bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the Binding of Urokinase‐type Plasminogen Activator (u‐PA) to Plasminogen, to Plasminogen‐activator Inhibitor‐1 and to the u‐PA Receptor
Authors: Lijnen H. Roger , De Cock Frans , Collen Désiré 
Published: March 2005

Binding parameters [association‐rate (kass and dissociation‐rate (kdiss constants, and affinity constants (KA=kass/kdiss)] for the interaction between urokinase‐type plasminogen activator (u‐PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor‐1 (PAI‐1) and its receptor (u‐PAR), were determined by real‐time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N‐terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site‐blocked plasmin (d‐ValPheLysCH2‐plasmin) to the 54‐kDa or 32‐kDa molecular forms of recombinant single‐chain u‐PA (rscu‐PA) ranged between 0.57×106 M−1 and 1.7×106M‐1, compared to 14–22×106 M‐1 for binding to the corresponding active site‐blocked recombinant two‐chain u‐PA (rtcu‐PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu‐PA (rtcu‐PA with the active site Ser356 converted to dehydroAla) were 81×106M‐1 and 670×106 M‐1 respectively. Binding of active site‐blocked LMM‐plasmin (a low‐molecular‐mass plasmin derivative lacking kringles 1–4) and of the plasmin B chain to [Ser356deHAla] rtcu‐PA occurred with KA values of 3.7×106M‐1 and 0.33×106 M‐1 compared to 670×106 M‐1 for the binding of intact d‐ValPheLysCH2‐plasmin to [Ser356deHala]rtcu‐PA.The KA values for binding of latent PAI‐1 to 54‐kDa or 32‐kDa molecular forms of rscu‐PA and rtcu‐PA were in the range 0.34–2.1×106 M‐1. Reactivated PAI‐1 bound to 54‐kDa and 32‐kDa rtcu‐PA moieties with KA values of 26×106M‐1 and 28×106 M‐1, compared to 0.77×106M‐1 and 3.2×106 M‐1 for binding to the corresponding single‐chain u‐PA species, and 450×106 M‐1 for binding to [Ser356deHAla]rtcu‐PA. KA values for binding of plasmin(ogen) to the covalent rtcu‐PA/PAI‐1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu‐PA.Single‐chain and two‐chain 54‐kDa u‐PA moieties bound with a 1:1 stoichiometry and with very high affinity to u‐PAR (KA of 4.6–8.5×109 M‐1), whereas no significant binding of 32‐kDa u‐PA moieties was observed (KA≤0.2×106M‐1). Binding of the covalent rtcu‐PA/PAI‐1 complex to u‐PAR was indistinguishable from that of rtcu‐PA.These results indicate that plasminogen and plasmin bind with higher affinity to two‐chain u‐PA than to single‐chain u‐PA moieties, whereas binding requires the presence of both the A and the B chain; reactivated PAI‐1 binds to rtcu‐PA moieties with a higher affinity than latent PAI‐1, but has a very low affinity for rscu‐PA; 54‐kDa u‐PA moieties bind in a 1:1 stoichiometry to u‐PAR with high affinity which does not require the u‐PA active site; and the binding sites of u‐PA for PAI‐1 and for plasminogen are within the 32‐kDa u‐PA moiety (Leu144–Leu411), they do not overlap and are fully accessible when 54‐kDa u‐PA is bound to its receptor.
Elongation on the Amino‐terminal Part of Stefin B Decreases Inhibition of Cathepsin H
Authors: Jerala Roman , Kroon‐žitko Louisa , Popovič Tatjana , Turk Vito 
Published: March 2005

Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino‐terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily. The mutant proteins were expressed in Escherichia coli and purified to homogeneity. Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H. Inhibition of both amino‐terminal elongated stefin B mutants was decreased particularly for cathepsin H.A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed. The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases. Variable regions were selected from fragments of other proteins from the protein data base. We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes. This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H. We conclude that the amino‐terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H.
The Modulation of Enzyme Reaction Rates Within Multi‐Enzyme Complexes
Authors: Gontero Brigitte , Giudici‐Orticoni Marie‐Thérèse , Ricard Jacques 
Published: March 2005

