bluuebunny/crossref_metadata_2025 huggingface dataset:
Spinach Chloroplast O‐Acetylserine (thiol)‐Lyase Exhibits two Catalytically Non‐Equivalent Pyridoxal‐5′‐Phosphate‐Containing Active Sites
Authors: Rolland Norbert , Ruffet Marie‐Line , Job Dominique , Douce Roland , Droux Michel
Published: August 2004

A synthetic gene encoding the mature spinach‐chloroplast O‐acetylserine (thiol)‐lyase was constructed and expressed in an Escherichia coli strain carrying the T7 RNA polymerase system. The pure recombinant protein was obtained at high yield (6 mg/l cell culture) using a new purification procedure that includes affinity chromatography on Green A agarose. Its specific activity was of the order of 1000 U/mg, and its physical properties were similar to those previously reported for the natural enzyme isolated from spinach chloroplasts. In particular the recombinant enzyme, as for the natural enzyme, behaved as a homodimer composed of two identical subunits each of Mr 35000. From steady‐state kinetic studies using sulfide or 5‐thio(2‐nitrobenzoate) (Nbs) as alternative nucleophilic co‐substrates, the enzyme exhibited positive kinetic co‐operativity with respect to O‐acetylserine [Ser(Ac)] in the presence of sulfide and a negative kinetic co‐operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by absorbance and fluorescence measurements to obtain insight into the role of pyridoxal 5′‐phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme provoked the disappearance of the 409‐nm absorbance band of the pyridoxal 5′‐phosphate Schiff base and the appearance of two new absorbance bands, the one located between 320 nm and 360 nm and the other centered at 470 nm. Also, the fluorescence emission of the pyridoxal 5′‐phosphate Schiff base was quenched upon addition of Ser(Ac) to the enzyme. These changes were most presumably due to the formation of a Schiff base intermediate between α‐aminoacrylate and the pyridoxal 5′‐phosphate cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbance and fluorescence equilibrium data disclosed a cooperative behavior in Ser(Ac) binding, in agreement with the steady‐state kinetic results. Fluorescence quenching experiments with acrylamide and iodide revealed that the indole ring of Trp176 was largely exposed and located within the pyridoxal 5′‐phosphate active site. These results are consistent with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit‐subunit interactions, the enzyme exhibits two non‐equivalent pyridoxal‐5′‐phosphate‐containing active sites.
Catabolic Ornithine Carbamoyltransferase of Pseudomonas aeruginosa
Authors: Nguyen Van Thanh , Baker Darren P. , Tricot Catherine , Baur Heinz , Villeret Vincent , Dideberg Otto , Gigot Daniel , Stalon Victor , Haas Dieter
Published: August 2004

Pseudomonas aeruginosa, has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu105 and the C‐terminus are known to be essential for this cooperativity. When Glu105 and nine C‐terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis‐Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF, gene to the P. aeruginosa arcB, gene. A hybrid enzyme consisting of 101 N‐terminal ArgF amino acids fused to 233 C‐terminal ArcB residues and the reciprocal ArcB‐ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N‐terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N‐terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N‐terminal ArgF amino acids and the corresponding C‐terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N‐terminal ArcB region, a replacement of motif 28–33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three‐dimensional structure of catabolic OTCase, which allows predictions about trimer–trimer interactions. Dodecameric assembly appears to require at least three regions: the N‐ and C‐termini (which are close to each other in a monomer), residues 28–33 and residues 147–154.

Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu105→Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.
Quantitative Analysis of the Targeting of Mannose‐Terminal Glucocerebrosidase
Authors: Bijsterbosch Martin K. , Donker Wilma , Van De Bilt Hendrika , Van Weely Sonja , Van Berkel Theo J. C. , Aerts Johannes M. F. G.
Published: August 2004

Gaucher's disease is an inherited lysosomal storage disorder that is caused by a deficiency of glucocerebrosidase. The resulting accumulation of the substrate glucosylceramide in macrophages of liver, spleen, and bone marrow causes severe clinical symptoms. Gaucher's disease is treated by intravenous administration of a modified glucocerebrosidase (Alglucerase), which has exposed mannose residues to promote uptake by target macrophages. To evaluate the effectiveness of the targeting of Alglucerase, we studied the fate of the enzyme in the rat. Intravenously injected Alglucerase was rapidly cleared from the circulation (half‐life 2.0±0.5 min). The liver was the main site of uptake, with 65.6±1.2% of the dose present at 10 min after injection. Smaller amounts (<3% of the dose) were taken up by spleen and bone marrow. Previous injection with mannan substantially increased the plasma half‐life of the enzyme (14.8±3.2 min versus 1.7±0.3 min in solvent‐preinjected controls) and uptake of the enzyme by liver, spleen and bone marrow was reduced by >90%. These findings indicate that the enzyme is taken up by these organs via mannose‐specific receptors. Subcellular fractionation of the liver indicated that the enzyme is internalized and transported to the lysosomes. By isolating various liver cell types after injection of the Alglucerase, it was found that endothelial cells are the main site of uptake of the enzyme: 60.8±3.4% of the total liver uptake. Parenchymal and Kupffer cells were responsible for 31.0±3.1% and 8.2±0.7% of the hepatic uptake, respectively.

