities; this has been thought to arise due to the variations in the sequences of the loop regions where the ion binds. We focus attention here on the role of the flanking helical regions on the calcium‐binding affinities. Peptides were synthesized in a manner that simulates the E and F helical flanks of site 4 (the strongest calcium‐binding site of the calmodulin) to sandwich the loop sequences of sites 1, 2, 3 and 4 so as to produce peptides named 414, 424, 434 and 444, as well as using the helical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium‐mimic dye Stains‐all (4,4,4′,5′‐dibenzo‐3,3′‐diethyl‐9‐methyl‐thiacarbocya‐nine bromide). Binding abilities were seen to increase several‐fold when the E and F helices of site 1 were replaced by those of site 4 (i.e., 111–414). In contrast, the intensity of circular dichroism induced in the absorption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 444–141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weaken it.
Mutational Analysis of a Surface Area that is Critical for the Thermal Stability of Thermolysin‐Like Proteases
Authors: Veltman Oene R. , Vriend Gert , Hardy Florence , Mansfeld Johanna , Van Den Burg Bertus , Venema Gerard , Eijsink Vincent G. H.
Published: July 2004

Site‐directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55–69 region of the thermolysin‐like protease of Bacillus steawthermophilus (TLP‐ste) to thermal stability. The importance of the 55–69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55–69 region and the rest of the protease molecule. The calcium‐dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57→Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability‐determining region of TLP‐ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.
Stereochemistry of Peroxisomal and Mitochondrial β‐oxidation of α‐methylacyl‐CoAs
Authors: Schmitz Werner , Conzelmann Ernst
Published: July 2004

The stereochemistry of β‐oxidation of α‐methyl‐branched fatty acids was analyzed, in rat liver and in human cells, with (2R)‐ and (2S)‐2‐methyltetradecanoic acid as model substrates. In rat liver, formation of the α,β‐unsaturated compound was found to be concentrated in mitochondria while in human cells, this activity co‐distributed mainly with peroxisomal marker enzymes. In both cases, the dehydrogenating enzymes were absolutely specific for the (2S)‐enantiomer. In human liver, activation was some three times faster with the (2R)‐ than with the (25)‐isomer while in rat liver both were activated at about the same rate.
Ala217 is Important for the Catalytic Function and Autoactivation of Prostate‐Specific Human Kallikrein 2
Authors: Mikolajczyk Stephen D. , Millar Lisa S. , Marker Kathy M. , Grauer Lana S. , Goel Amita , Cass Michelle M. J. , Kumar Abhay , Saedi Mohammad S.
Published: July 2004

Prostate‐specific human kallikrein, hK2, is a serine protease found in prostate tissues that has 78% amino acid sequence identity with prostate‐specific antigen (PSA). We have previously reported the affinity purification of hK2 heterologously expressed in a hamster cell line and demonstrated an arginine‐restricted substrate specificity. Here, we describe the cloning, expression, purification, and enzymatic activity of a mutant form of hK2 containing an alanine to valine substitution at residue 217 ([Val217]hK2). This mutant form was secreted into the serum‐free spent media of recombinant cells as the stable proenzyme form ([Val217]phK2). Mild trypsin treatment was used to convert [Val217]phK2 to the active form, which had reduced catalytic function compared to the wild‐type hK2. Kinetic studies using the chromogenic substrate D‐H‐Pro‐Phe‐Arg‐4‐nitroanilide showed that [Val217]hK2 has significantly decreased substrate binding, with aKmof 4200 μM compared to 11 μM for wild‐type hK2. ThekMfor [Val217]hK2 was more than 100‐fold lower than for hK2. hK2, but not [Val217]hK2, was able to activate [Val217]phK2. [Val217]hK2 also showed altered specificity on a synthetic peptide substrate compared to wild‐type hK2, which exhibited partial hydrolysis at a PSA chymotrypsin‐like cleavage site as well as the trypsin‐like site cleaved by hK2. These results indicate that Ala217 is a key residue affecting the catalytic properties of hK2.
Role of Tyr518 and Tyr519 in the Regulation of Catalytic Activity and Substrate Phosphorylation by Syk Protein‐Tyrosine Kinase
Authors: Couture Clement , Williams Scott , Gauthier Nathalie , Tailor Pankaj , Mustelin Tomas
Published: July 2004