Octameric ribulose bisphosphate carboxylase‐oxygenase binds in an independent manner its substrate (ribulose bisphosphate) and a substrate analog (6‐phosphogluconate). The eight active sites of the free enzyme are thus independent. The kinetic behaviour of the active site becomes different if ribulose bisphosphate carboxylase‐oxygenase is inserted in the five‐enzyme complex previously isolated from chloroplasts. Ribulose bisphosphate carboxylase‐oxygenase then becomes more active than the corresponding free enzyme form. By comparing the behaviour of the same enzyme in the free state and in the associated state it then becomes possible to study how the thermodynamics of protein–protein interactions alters the kinetic behaviour of ribulose bisphosphate carboxylase‐oxygenase. This alteration may be expressed in terms of stabilization‐destabilization energies exerted upon the various intermediate states of the enzyme reaction, within the multi‐protein complex. Heterologous interactions within this complex exert a constant stabilization energy on the enzyme ground states along the reaction co‐ordinate of−1.68 kJ/mol and a constant stabilization energy of –3.79 kJ/mol on the enzyme transition states. These stabilization energies express how information propagates within the multi‐enzyme complex as to increase the apparent affinity of the substrate for the active sites of ribulose bisphosphate carboxylase‐oxygenase, as well as to increase the catalytic rate constant.The binding of the substrate analog 6‐phosphogluconate to free ribulose bisphosphate carboxylase‐oxygenase is non‐co‐operative. It becomes positively co‐operative if this enzyme is inserted in the multi‐protein complex. Under these conditions, only one type of enzyme‐inhibitor complex is detected experimentally. Here again heterologous interactions stabilize this enzyme‐inhibitor complex relative to that expected if ribulose bisphosphate carboxylase oxygenase is free. The extent of stabilization is –1.03 kJ/mol.Neither free nor associated ribulose bisphosphate carboxylase‐oxygenase display any co‐operativity relative to substrate binding. However, in the presence of the substrate analog 6‐phosphogluconate, this enzyme displays positive co‐operativity relative to substrate, although not if it is naked. These results can be explained theoretically and show that the maximum value of the Hill coefficient is ≤2. As 6‐phosphogluconate and other substrate analogs are present in chloroplasts under normal conditions, this co‐operativity might be of functional importance in vivo.
Covalent Modification of Catalytic Sites on Membrane‐bound Beef Heart Mitochondrial ATPase by 2‐Azido‐adenine Nucleotides
Authors: Martins Ione S. , Penefsky Harvey S. 
Published: March 2005

Incubation in the dark of 32P‐labeled 2‐azido‐adenine nucleotides with submitochondrial particles from beef heart led to tight binding of the label by membrane‐bound F1. That is, the label remained with the particles following two passages through centrifuge columns. After removal of free nucleotides and ultraviolet irradiation, the radioactive label was covalently bound exclusively to the β subunit of the ATPase. Extraction of the modified enzyme from the membrane with chloroform followed by tryptic digestion and separation of peptides by reverse‐phase high‐pressure liquid chromatography indicated that the radioactive label had been inserted into a peptide fragment that included part of the catalytic site.Covalent modification of catalytic sites by 2‐azido‐ADP was accompanied by parallel inhibition of both ATP synthesis and ATP hydrolysis by submitochondrial particles. Estimation of the likely amount of F1 participating in the reaction and extrapolation to complete inhibition suggested that modification of no more than a single site was sufficient to block both reactions. The results support suggestions of cooperative interactions between catalytic sites as well as a single catalytic pathway for both enzymic reactions.
The Solution Structure of a DNA Duplex Containing the cis ‐Pt(NH3)2[d(‐GTG‐)‐N 7(G), N 7(G)] Adduct, as Determined with High‐Field NMR and Molecular Mechanics/Dynamics
Authors: Van Garderen Carla J. , Van Houte Leo P. A. 
Published: March 2005

The solution structure of the cis ‐Pt‐GTG adduct in the double‐stranded oligomer d(CTCTAGTG‐CTCAC) · td(GTGAGCACTAGAG) was studied with high‐resolution NMR techniques. For model building, the distance information obtained from two‐dimensional NOE experiments was used in molecular mechanic computations and molecular dynamic structure refinements.The structural distortion upon platination appears to be restricted to the base pairs Pt‐G6 · C21 and T7 · A20; Pt‐G8 · C19 forms a normal Watson‐Crick base pair. T7 is positioned in the minor groove and stacks with the highly propeller‐twisted Pt‐G6. There is no hydrogen bonding between T7 and A20. The complementary strand is undistorted; A20 stacks with its flanking cytidines (C19 and C21) as in regular B‐DNA. The duplex is locally unwound (from base pair A5 · T22 to G8 · C19: 19°) and is slightly kinked (20°) at the platination site. The platinum coordination distorts the DNA structure at the 5′ side of the platinated ‐GTG‐ sequence and changes the minor groove face.
CMP‐N‐acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands
Authors: SHAW Lee , SCHNECKENBURGER Petra , SCHLENZKA Wiebke , CARLSEN Jens , CHRISTIANSEN Kirsten , JÜRGENSEN Dorthe , SCHAUER Roland 
Published: March 2005