We conclude that Alglucerase is rapidly cleared from the circulation by mannose‐specific receptors in liver, spleen, and bone marrow. However, less than 10% of the enzyme taken up by the liver is accounted for by Kupffer cells, the hepatic target cells for therapeutic intervention. It is suggested that alterations of the formulation of the therapeutic enzyme may lead to a higher uptake by Kupffer cells and other macrophages, and thus to a more (cost)effective therapy of Gaucher's disease.
Catalytic Properties, Molecular Composition and Sequence Alignments of Pyruvate: Ferredoxin Oxidoreductase from the Methanogenic Archaeon Methanosarcina Barkeri (Strain Fusaro)
Authors: Bock Anne‐Katrin , Kunow Jasper , Glasemacher Jürgen , Schönheit Peter
Published: August 2004

Methanosarcina barkeri (strain Fusaro) was grown on pyruvate as methanogenic substrate [Bock, A. K., Prieger‐Kraft, A. & Schönheit, P. (1994) Arch. Microbiol. 161, 33–46]. The first enzyme of pyruvate catabolism, pyruvate oxidoreductase, which catalyzes oxidation of pyruvate to acetyl‐CoA was purified about 90‐fold to apparent electrophoretic homogeneity. The purified enzyme catalyzed the CoA‐dependent oxidation of pyruvate with ferredoxin as an electron acceptor which defines the enzyme as a pyruvate: ferredoxin oxidoreductase. The deazaflavin, coenzyme F420, which has been proposed to be the physiological electron acceptor of pyruvate oxidoreductase in methanogens, was not reduced by the purified enzyme. In addition to ferredoxin and viologen dyes, flavin nucleotides served as electron acceptors. Pyruvate: ferredoxin oxidoreductase also catalyzed the oxidation of 2‐oxobutyrate but not the oxidation of 2‐oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, 3‐hydroxypyruvate and oxaloacetate. The apparent Km values of pyruvate: ferredoxin oxidoreductase were 70 μM for pyruvate, 6 μM for CoA and 30 μM for clostridial ferredoxin. The apparent Vmax with ferredoxin was about 30 U/mg (at 37°C) with a pH optimum of approximately 7. The temperature optimum was approximately 60°C and the Arrhenius activation energy was 40 kJ/mol (between 30°C and 60°C). The enzyme was extremely oxygen sensitive, losing 90% of its activity upon exposure to air for 1 h at 0°C. Sodium nitrite inhibited the enzyme with a Ki, of about 10 mM. The native enzyme had an apparent molecular mass of approximately 130 kDa and was composed of four different subunits with apparent molecular masses of 48, 30, 25, and 15 kDa which indicates that the enzyme has an αβγδ structure. The enzyme contained 1 mol/mol thiamine diphosphate, and about 12 mol/mol each of non‐heme iron and acid‐labile sulfur. FAD, FMN and lipoic acid were not found. The N‐terminal amino acid sequences of the four subunits were determined. The sequence of the α‐subunit was similar to the N‐terminal amino acid sequence of the α‐subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductases of the hyperthermophiles Archaeoglobus fulgidus, Pyrococcus furiosus and Thermotoga maritima and of the mesophile Helicobacter pylori, and to the N‐terminal amino acid sequence of the homodimeric pyruvate: ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the α‐subunit of the M. barkeri enzyme and the heterodimeric pyruvate: ferredoxin oxidoreductase of the archaeon Halobacterium halobium.
Nerve Growth Factor Treatment of Sensory Neuron Primary Cultures Causes Elevated Levels of the mRNA Encoding the ATP Synthase β‐Subunit as Detected by a Novel PCR‐Based Differential Cloning Method
Authors: Kendall Giles , Ensor Elizabeth , Crankson Herbert D. , Latchman David S.
Published: August 2004

The mRNA encoding the rat ATP synthase β‐subunit was rapidly induced by nerve growth factor, within 60 min, in cultured adult rat dorsal root ganglion neurons. ATP synthase β‐subunit cDNA clones were isolated from a λ library. The library was constructed using rat dorsal root ganglion mRNA that was differentially screened with cDNA‐derived probes from untreated and nerve‐growth‐factor‐treated primary cultures of adult rat dorsal root ganglion sensory neurons. Radiolabelled probes were made from submicrogram quantities of RNA, by a novel PCR‐based technique, which allows small amounts of primary tissue to be used for library screening. The use of this technique in isolating novel differentially expressed mRNAs is discussed.
Oxidative Inactivation of Human 5‐Lipoxygenase in Phosphatidylcholine Vesicles
Authors: De Carolis Emidio , Denis Danielle , Riendeau Denis
Published: August 2004