The Syk protein‐tyrosine kinase is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. After cross‐linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues. We have previously reported that Syk expressed in COS cells is predominantly phosphorylated at both Tyr518 and Tyr519 at its putative autophosphorylation site. In this study, we have examined the role of each of these two residues for the catalytic activity of Syk in vitro and for the Syk‐induced phosphorylation of cellular proteins in intact cells. Mutation of either residue had minor effects on the catalytic activity of Syk, and even the double mutant [F518, F519]Syk was about 60% as active as the wild‐type enzyme. In intact cells, however, all three mutants consistently failed to induce the extensive tyrosine phosphorylation of cellular proteins typically observed with wild‐type Syk. We have recently shown that the doubly phosphorylated Y518/Y519 site is also the site for association of Syk with the SH2 domain of the Lck kinase, which suggests that although phosphates at Y518/Y519 may enhance the catalytic activity of Syk, its interaction with Src family protein‐tyrosine kinases is at least equally important for the induction of downstream substrate phosphorylation.
Lipoxygenase‐2 Oxygenates Storage Lipids in Embryos of Germinating Barley
Authors: Holtman Wessel L. , Vredenbregt‐Heistek Jolanda C. , Schmitt Nathalie F. , Feussner Ivo
Published: July 2004

Besides the pre‐existing lipoxygenase (LOX‐1) present in quiescent grains, a new lipoxygenase (LOX‐2) is induced in embryos of germinating barley [Holtman, W. L., Van Duijn, G., Sedee, N. J. A. & Douma, A. C. (1996)Plant Physiol. III, 569–576]. The fact that LOX‐I and LOX‐2 form different products after incubation with linoleic acid, the (9S)‐ and (13S)‐hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M. M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1991)FEBS Lett. 280, 159–162; Doderer, A., Kokkelink, I., Van der Veen, S., Valk, B. E., Schram, A. W. & Douma, A. C. (1992)Biochim. Biophys. Acta 1120, 97–104], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX‐I oxygenated at C9, and LOX‐2 at C13. When testing more complex substrates, it was found that both LOX‐1 and LOX‐2 were capable of metabolizing esterified fatty acids. Km values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7‐35‐fold for LOX‐1 versus 2‐8‐old for LOX‐2). Interestingly, LOX‐1 showed significantly higher Km values for esterified fatty acids than did LOX‐2. This was reflected by analyses of the products formed from di‐ and tri‐linoleoylglycerol; LOX‐2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX‐1, with a corresponding larger extent of oxygenation.

In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX‐derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX‐2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)‐hydroxy derivatives were formed, indicating the in vivo action of LOX‐2. These data show that LOX‐2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX‐2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)‐13‐hydroxy‐octadecadienoic acid, serves as an endogenous substrate for β‐oxidation and therefore as a carbon source for the growing barley embryo.
Characterization of Four Toxins from Buthus Martensi Scorpion Venom, which Act on Apamin‐Sensitive Ca2+‐Activated K+ Channels
Authors: Romi‐Lebrun Régine , Martin‐Eauclaire Marie‐France , Escoubas Pierre , Wu Fang Qi , Lebrun Bruno , Hisada Miki , Nakajima Terumi
Published: July 2004

Four peptidyl inhibitors of the small‐conductance Ca2+‐activated K+ channels (SKCa) have been isolated from the venom of the Chinese scorpion Buthus martensi. These peptides were identified by screening C18HPLC fractions of the crude venom by means of mass analysis by matrix‐assisted‐laser‐desorption/ionization time‐of‐flight mass spectrometry, and toxicological tests in mice. Edman degradation analysis of the purified peptides showed sequences of 28–31 amino acids including 6 cysteine residues. Three of the sequences were similar to the P01 peptides from Androctonus scorpions, showing 76% sequence similarity for the most closely related, named BmPOl, and 46% for the other two, named BmP02 and BmP03. Like the P01 peptides, these molecules showed a low toxic activity in mice after intracerebro‐ventricular injection, and competed (K0.5 > 1 μM) with iodinated apamin for binding to its receptor site from rat brain, which has been proved to be the SKCa channels. The fourth toxin was structurally related to the P05/leiurotoxin I toxin family, with 90% similarity, and was named BmP05. This toxin exhibited a high toxic activity with lethal effects in mice. Due to its small representation in the venom [less than 0.01% (by mass)], its biological properties have been assessed on the synthetic analogue of BmP05, which was assembled on a solid phase by means of Fmoc methodology. The synthetic peptide was physicochemically identical to the natural peptide, as shown by comparison of their molecular masses and amino acid compositions, and by their coelution after coinjection on capillary electrophoresis. These results confirmed the primary structure of BmP05 including an amidated C‐terminus. Similarly to natural BmP05, synthetic BmP05 produced toxic and lethal effects after intracerebroventricular injection in mice (LD50= 37 ng), and was able to compete with iodinated apamin for binding to its receptor in rat brain (K0.5= 20 pM).
Structural Requirements of Phospholipase C δ1 for Regulation by Spermine, Sphingosine and Sphingomyelin
Authors: Pawelczyk Tadeusz , Matecki Andrzej
Published: July 2004