In this report, the nature of the protein components involved in the functioning of cytidine‐5′‐monophosphate‐N‐acetylneuraminic acid (CMP‐Neu5 Ac) hydroxylase in high‐speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH–cytochromeb5reductase, cytochromeb5and a 56‐kDa terminal electron acceptor having the CMP‐Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction; however, the amphipathic, usually membrane‐associated, forms of cytochromeb5and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylasein vivo.Although the majority of cellular cytochromeb5and cytochromeb5reductase is membrane‐bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent‐solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochromeb5. Accordingly, in reconstitution experiments, pure hydrophilic cytochromeb5interacts more effectively with the hydroxylase than isolated amphipathic cytochromeb5.Studies on the CMP‐Neu5Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containingN‐glycoloylneuraminic acid.
Complex formation between protein C inhibitor and prostate‐specific antigen in vitro and in human semen
Authors: CHRISTENSSON Anders , LILJA Hans 
Published: March 2005

Protein C inhibitor (PCI), a serine‐proteinase inhibitor first purified from human blood plasma, occurs at high concentrations (3–4 μM) in seminal fluid in both a high‐molecular‐mass and low‐molecular‐mass form. Immunochemical data have previously suggested that PCI in seminal plasma forms complexes with the most abundant serine proteinase in semen, prostate‐specific antigen (PSA). To provide a structural characterization of the PCI target, immunodetected as PSA, a procedure was developed to isolate low‐molecular‐mass and high‐molecular‐mass‐forms of PCI from seminal fluid. The high‐molecular‐mass form of PCI, recognized by monoclonal antibodies against PSA, was dissociated by alkaline treatment into the low‐molecular‐mass form of PCI and a 33‐kDa protein identified as PSA by 25 conclusive steps of N‐terminal sequence analysis. We developed a sensitive immunofluorometric assay (IFMA) to measure PCI‐PSA complexes in body fluids and investigated the rate at which purified PSA may form complexes with purified PCI. Formation of complexes detected by this IFMA and the appearance of SDS‐stable approximately 90‐kDa complexes paralleled loss of PSA activity recorded with chromogenic substrates. The rate of complex formation was slow compared to that reported for PCI and activated protein C, but was enhanced up to sixfold in the presence of heparin. Less than 10% of the initial PSA activity remained after 3 h incubation with a sevenfold molar excess of PCI and in the presence of heparin.In freshly collected ejaculates, the rate of PCI‐PSA complex formation measured by IFMA was similar to that observed between the purified proteins, and paralleled the appearance of SDS‐stable complexes by immunoblotting. During gel dissolution in freshly collected ejaculates, approximately 40% of immunodetected PCI becomes complexed to PSA. Although PCI is a slow inhibitor of PSA, complexes between PCI and PSA are detected at levels that correspond to an inactivation of up to 5% of the PSA activity in the ejaculate.
Preparation of cyclic 2′,3′‐monophosphates of oligoadenylates (A2′p)nA > p and A3′p(A2′p)n‐1A>p
Authors: BUDOWSKY Edward I. , KAYUSHINA Elena N. , TARASOV Andrew K. , ORLENKO Svetlana A. , CHERKASOV Igor A. , GAVRILOV Alexander E. , STRELENKO Yuri A. 
Published: March 2005