Human 5‐lipoxygenase is a non‐heme iron protein which possesses 5‐oxygenase, leukotriene A4 synthase and pseudoperoxidase activities and which undergoes a rapid irreversible inactivation during these reactions. The inactivation of the enzyme was dependent on the structural characteristics of the substrate for the reaction, on O2 concentration and on exposure to phospholipids and calcium. The apparent first‐order rate constant for enzyme inactivation (km) was 0.6 min−1 during the oxygenation of arachidonic acid in air‐saturated buffer containing phosphatidylcholine vesicles and Ca2+. The rate of enzyme inactivation was dependent on the substrate for the reaction and was about threefold slower during the oxygenation of 5,8‐icosadienoic acid and 12(S)‐hydroxyicosatetraenoic acid compared with arachidonic acid. Lowering the O2 concentration to 60 μM during the oxygenation of arachidonic acid also caused a 2.5‐fold decrease in km without affecting the initial rate of the reaction resulting in an increase in both 5‐hydroperoxyicosatetraenoic acid (5‐HPETE) and leukotriene A4 accumulation. The concentration of O2 for half‐maximal activity (initial rate and product accumulation) was approximately 10 μM. In contrast, the activity and the rate of inactivation during the leukotriene A4 synthase reaction with exogenous 5‐HPETE (km= 2.0 min−1) were independent of O2 concentration. A rapid inactivation of the enzyme was also observed during aerobic incubation with phosphatidylcholine vesicles and Ca2+ in the absence of substrate, with a sequential loss of the oxygenase (t1/2= 0.5 min) and pseudoperoxidase (t1/2= 7 min) activities. Protection against this turnover‐independent inactivation was observed in the presence of the selective reversible 5‐lipoxygenase inhibitor L‐739,010 (1S, 5R] 3‐cyano‐1‐(3‐furyl)‐6‐(6‐[3‐(3rα‐hydroxy‐6,8‐dioxyabicyclo [3.2.1] octanyl)] pyridin‐2‐ylmethoxy) naphthalene) and by prior treatment of vesicles with sodium borohydride and, to a lesser extent, by glutathione peroxidase. The results show that the inactivation of 5‐lipoxygenase in phospholipid vesicles is dependent on the structure of the unsaturated fatty acid substrate for the reaction, on the concentration of oxygen and on a turnover‐independent oxidation at the active‐site leading to the sequential loss of the oxygenase and pseudoperoxidase activities of the enzyme.
The Cloning of a Rapidly Evolving Seminal‐Vesicle‐Transcribed Gene Encoding the Major Clot‐Forming Protein of Mouse Semen
Authors: Lundwall Åke
Published: August 2004

Approximately 30 kb of the mouse genome, containing the gene for a major seminal vesicle transcript, has been cloned. The gene was identified by the similarity to members of a family with rapidly evolving genes that includes the gene encoding the major clot protein in rat semen, SVS II, and the human semenogelin genes. The nucleotide sequence of 16.9 kb was determined; this sequence encompasses the gene of 2215 bp plus 9‐kb and 5.6‐kb regions flanking the 5′ and 3′ ends of the gene. The transcription unit is divided into three exons, of which the first encodes the signal peptide, the second the secreted protein, while the third exon' contains 3′‐nontranslated nucleotides only. The transcript encodes a protein of 375 amino acid residues, including a signal peptide of 22 residues. The secreted polypeptide is a protein of Mr 38442 and is similar in sequence but smaller than the major clot‐forming protein of rat semen, SVS II. It is highly charged at pH 7 and it has an isoelectric point of 10.68. The central part of the protein consists of tandem repeats that might serve as a substrate for transglutaminase.
A Note on Circular‐Dichroic‐Constrained Prediction of Protein Secondary Structure
Authors: Galat Andrzej
Published: August 2004

Circular dichroic (CD) spectra of bovine immunosuppressant binding proteins FKBP12 and FKBP25, and cyclophilins (peptidylprolyl isomerases) A (bCyP‐18) and B (bCyP‐20), the immunophilins which selectively bind the clinically useful immunosuppressants FK506, rapamycin and cyclosporin A, respectively, were analysed using the singular‐value‐decomposition algorithm augmented by a simplified variable selection method. The differences between the CD‐estimated values of α‐helix, β‐structure and β‐turn and those predicted by the Chou‐Fasman algorithm were minimized using the CD data as constraints of an algorithm which utilizes the method of hierarchical updating of quasi‐equipotential peptide segments of the Chou‐Fasman prediction. The method allows one to correct the Chou‐Fasman prediction of secondary structures in globular proteins.
Energizing Effects of Illumination on the Reactivities of Lysine Residues of the γ Subunit of Chloroplast ATP Synthase
Authors: Komatsu‐Takaki Mizuho
Published: August 2004