We studied the relationship between sphingomyelin, calcium, spermine and sphingosine in regulation of phospholipase C (PLC) δ1 activity. Inhibition of PLC δ1 by sphingomyelin was promoted by spermine and Ca2+ and was partially abolished by sphingosine. The effect of sphingosine and spermine entirely depended on Ca2+. In the absence of Ca2+, no effect of these substances on PLC δ1 activity was observed. Using deletion mutants and active fragments of PLC δ1 generated by limited proteolysis, we have studied the structural requirements of the enzyme for regulation by these compounds. The deletion mutant of PLC δ1 lacking the first 58 amino acids and the mutant lacking the entire pleckstrin homology (PH) domain were fully active in the detergent assay, and their activities were affected by spermine, sphingosine, Ca2+ and sphingomyelin to the same extent as the native enzyme. The limited proteolysis of PLC δ1 generated two fragments of 40 kDa and 30 kDa, which formed a stable active complex. The relationship between Ca2+ concentration and enzymatic activity was almost identical for the native PLC δ1 and the proteolytic complex. The activity of the proteolytic complex formed by the 40 kDa and 30 kDa peptides was not affected by spermine and sphingosine. Sphingomyelin inhibited the complex slightly less than the native PLC δ1, and this inhibition was not promoted by spermine. These observations suggest that for activation of PLC δ1 by spermine and sphingosine, the region spanning domains of high conservation, named X and Y, must be intact. In contrast, the PH domain and the intact spanning region of the X and Y domains are not essential for inhibition of PLC δ1 by sphingomyelin.
The AlkB Monooxygenase of Pseudomonas oleovorans
Authors: Staijen Ivo E. , Hatzimanikatis Vassily , Witholt Bernard
Published: July 2004

We have studied the synthesis and stability of the monooxygenase AlkB of Pseudomonas oleovorans in its natural host and in recombinant Escherichia coli. Three strains were investigated: the prototype strain P. oleovorans and the E. coli alk + recombinants HB101 (pGEc47) and W3110 (pGEc47). Plasmid pGEc47 allows regulated expression of alkB and synthesis of active AlkB in E. coli.

The E. coli strains were selected because E. coli HB101 (pGEc47) produces similar amounts of AlkB as P. oleovorans (1.5–2% of total cell protein), whereas E. coli W3110 (pGEc47) is able to make substantially (about fivefold) more AlkB. The AlkB synthesis and degradation rates in batch cultures of the three strains were determined by means of isotopic‐labeling and immunological techniques. The mean specific AlkB synthesis rates in P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47) were approximately 7, 12.5 and 45 μg · mg protein‐−1· h−1, respectively. The half‐lives of AlkB were estimated to be 80, 3 and 15 for P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47), respectively. Thus, the intracellular AlkB level in each of the three strains was the result of their AlkB synthesis and degradation rates.

The AlkB level during batch growth was modelled by means of experimentally derived parameters for AlkB synthesis and degradation, and showed good agreement with AlkB levels determined by means of immunoblotting in all strains investigated.
Ornithine Carbamoyltransferase from the Extreme Thermophile Thermus Thermophilus Analysis of the Gene and Characterisation of the Protein
Authors: Sanchez Rony , Baetens Margot , Van De Casteele Mark , Roovers Martine , Legrain Christiane , Glansdorff Nicolas
Published: July 2004