The action of the guanylyl‐preferring RNase from Bacillus intermedius (binase) on a mixture of oligoadenylates with randomly distributed 2′‐5′ and 3′‐5′ internucleotide bonds [(A2′/3′p)n] under conditions sufficient for complete hydrolysis of poly(A) results in a mixture containing a single circular oligoadenylate and two series of linear oligoadenylates ending in cyclic 2′,3′‐phosphate.Individual compounds formed upon digestion of (A2′/3′p)n with binase have been isolated. Their structure was determined on the basis of their chemical and enzymatic conversions and confirmed by 1H‐, 13C‐ and 31P‐NMR spectra. According to these data, the circular triadenylate contains one 2′‐5′ and two 3′‐5′ internucleotide bonds, linear oligoadenylates of one series contain exclusively 2′‐5′ internucleotide bonds [(A2′p)nA>p], while each compound of the other series contains a single 3′‐5′ internucleotide bond connecting the 5′‐ultimate nucleotide residue with the penultimate one [A3′p(A2′p)n‐1A>p].The incubation of compounds of the former series A3′p(A2′p)n>p at pH 1.0 and the subsequent action of phosphatase results in successive formation of compounds of two other new series: A3′p(A2′p)nA2′(3′)p and A3′p(A2′p)nA.
Triose‐phosphate isomerase of Leishmania mexicana mexicana Cloning and characterization of the gene, overexpression in Escherichia coli and analysis of the protein
Authors: KOHL Linda , CALLENS Mia , WIERENGA Rik K. , OPPERDOES Fred R. , MICHELS Paul A. M. 
Published: March 2005

The gene of triose‐phosphate isomerase in Leishmania mexicana has been cloned and characterized. The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27561 Da and a net charge of +2. Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody‐like organelles called glycosomes and in the cytosol. The primary structure of the enzyme has many features in common with that of triosephosphate isomerase in the related organism Trypanosoma brucei. Their sequences are 68% identical. The residues consituting the subunit interface are highly conserved between the enzyme of L. mexicana and T. brucei, but are mostly different from those in the enzyme of other organisms. One major substitution was detected in the interface region of the L. mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences. The glutamine is thought to be important for the stability of the dimeric enzyme. L. mexicana triose‐phosphate isomerase has been overexpressed in Escherichia coli. Growth conditions were established to obtain high levels of soluble and active protein. The enzyme has been purified to near homogeneity. It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T. brucei enzyme. The kinetics of the different enzyme preparations were very similar. For the recombinant enzyme the following values were measured: with glyceraldehyde 3‐phosphate as substrate Km= 0.30±0.05 mM and kcat= 2.5X105 min−1; with dihydroxyacetone phosphate as substrate Km= 1.3±0.3 mM and kcat= 2.8X104 min−1.
Transport of citrate catalyzed by the sodium‐dependent citrate carrier of Klebsiella pneumoniae is obligatorily coupled to the transport of two sodium ions
Authors: LOLKEMA Juke S. , ENEQUIST Hans , VAN DER REST Michel E. 
Published: March 2005

Aerobically grown Escherichia coli GM48 harboring plasmid pKScitS that codes for the sodium‐dependent citrate carrier from Klebsiella pneumoniae (CitS) allows initial‐rate measurements of citrate uptake in whole cells. The cation stoichiometry and selectivity of CitS was studied using this experimental system. The relationship between the initial rate of uptake of citrate and the Na+ concentration was sigmoidal at pH values between 5 and 7 suggesting a Na+ stoichiometry higher than 1. Rates of uptake increased quadratically in a range of non‐saturating Na+ concentrations showing that two Na+ are translocated/catalytic cycle. Symport of Na+ is absolutely required in the range pH 5–7 because no uptake could be detected in the absence of Na+. Protons cannot replace Na+ in the translocation step but the decrease in apparent affinity for Na+ towards lower pH suggests that protons can compete with Na+ for the cation‐binding sites.Li+ can replace Na+ in the symport reaction but it takes about a 200‐fold higher concentration of Li+ over Na+ to achieve the same rate of uptake, showing that the affinity of CitS for Li+ is much lower than for Na+. Though high Li+ concentrations have an inhibitory effect on citrate uptake, the data suggest that the Li+ stoichiometry is also 2.
The C‐terminus of the 14‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae is involved in the assembly of a functional enzyme
Authors: HEMRIKA Wieger , DE JONG Marian , BERDEN Jan A. , GRIVELL Leslie A. 
Published: March 2005