Incubation of chloroplast thylakoids with pyridoxal 5′‐phosphate for a short time (5 s) modified the lysine residues of the γ subunit of ATP synthase. Energization of thylakoids by illumination increased the reactivity of Lys24 by a factor of three and decreased the reactivity of Lys30 to 60%. The reactivities of these residues reached their maximum and minimum values, respectively, within 1 s after the onset of illumination. Illumination of thylakoids increased the reactivities of Lys222 and Lys231 in two steps by a factor of three. The first step was completed within 1 s and the second step was completed 20–30 s after the onset of illumination. In the presence of 10 mM NH4CI, illumination of thylakoids did not change the reactivities of these lysine residues.

These results suggest that the Lys24‐ and Lys30‐containing region of the γ subunit changes its conformation rapidly in response to μH+ and that the Lys222‐ and Lys231‐containing region of the γ subunit changes its conformation in two steps in response to μH+ formation.
Molecular Cloning of Mouse 17β‐Hydroxysteroid Dehydrogenase Type 1 and Characterization of Enzyme Activity
Authors: Nokelainen Pasi , Puranen Terhi , Peltoketo Hellevi , Orava Mauri , Vihko Pirkko , Vihko Reijo
Published: August 2004

The biological activity of certain estrogens and androgens is modulated by enzymes called 17β‐hydroxysteroid dehydrogenases (17β‐HSDs), which catalyze the interconversion between less active 17‐oxosteroid and more active 17β‐hydroxysteroid forms. In the present report, we describe cloning of mouse 17β‐HSD type‐1 cDNA from an ovarian library generated from 4,4′‐(1,2‐diethyl‐1,2‐ethenediyl)bisphenol‐(diethylstilbestrol)‐treated mice, and characterization of the corresponding enzyme. The open reading frame of the mouse 17β‐HSD type‐1 cDNA encodes a peptide of 344 amino acid residues with a predicted molecular mass of 3678.5 Da. The mouse 17β‐HSD type‐1 enzyme shares 63% and 93% overall identity with human and rat 17β‐HSD type‐1 enzymes, respectively, and the most striking differences between the mouse and human type‐1 enzymes are between the amino acid residues 197 and 230 and in the carboxy terminus of the enzymes. Similarly to the human 17β‐HSD type‐1 enzyme, the mouse type‐1 enzyme primarily catalyzes reductive reactions from 17‐oxo forms to 17β‐hydroxy forms in intact cultured cells, but unlike the human type‐1 enzyme, the mouse enzyme does not prefer phenolic over neutral substrates. Thus, mouse 17β‐HSD type 1 catalyzes reduction of androst‐4‐ene‐3,17‐dione (androstenedione) to 17β‐hydroxyandrost‐4‐en‐3‐one (testosterone) as efficiently as 3β‐hydroxyestra‐1,3,5(10)‐trien‐17‐one (estrone) to estra‐1,3,5(10)‐triene‐3β,17β‐diol (estradiol). 17β‐HSD type 1 is predominantly expressed in mouse ovaries, in which it is located in granulosa cells.
The Proteasome Subunit, C2, Contains an Important Site for Binding of the PA28 (11S) Activator
Authors: Kania Mary A. , Demartino George N. , Baumeister Wolfgang , Goldberg Alfred L.
Published: August 2004

The PA28, or 11S regulatory complex, stimulates the peptidase activities of the 20S proteasome. Monoclonal antibodies were screened for their ability to inhibit the activation by PA28 of proteasomes from rabbit reticulocytes. We identified one antibody that inhibited proteasome activation by PA28 and dissociated formed proteasome‐PA28 complexes. A fourfold molar excess of antibody to proteasome markedly reduced the PA28 activation of three peptidase activities. Examination of proteasome‐antibody mixtures by electron microscopy revealed that the antibody formed chains of proteasomes, and digital image analysis of individual proteasomes demonstrated that the antibody binds to the outer α rings. This antibody recognizes proteasome subunit C2, which we conclude contains an important contact site for the PA28 activator. However, the antibody did not block proteasome activation by PA700, or 19s regulator, which also associates with the α rings. Thus, these two regulators appear to bind to the proteasome at different sites.
Role of Insulin‐Like Growth Factors in Autocrine Growth of Human Retinoblastoma Y79 Cells
Authors: Giuliano Michela , Vento Renza , Lauricella Marianna , Calvaruso Giuseppe , Carabillo' Maria , Tesoriere Giovanni
Published: August 2004

In this study, we have demonstrated that human retinoblastoma Y79 cells produce insulin‐like growth factors (IGFs) type I and type II and release them into the medium. We have also ascertained, by means of competitive studies and cross‐linking procedure, that Y79 cells contain the type‐I IGF receptor (IGF‐IR). Furthermore, surface‐bound IGF‐I is internalised by the receptor, then degraded to amino acids.