The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a λ‐ZAP genomic library. An ORF of 903 bp was found coding for a protein of M, 33200. The coding region has a very high overall G+C content of 68.0%. T. thermophilus OTC displays 38–48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus furiosus and from Bacillus subtilis. The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on δ‐N‐phosphonoacetyl‐L‐ornithine‐Sepharose. The native enzyme has an M, of about 110000, suggesting a trimeric structure, as for most anabolic OTC from various organisms. T. thermophilus OTC exhibits Michaelis‐Menten kinetics for carbamoyl phosphate and ornithine with a Kmapp of 0.10 mM for both substrates. The pH optimum was dependent on ornithine concentration with an optimum at pH 8 for ornithine concentrations around Km values. Higher concentrations shift the optimum towards lower pH. The optimal temperature was above 65°C and the activation energy 39.1 kJ/mol. The enzyme is highly thermostable. In the presence of its substrates the half‐life time was several hours at 85°C. Ionic and hydrophobic interactions contribute to the stability. The expression of T. thermophilus OTC was negatively regulated by arginine.
Isolation and Properties of Drosophila Melanogaster Ferritin — Molecular Cloning of a cDNA that Encodes One Subunit, and Localization of the Gene on the Third Chromosome
Authors: Charlesworth Alexandra , Georgieva Teodora , Gospodov Ivan , Law John H. , Dunkov Boris C. , Ralcheva Natalia , Barillas‐Mury Carolina , Ralchev Kiril , Kafatos Fotis C.
Published: July 2004

Ferritin was purified from iron‐fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2‐mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N‐terminal amino acid sequences were identical for the 25‐kDa and 26‐kDa subunits, but different for the 28‐kDa subunit. Conserved ferritin PCR primers were used to amplify a 360‐bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed.
Structural Characterization of Four Genetic Variants of Human Serum Albumin Associated with Alloalbuminemia in Italy
Authors: Minchiotti Lorenzo , Watkins Scott , Madison Jeanne , Putnam Frank W. , Kragh‐Hansen Ulrich , Amoresano Angela , Pucci Piero , Cesati Roberto , Galliano Monica
Published: July 2004

A long‐term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C‐terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single‐point mutations: albumins Tregasio, Val122→ Glu, Bergamo, Asp314→ Gly, and Maddaloni, Va1533→ Met. The fourth, albumin Besana Brianza, has the same Asp494→Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N‐glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.
Identification of Human Common Nuclear‐Matrix Proteins as Heterogeneous Nuclear Ribonucleoproteins H and H′ by Sequencing and Mass Spectrometry
Authors: Holzmann Klaus , Korosec Thomas , Gerner Christopher , Grimm Rudolf , Sauermann Georg
Published: July 2004

Nuclear‐matrix proteins were prepared from different rat and human cells and separated by two‐dimensional gel electrophoresis. By computer‐assisted analysis of the images, two of the proteins were identified as ubiquitously occurring (common) nuclear‐matrix proteins, which appeared in tissue‐dependent concentrations. The two proteins that originated from human blood mononuclear cells were analyzed further. Tryptic digests of the blotted proteins were analyzed by partial peptide sequencing and matrix‐assisted laser‐desorption ionization‐time‐of‐flight mass spectrometry. The two human common nuclear‐matrix proteins were identified as heterogeneous nuclear ribonucleoproteins (hnRNP) H and H′ or their variants. Furthermore, mass analysis revealed details on the N terminus of hnRNP H.
Studies of Mice Lacking Plasminogen Activator Gene Function Suggest that Plasmin Production Prior to Ovulation Exceeds the Amount Needed for Optimal Ovulation Efficiency
Authors: Ny Annelii , Nordström Lena , Carmeliet Peter , Ny Tor
Published: July 2004

Many studies suggest that the plasminogen activator (PA) system plays a role in the proteolytic degradation of the follicle wall at the time of ovulation. Consistently, the ovulation efficiency is reduced by 26% in mice where both physiological PA genes have been inactivated. To reveal the mechanism behind reduced ovulation efficiency in PA‐deficient mice and its effect on ovarian proteolysis, we have studied the regulation of plasmin activity in the ovaries of 25‐day‐old wild‐type mice and mice with deficient PA gene function during gonadotropin‐induced ovulation. In wild‐type mice the plasmin activity was low in ovarian extracts from mice treated with pregnant mare's serum gonadotropin. However, this activity was increased between 2–8 h after an ovulatory dose of human choriogonadotropins. In mice lacking either tissue‐type PA (tPA) or PA inhibitor type 1 (PAI‐1) the plasmin activity levels prior to ovulation were similar to wild‐type mice, while extracts prepared from urokinase‐type PA (uPA) deficient mice had 10% or less of the plasmin activity. This indicates that most of the plasmin activity in the mouse ovary is generated by uPA. In addition, as the ovulation efficiency is impaired in tPA/uPA‐deficient mice but appears normal in uPA‐deficient mice, our data indicates that the amount of plasmin generated by PAs prior to ovulation in wild‐type mice greatly exceeds the amount required for efficient ovulation.
Extensive Random Mutagenesis Analysis of the Na+/K+‐ATPase α Subunit Identifies Known and Previously Unidentified Amino Acid Residues that Alter Ouabain Sensitivity Implications for Ouabain Binding
Authors: Croyle Michelle L. , Woo Alison L. , Lingrel Jerry B.
Published: July 2004