Disruption of QCR7, the gene encoding the 14‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisie,, results in an inactive enzyme which lacks holo‐cytochrome b and has severely reduced levels of apo‐cytochrome b, the Rieske Fe‐S protein and the 11‐kDa subunit [Schoppink, P. J., Berden, J. A. & Grivell, L. A. (1989) Eur. J. Biochem. 181, 475–483].An episomal system was developed to study the effect on complex III of transformation of in vitro mutagenised QCR7 genes to a QCR7° mutant. Transformation of a gene (TNT1) in which the 12 C‐terminal residues are replaced by 3 amino acids encoded by an oligonucleotide containing a stop codon in all three reading frames (STOP‐oligonucleotide), only leads to partial complementation of the respiratory capacity of the yeast strain. The amounts of apo‐cytochrome b, the Rieske Fe‐S protein and the 11‐kDa subunit are reduced and enzymic activity, together with the amount of holo‐cytochrome b, is lowered to about 40% of that of the wild type, indicating a normal turnover number of the mutant enzyme.Transformation of the QCR7° mutant with another gene (TNT2) encoding the first 96 residues of the 14‐kDa subunit fused to 9 amino acids encoded by the STOP‐oligonucleotide, leads to a phenotype almost indistinguishable from that of the QCR7° mutant.The role of the charged C‐terminus of the 14‐kDa (and the 11‐kDa) subunit in the assembly of a functional complex III is discussed.
Thermodynamic stability and solution conformation of tandem G · A mismatches in RNA and RNA · DNA hybrid duplexes
Authors: EBEL Susanne , BROWN Tom , LANE Andrew N. 
Published: March 2005

G · A mismatches form a variety of hydrogen‐bonded structures in DNA, most of which destabilise the duplex. Tandem G · A mismatches in the context YGAR (Y = pyrimidine, R = purine), however, form base pairs using the amino group of the guanine residue [Li, Y., Zon, G. & Wilson, W. D. (1991) Proc. Natl Acad. Sci. USA 88, 26–30], which permits extensive base‐base stacking, leading to a slight stabilisation of the helix [Ebel, S., Lane, A. N. & Brown, T. (1992) Biochemistry 31, 12083–12086]. We have measured the thermodynamic stability of several RNA and RNA · DNA hybrid duplexes containing tandem G · A mismatches. The RNA duplexes are intrinsically much more stable than the corresponding DNA duplexes and the mutations are destabilising in all cases.NOE and coupling‐constant data show that all of the sugars are in the C3′‐endo range of conformations, and glycosidic torsion angles are in the range ‐160° to ‐180° in r(CCACGAGUGG) · r(CCACGAGUGG). Both sequential NOE intensities and circular‐dichroism measurements indicate that the global conformation of the mismatched RNA is A‐like. The N1H group of the mismatched guanine residue is not involved in hydrogen bonding with the adenine residue, indicating the presence of the amino‐pairing scheme. Determination of the structure using ‘loose’ NMR‐derived constraints shows that the potential energies of the imino‐paired and amino‐paired forms are similar, but substantially higher than energy‐minimised RNA. Using tighter constraints derived from more extensive analysis of one‐dimensional and two‐dimensional NOE data showed that the amino‐paired structure agrees with the constraint data better than the imino‐paired structure, and also accounts for unusual chemical shifts and the lack of hydrogen bonding of the guanine N1H group. Resulting molecular models show that the amino‐paired mismatches are not as extensively stacked on the neighbouring part of the duplex as in the B‐DNA analogues, largely accounting for the lower thermodynamic stability in the RNA duplexes.
Escherichia colielongation‐factor‐Tu mutants with decreased affinity for aminoacyl‐tRNA
Authors: ANDERSEN Carsten , WIBORG Ove 
Published: March 2005

The two evolutionary well‐conserved histidine residues, His66 and His118, ofEscherichia colielongation factor Tu have been subjected to mutational analysis. The two histidines have each been replaced by alanines, denoted H66A and H118A, respectively. His 118 has also been substituted by glutamate, H118E. The three mutants have been characterized with respect to thermostability, GTPase activity and affinity for aminoacylated tRNA. Most conspicuously, the tRNA affinity is reduced or almost abolished.k−1for dissociation of the ternary complex increases by factors of 14, 40 and 48 for H66A, H118A and H118E, respectively, when compared to the wild type. The half‐lives for the non‐enzymic deacylation of aminoacylated tRNA in the ternary complex are 391, 107, 69, 54 and 61 min for wild type, H66A, H118A, H118E and free aminoacylated tRNA, respectively. TheKdis about 20‐times higher for H66A compared to wild type.Our results strongly suggest that His66 and His118 play major roles in stabilization of the ternary complex.