Insulin, IGF‐I and IGF‐II caused down‐regulation of IGF‐IR; the effect is concentration and time dependant. Scatchard analysis demonstrated that incubation with insulin markedly decreased the binding capacity measured for IGF‐I while the apparent Kd value calculated for IGF‐I binding was not significantly modified. IGF‐I, IGF‐II and insulin induced tyrosine phosphorylation of IGF‐IR. Tyrosine phosphorylation of this receptor with, however, a less strong signal, was detectable even in cells cultured in serum‐free medium without the addition of any exogenous growth factor. Similar results have been found concerning the tyrosine phosphorylation of insulin receptor substrate‐1 (IRS 1). Tyrosine phosphorylation of both IGF‐IR and IRS 1, either under basal conditions or after stimulation with growth factors, was strongly inhibited when α‐IR3, a monoclonal antibody to IGF‐IR, was added to the culture.

IGF‐I was capable of inducing Y79 cell proliferation and its effect was entirely inhibited by the addition of α‐IR3. This antibody also markedly reduced the proliferation of Y79 cells cultured in serum‐free medium not supplemented with stimulatory factors. Our results indicate that IGF‐I and IGF‐IR mediate an autocrine growth mechanism in Y79 cells.
Hybrids of Chicken Cystatin with Human Kininogen Domain 2 Sequences Exhibit Novel Inhibition of Calpain, Improved Inhibition of Actinidin and Impaired Inhibition of Papain, Cathepsin L and Cathepsin B
Authors: Auerswald Ennes A , Nägler Dorit K. , Gross Stefan , Assfalg‐Machleidt Irmgard , Stubbs Milton T. , Eckerskorn Christoph , Machleidt Werner , Fritz Hans
Published: August 2004

Chicken cystatin and human kininogen domain 2 are members of the cystatin superfamily of protein‐type cysteine proteinase inhibitors. They show structural and functional similarities, but only human kininogen domain 2 inhibits calpain. Using recombinant chicken cystatin as a scaffold for hybrid cassette analysis, the known reactive‐site regions (N‐terminus, first hairpin loop and second hairpin loop) were substituted by the corresponding sequences of human kininogen domain 2 in a single and combined manner. Seven hybrids were expressed, purified to homogeneity, characterized protein‐chemically, and their inhibition of papain, actinidin, human cathepsin B, human cathepsin L and calpain (80‐kDa subunit of rabbit skeletal muscle calpain II and porcine erythrocyte calpain I) was determined. Strong but temporary inhibition of calpain by chicken cystatin hybrids carrying the N‐terminus alone (variant sc1‐KD2) or the N‐terminus together with the first hairpin loop (variant sc1/2‐KD2) was observed; hybrids of the second hairpin loop (sc3‐KD2, sc1/3‐KD2, sc2/3‐KD2, sc1/2/3‐KD2) were less strong calpain inhibitors. These data indicate that the inhibition of calpain by human kininogen domain 2 requires the correct conformation and combination of several contact sites, and suggest that the N‐terminus and the first hairpin loop play a major role in this ensemble. Remarkably, hybrid sc2‐KD2 exhibited 5 or 150 times stronger inhibition of actinidin compared to native chicken cystatin or to proteolytically isolated human kininogen domain 2, respectively. This indicates an important role of the first hairpin loop of cystatins in the interaction with actinidin. Along with the impaired inhibition of cathepsin L, papain, actinidin, cathepsin B and calpain by the hybrids sc1/3‐KD2, sc2/3‐KD2 and sc1/2/3‐KD2, these results support our hypothesis that all three predicted contact regions of kininogen domain 2 contribute to binding in the active‐site clefts of papain‐like enzymes in a finely balanced manner.
Backbone Dynamics of a Domain of Protein L Which Binds to Immunoglobulin Light Chains
Authors: Wikström Mats , Forsén Sture , Drakenberg Torbjörn
Published: August 2004

Protein L is a multidomain protein expressed at the surface of some strains of the anaerobic bacterial species Peptostreptococcus magnus. The molecule interacts with the variable domain of immunoglobulin (Ig) light chains through five repeated homologous domains denoted B1 to B5. The fold of the Ig‐light‐chain‐binding B1 domain of protein L (PLB1) has been shown to comprise an α‐helix packed against a four‐stranded β‐sheet and therefore resembles the structure of the IgG‐binding domains of streptococcal protein G. In the present study, amide‐proton exchange and 15N‐relaxation NMR measurements were performed on the B1 domain to investigate its backbone mobility. It was shown that the folded portion of PLB1 is rigid with no regions of significantly higher flexibility than average. The N‐terminus, however, is highly flexible consistent with earlier studies on the solution structure of PLB1. Comparison of the amide‐proton‐exchange data with similar measurements performed on the IgG‐binding domains of protein G indicates that the two proteins have different exchange behaviors in heir second β‐strands. Both protein G and L employ this region of their structures for binding to immunoglobulins since the interaction of protein G and protein L with IgG Fab and the Ig light chain, respectively, involves residues from the second β‐strand.
A Comparative study of the Phosphotyrosyl Phosphatase Specificity of Protein Phosphatase Type 2A and Phosphotyrosyl Phosphatase Type 1B Using Phosphopeptides and the Phosphoproteins p50/HS1, c‐Fgr and Lyn
Authors: Agostinis Patrizia , Donella‐Deana Arianna , Van Hoof Christine , Cesaro Luca , Brunati Anna Maria , Ruzzene Maria , Merlevede Wilfried , Pinna Lorenzo A. , Goris Jozef
Published: August 2004