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the α1 subunit of the sheep Na+/K+‐ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain‐resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111→Arg, Asp121→Gly, Asp121→Glu, Asn122→Asp, and Thr797→a were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+‐ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330→Gln, Ala331→Gly, Thr338→a, and Thr338→Asn in the H4 transmembrane domain and Phe982→Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1–‐7.9‐fold difference. The results of this extensive screening of the Na+/K+‐ATPase α1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1‐H2 and H4‐H8 regions represent the major binding sites for the cardiac glycoside class of drugs.
Partial Identification of the Zn2+‐Binding Sites in Factor XII and its Activation Derivatives
Authors: Røjkjær Rasmus , Schousboe Inger
Published: July 2004

With the purpose of identifying the Zn2+–binding sites in factor XII, the effect of chemical modification of His, Glu and Asp residues, amino acids known to participate in the catalytic coordination binding of Zn2+ in a number of Zn2+–binding proteins, was analysed. The number of modifiable His residues in factor XII and α–factor XIIa was 16.0±0.7 and 17.3±0.7, respectively. When factor XII/α–factor XIIa was incubated with saturating concentrations of Zn2+ before the diethylpyrocarbonate modification of the His residues, these numbers were reduced to 6.3±0.1 and 8.2±0.5, indicating that ten and nine His residues, respectively, are involved in the binding.

Analysis of the Zn2+–binding capacity of factor XII, α–factor XIIa and β–factor XIIa showed that while factor XII contains four Zn2+–binding sites, α–factor XIIa had only three and β–factor XIIa had none. Modification of the His residues resulted in a complete loss of Zn2+‐binding while Asp/Glu modification resulted in loss of two and one Zn2+‐binding sites in factor XII and α‐factor XIIa, respectively. This suggests that two of the four sites in factor XII contain His residues, exclusively, while the two others are comprised of two His residues and one Asp/Glu residue. One of the latter is lost when factor XII is activated to α‐factor XIIa. Two of the sites are suggested to be located at positions His40‐His44 and His78–His82. The location of the remaining two sites are reduced to four possible positions.
Chromatin Structure of the Rat Somatotropin Gene Locus and Physical Linkage of the Rat Somatotropin Gene and Skeletal‐Muscle Sodium‐Channel Gene
Authors: Kazahari Koji , Sano Eiji , Matsuura Nobuo , Ono Masao
Published: July 2004

Cosmid clones from –32 kb to +74 kb region of the rat somatotropin gene locus were isolated for examination of the chromatin structure in the region from –39kb to +47 kb by DNase I‐sensitivity analysis using rat pituitary‐derived GC (somatotropin+, prolactin–), and 235 (somatotropin–, prolactin+) cells, and liver‐derived BRL (somatotropin–, prolactin–) cells. DNase I‐hypersensitive sites (DHS) specific for somatotropin‐producing cells were previously shown to be located exclusively in the −2kb to +9kb region [Aizawa, A., Yoneyama, T., Kazahari, K. & Ono, M. (1995)Nucleic Acids Res. 23,2236–2244]. No other DHS having this specificity was found in the region examined in this study. Except for these and two other DHS located in a cluster in this region, no DHS could be found from –23 kb to +22 kb. DHS having no or less cell‐type specificity were scattered about in the –39 kb to –23 kb and +22 kb to +47 kb regions. The polyadenylation site of the human skeletal‐muscle Na‐channel a‐subunit gene has been shown present 22 kb upstream from the somatotropin gene [Bennani‐Baiti, I. M., Jones, B. K., Liebhaber, S. A. & Cooke, N. E. (1995)Genomics 29,647–652]. Polyadenylation site of the rat skeletal‐muscle Na‐channel gene was shown in this study to be at –15.7kb. The skeletal‐muscle Na‐channel gene was specifically expressed in skeletal‐muscle cells but not in somatotropin‐producing cells, and thus the boundary region that ensures the cell‐type‐specific expression of each gene would appear to be situated between two genes. The region prerequisite for cell‐type‐specific expression of the rat somatotropin gene was estimated based on the present findings.
Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by ΔH+
Authors: Cappellini Paolo , Turina Paola , Fregni Valeria , Melandri B. Andrea
Published: July 2004

Sulfite stimulates the rate of ATP hydrolysis by the ATP synthase in chromatophores of Rhodobacter capsulatus. The stimulated activity is inhibited by oligomycin. The activation takes place also in uncoupled chromatophores. The activation consists in an increase of about 12–15‐fold of the Vmax for the ATP hydrolysis reaction, while the Km for MgATP is unaffected at 0.16±0.03 mM. The dependence of Vmax on the sulfite concentration follows a hyperbolic pattern with half maximum effect at 12 mM.

Sulfite affects the ability of the enzyme in translocating protons. Concomitant measurements of the rate of ATP hydrolysis and of ATP‐induced protonic flows demonstrate that at sulfite concentrations of greater than 10 mM the hydrolytic reaction becomes progressively uncoupled from the process of proton translocation. This is accompanied by an inhibition of ATP synthesis, either driven by light or by artificially induced ionic gradients. ATP synthesis is totally inhibited at concentrations of at least 80 mM.

Sulfite interferes with the mechanism of activation by ΔH+. Low concentrations of this anion (≤ 2 mM) prevent the activation by ΔH+. At higher concentrations a marked stimulation of the activity prevails, regardless of the occurrence of a ΔH+ across the membrane. Phosphate at millimolar concentrations can reverse the inhibition by sulfite.

These experimental results can be simulated by a model assuming multiple and competitive equilibria for phosphate or sulfite binding with two binding sites for the two ligands (for sulfite K1S= 0.26 and K2S= 37 mM, and for phosphate K1P= 0.06 and K2S= 4.22 mM), and in which the state bound only to one sulfite molecule is totally inactive in hydrolysis. The competition between phosphate and sulfite is consistent with the molecular structures of the two ligands and of the enzyme.
Transcriptional Regulatory Region for Expression of the Rat ATP Citrate‐Lyase Gene
Authors: Fukuda Hitomi , Iritani Nobuko , Noguchi Tamio
Published: July 2004

We previously mapped the sequences responsive to insulin/glucose stimulation and polyunsaturated fatty‐acid suppression in the proximal promoter region between positions –104 and –20 of the ATP citrate‐lyase (ACL) gene [Fukuda, H., Iritani, N., Katsurada, A. & Noguchi, T. (1996) FEBS Lett. 380, 204–207]. To investigate further the regulatory DNA sequences required for stimulation and suppression of this gene, primary cultured hepatocytes were transfected with plasmids containing the 5′‐flanking sequences of the rat ACL gene fused to the chloramphenicol acetyltransferase (CAT) gene. When two copies of the sequences spanning –64 to –41 (linked to ACLcat20) were used for transfection, CAT activity significantly increased in response to insulin/glucose treatment. This increase was inhibited by addition of polyunsaturated fatty acid. Mutational analysis of this region showed that sequences between –55 and –51 are essential for recognition and interaction with trans‐acting factors. Gel mobility shift assays using the sequence from –64 to –41 as a probe revealed nuclear factor(s) from rat liver that specifically complexed with the sequences. In addition, by antibody supershift assays, we have detected the binding of the transcriptional factor Sp1 at the G+C‐rich region located within –64 to –41 of the ACL promoter. On the other hand, the formations of DNA‐protein complexes with Sp1 binding site or ACL(–64 to –41) were decreased in rats fed a high‐carbohydrate diet in comparison with those in rats fasted or fed a polyunsaturated fatty‐acid‐rich diet. Cotransfection studies in rat hepatocytes, with the Sp1 expression vector and ACLcat constructs, showed the inactivation of the promoter. These results demonstrated that the region from –64 to –41 of the ACL gene was responsible for stimulation due to insulin/ glucose, the stimulation was suppressed by polyunsaturated fatty acid, and Sp1 may be involved in the regulation.
Constitution and Solution Conformation of the Antibiotic Mersacidin Determined by NMR and Molecular Dynamics
Authors: Prasch Thomas , Naumann Thorsten , Marker Robert L. M. , Sattler Michael , Schubert Wolfgang , Schaal Susanne , Bauch Markus , Kogler Herbert , Griesinger Christian
Published: July 2004