The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl‐phosphatase‐activator‐(PTPA)‐stimulated protein phosphatase (PP)2AD (rabbit muscle) and a bona fide PTP‐1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine‐containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c‐Src and c‐Fgr, as well as the intact phosphoprotein p501 HS1 and the Src‐related tyrosine kinases, Lyn and c‐Fgr.

The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2AD than for PTP‐1B, C‐terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2AD, but they do not affect the PTP‐1B activity. Acidic residues at the –2 and –3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP‐1B.

The higher‐order structure of the protein substrates also differentially influences both enzymes: the phospho‐octapeptide KDDEYp NPA, which reproduces the autophosphorylation site in c‐Fgr (Tyr400), is only dephosphorylated by PP2AD if embedded in the intact protein, whereas the opposite is true for PTP‐1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP‐1B and not for PP2AD. Lyn and c‐Fgr phosphorylated by C‐terminal Src kinase (CSK) at their down‐regulatory site are resistant to the action of both PTPases while the [Phe6]Src‐(514–533) phosphopeptide, representing the highly similar site affected by CSK in c‐Src, is readily dephosphorylated by both PTPases, although to a different extent.

In vitro dephosphorylation of the c‐Fgr Tyr400 site by PP2AD is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down‐regulatory site) of c‐Fgr are phosphorylated, PP2AD can reverse both phosphorylations.
Purification and Characterization of a DNA Helicase from Pea Chloroplast that Translocates in the 3′‐to‐5′ Direction
Authors: Tuteja Narendra , Phan Tuan‐Nghia , Tewari Krishna K.
Published: August 2004

An ATP‐dependent DNA hecase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68‐kDa subunits. The purified enzyme shows DNA‐dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA gase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utized. Pea chloroplast DNA hecase can unwind a 17‐bp duplex whether it has unpaired single‐stranded tails at both the 5′ end and 3′ end, at the 5′ end or at the 3′ end only, or at neither end. However, it fails to act on a blunt‐ended 17‐bp duplex DNA. The enzyme moves unidirectionally from 3′ to 5′ along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.
Folding and Activation of Human Procathepsin S from Inclusion Bodies Produced in Escherichia coli
Authors: Kopitar Gregor , Dolinar Marko , Štrukelj Borut , Pungerčar Jože , Turk Vito
Published: August 2004

Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7‐based expression system. After cell disruption, the dissolved inclusion body proteins were S ‐sulphonated with 2‐nitro‐5‐thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two‐step dilution which significantly increased the yield of production compared to single‐step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 μg/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/I fermentation broth.
Linking of Isozyme and Class Variability Patterns in the Emergence of Novel Alcohol Dehydrogenase Functions
Authors: Hjelmqvist Lars , Shafqat Jawed , Siddiqi Abdur Rehman , Jörnvall Hans
Published: August 2004

The nature of the isozyme differences in the class‐I alcohol dehydrogenase structure from the lizard, Uromastix hardwickii, was determined and related to those in the human and horse enzymes, for which isozyme structures have also been established. The Uromastix isozymes differ much (at a total of 72 positions, 19%) but, in spite of this, have similar properties and were not obtained resolved. Their structures were analyzed in mixture, and the two sub‐sets of peptides obtained could be distinguished by evaluation of the recovery ratios within the peptide pairs. The isozymes have class‐I activities, with an ethanol dehydrogenase activity of 0.6 U/mg and no formaldehyde dehydrogenase activity, have typical class‐I structures, and are composed of N‐terminally acetylated 375‐residue subunits (a and b). Importantly, variability patterns between the isozymes are reminiscent of those both in the other two lines with isozymes (primates and horse) and in the class distinctions of the enzyme. Hence, the variability pattern since the distant stage of class‐I emergence is also visible within the more recent isozyme divergence, illustrating a continuity in the evolution of isozymes to classes (and then to enzymes). The pattern also links the different levels of multiplicity and may suggest an acceptability in common to duplications and mutations, compatible with the emergence of novel functions.
Liver Class‐I Alcohol Dehydrogenase Isozyme Relationships and Constant Patterns in a Variable Basic Structure
Authors: Shafqat Jawed , Hjelmqvist Lars , Jörnvall Hans
Published: August 2004

The major ethanol dehydrogenase of cobra liver was characterized in order to clarify isozyme relationships and functional motifs of the vertebrate enzyme. The cobra protein is a class‐I form, most related to one of the isozyme subunits (the a form) in Uromastix (lizard) liver. This positions the isozyme duplication and defines the main‐line alternative. The new structure also allows extensive correlations with structure/function relationships for alcohol dehydrogenases in general, of which 38 animal variants (still disregarding strain and allelic differences) now have been characterized. Architectural features are discerned, distinguishing the enzyme at large, the classes, and the functional interactions at the sites of substrate binding and coenzyme binding. Variability is greater at the substrate‐binding site, with only one of 13 residues strictly conserved (His67, one of the active‐site zinc ligands) but all other residues differing among and frequently within classes. However, many substrate‐interacting residues are class preferential and may be used in predictive assignments. Class‐I/III differences concern position 48 (typically Ser in class I, Thr in class III), position 93 (Phe versus Tyr), position 141 (branch‐chained aliphatic residue versus methionine), position 57 (hydrophobic residue versus Asp), position 115 (Asp versus Arg), position 116 (Leu or IIe versus Val), position 306 (Met or Leu/IIe versus Phe), position 309 (Phe or Leu/IIe versus Val) and position 318 (Val or IIe versus Ala). In contrast, coenzyme binding is more conserved. A characteristic coenzyme‐binding motif, covering only a 50‐residue stretch, is defined as tVDiK (residues 178, 203, 223, 224, 228; capital letters for residues strictly conserved and small‐cases letters for residues nearly so). This motif is class independent and unique to animal alcohol dehydrogenases. Therefore, the novel enzyme structure establishes class‐I isozyme relationships, shows characteristic ‘constant’ residues also in the ‘variable’ class‐I line, and defines residue‐specific patterns which may have a predictive value in functional assignments of an increasing number of undefined further forms expected to result from gene projects.
Depolarization‐induced Tyrosine Phosphorylation of Paxillin in PC12h Cells
Authors: Khan Muhammad Ashraf , Okumura Nobuaki , Okada Masato
Published: August 2004

Treatment of PC12h cells with a high concentration of KCl induces depolarization of the plasma membrane and Ca2+ influx into the cells. We have previously shown that KCl induced tyrosine phosphorylation of cellular proteins of 120, 110, 68, 44 and 42 kDa. In the present study, we found that the 68‐kDa protein is paxillin, a tyrosine kinase substrate associated with the actin cyloskeleton. A calcium ionophore, A23187, also induced tyrosine phosphorylation of the 68‐kDa protein, while KCl did not in the presence of EGTA or nifedipine, indicating that the effect of KCl was due to the Ca2+ influx into the cells. Tyrosine phosphorylation of paxillin was also induced by nerve growth factor and epidermal growth factor, but its migration patterns on an SDS/polyacrylamide gel were different, that is, nerve growth factor and epidermal growth factor caused upward shifts of the bands, while KCl did not. However, both forms could associate with Csk and Crk. The effect of KCl was blocked by cytochalasin D, indicating that tyrosine phosphorylation required the integrity of actin filaments. These results suggest that tyrosine phosphorylation of paxillin may be involved in Ca2+ ‐dependent events in neuronal and neuroendocrine cells.
The Presence of Tandem Repeats and the Initiation of Replication in Rabbit Mitochondrial DNA
Authors: Dufresne Catherine , Mignotte Françoise , Guéride Monique
Published: August 2004

The non‐coding region of rabbit mitochondrial DNA (mtDNA) exhibits two sets of tandem repeats between conserved sequence block 1 (CSB1) and the tRNAPhe gene. Both repeated sequences, short repeats (SR) and long repeats (LR), which contain 20 and 153 nucleotides, respectively, are involved in the generation of a high degree of mitochondrial heteroplasmy. Due to the location of these sequences in the regulatory region and their properties in terms of variable conformations, they could affect the initiation of replication of the heavy‐strand DNA (H‐strand DNA) and subsequently would influence the efficiency of mtDNA replication. The extremities of the displacement loop (D‐loop) DNA strand and the 5′ ends of RNA primers initiating the H‐strand DNA synthesis were characterized in individual rabbits. Mapping at the nucleotide level of the 5′ and 3′ ends of the D‐loop DNA strands indicates that both extremities are heterogeneous. The H‐strand replication origin OH is located close to the conserved sequence block CSB1 as in other mammals. In all of the individuals studied so far, DNA molecules with a 5′ end 1–2 nucleotides downstream of CSB1 were always present. As H‐strand DNA replication is believed to be primed by RNA transcribed from the light‐strand promoter (LSP), RNA mapping was carried out to identify the 5′ end of H‐strand RNA. Neighbouring initiation sites were identified at the nucleotide level in an (A+T)‐rich region at nucleotide 1841 and in a stretch of cytosine residues at nucleotides 1849–1852, which are located at the beginning of the first long repeat. A detailed RNA analysis indicates that H‐strand RNA molecules are initiated in each long repeat. The amplification of the regulatory region has produced multiple initiation transcription sites and a family of RNA primers of various lengths. These variations in length and the ensuing secondary structures are not critical for their potential function as H‐strand DNA replication primers.
Construction of a Multifunctional Pneumococcal Murein Hydrolase by Module Assembly
Authors: Sanz Jesús M. , García Pedro , García José L.
Published: August 2004