The solution structure of the tetracyclic lantibiotic mersacidin in methanol (CD3OH) has been determined by NMR followed by distance bound driven dynamics and subsequent restrained molecular dynamics simulations combined with an iterative relaxation matrix approach and alternatively by a simulated annealing protocol. The molecular dynamics simulations were performed with the AMBER program system and with the INSIGHT program package. The distance bound driven dynamics calculation was conducted using a modified version of the DISGEO II program. The interproton distance restraints were derived from jump symmetrized rotating‐frame Overhauser enhancement and exchange (JS‐ROESY) spectra, which yield optimum sensitivity for medium‐sized molecules like mersacidin. The connectivities via the sulfide bridges were unambiguously confirmed by heteronuclear NMR techniques (heteronuclear single quantum coherence and heteronuclear multiple bond correlation methods). Due to the tetracyclic structure, mersacidin exhibits a rather rigid globular shape, which neither belongs to the duramycin nor to the nisin structure type lantibiotics. The resulting structures for the simulated annealing protocol of restrained and subsequent free molecular dynamics were compared and found to be very similar.
Characterisation of Macrophage Inflammatory Protein‐5/Human CC Cytokine‐2, A Member of the Macrophage‐Inflammatory‐Protein Family of Chemokines
Authors: Coulin Florence , Power Christine A. , Alouani Sami , Peitsch Manuel C. , Schroeder Jens‐Michael , Moshizuki Mizuru , Clark‐Lewis Ian , Wells Timothy N. C.
Published: July 2004

A human monocyte‐activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to the macrophage inflammatory protein (MIP) group of chemokines, particularly MIP‐3 (76.7%) and MIP‐1α (75.4%), and has been named MIP‐5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse‐transcriptase PCR show that the mRNA for MIP‐5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP‐5 induces chemotaxis of human monocytes, T‐lymphocytes and, to a lesser degree, eosinophils at nanomolar concentrations; it has no effect on neutrophil migration. In receptor‐binding assays, MIP‐5 shows IC50 values of 12 nM for competition with 125I‐MIP‐la for binding to CC‐chemokine receptor (CCR)l, and 2.5 nM for competition with 125I‐MCP‐3 for binding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)‐8 receptors (CXC‐chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP‐5 was only able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3.
Immunological Localization and Tissue Distribution of Alkyldihydroxyacetonephosphate Synthase and Deficiency of the Enzyme in Peroxisomal Disorders
Authors: De Vet Edwin C. J. M. , Biermann Jan , Van Den Bosch Henk
Published: July 2004

Alkyldihydroxyacetonephosphate synthase (alkylglycerone‐phosphate synthase) is a peroxisomal enzyme involved in ether phospholipid biosynthesis. The recent cloning of the cDNA encoding this enzyme from guinea pig liver enabled the raising of specific antisera against this enzyme. Both a synthetic peptide corresponding to a predicted epitope and a recombinant protein expressed in Escherichia coli were used for that purpose. Using western blot techniques, the solubilization of the enzyme from the peroxisomal membrane by Triton X‐100 in the presence of salt was confirmed. Neutral hydroxylamine treatment of peroxisomes resulted in almost no release of the protein from the membrane. The complete polypeptide chain of the enzyme was resistant to proteolysis by trypsin when intact peroxisomes were studied. Carbonate treatment released alkyldihydroxyacetonephosphate synthase from the membrane indicating that the enzyme is not an integral membrane protein. This idea is in accord with the absence of a clear hydrophobic transmembrane domain in the deduced amino acid sequence of the enzyme. Alkyldihydroxyacetonephosphate synthase, as well as its mRNA, could be detected in all five guinea pig tissues examined. When using the antiserum against guinea pig recombinant alkyldihydroxyacetonephosphate synthase, a cross‐reactive protein was detected in a human liver homogenate that runs at a slightly higher molecular mass. The absence of this band in liver of Zellweger syndrome and Rhizomelic chondrodysplasia punctata patients provides strong evidence that it represents the human homolog of this enzyme.