A chimeric trifunctional pneumococcal peptidoglycan hydrolase (CHL) has been constructed by fusing a choline‐binding domain with two catalytic modules that provide lysozyme and amidase activity. The chimeric enzyme behaves as a choline‐dependent enzyme and its activity is comparable to that of the parent enzymes. Site‐directed mutagenesis of CHL produced a mutated enzyme ([D9A, E36A]CHL) that only exhibited an amidase activity. Comparative biochemical analyses of CHL and [D9A, E36A]CHL strongly suggest that the lysozyme catalytic module confers 88% of the total activity of CHL, whereas 12% of the activity can be ascribed to the amidase module. Both enzymatic activities are affected by the process of activation or conversion induced by choline suggesting that the conversion process is produced by a conformational change in the choline‐binding domain. Our findings demonstrate that the three modules can acquire the proper folding conformation in the three‐domain chimeric CHL enzyme. This experimental evidence supports the modular theory of protein evolution, and demonstrates that modulor assembly of functional domains can be a rational approach to construct fully active chimeric enzymes with novel biological or biotechnological properties.
X‐ray Absorption Spectroscopy Study of Zinc Coordination in Tetanus Neurotoxin, Astacin, Alkaline Protease and Thermolysin
Authors: Morante Silvia , Furenlid Lars , Schiavo Giampietro , Tonello Fiorella , Zwilling Robert , Montecucco Cesare
Published: August 2004

Tetanus and botulinum neurotoxins constitute a new group of Zn‐endopeptidases which has recently been actively investigated with the purpose of correlating their biochemical properties to their neurobiocytosis inhibitory capacity. Crystallographic data show that Zn‐endopeptidases are characterized by an active site with a Zn atom coordinated to two histidines and a glutamate‐bound water molecule. The two histidines and the glutamate residues belong to the HEXXH motif which is characteristic of most Zn‐endopeptidases. A fourth metal ligand is a glutamate in thermolysin‐like proteinases, but it is an histidine in the astacin family of proteinases and in alkaline protease. Astacin and alkaline protease possess a tyrosine as fifth Zn ligand, whose position in the case of alkaline protease could not be determined by X‐ray crystallography. Not much is known about the atom arrangement around the active site in tetanus neurotoxin.

In this work X‐ray absorption spectroscopy has been used to obtain information on the Zn coordination mode in tetanus neurotoxin. The near‐edge and extended fine‐structure absorption spectra of this toxin are compared with those of astacin, alkaline protease and thermolysin. The present data and sequence information suggest a new pattern of Zn coordination in tetanus neurotoxin with one water molecule and three aromatic residues as metal ligands. These residues are the two histidines of the characteristic motif and a tyrosine which is tentatively identified with Tyr242, on the basis of sequence comparison and mutagenesis experiments. The mean distances of the Zn from the nearest coordinated atoms is reported. Our results indicate that alkaline protease, like astacin, also possesses a tyrosine as a fifth ligand.
A Two‐Hybrid System Analysis Shows Interactions Between 6‐Phosphofructo‐1‐Kinase and 6‐Phosphofructo‐2‐Kinase but not Between Other Glycolytic Enzymes of the Yeast Saccharomyces cerevisiae
Authors: Müller Susanne , Boles Eckhard , Zimmermann Friedrich K.
Published: August 2004

The yeast two‐hybrid system was used to investigate whether protein‐protein interactions between enzymes of the early reactions of glycolysis exist in the yeast Saccharomyces cerevisiae. Various glycolytic enzymes were fused either to the GAL4 transcription‐activating or DNA‐binding domain and were tested for their ability to restore GAL4‐dependent gene activation. All the different fusion proteins complemented the growth and enzymatic defects caused by the deletion of the respective genes, which indicates that these proteins are still functional. Interactions between the two phosphofructo‐1‐kinase subunits PFK1 and PFK2, interactions between the phosphofructo‐2‐kinase subunits, and dimerization of phosphoglucose isomerase were demonstrated. Dimerization of hexokinase 2, however, could not be demonstrated neither with N‐terminal nor C‐terminal fusions. A direct interaction between the hexose‐6‐phosphate interconverting enzymes hexokinase 2, phosphoglucose isomerase, and phosphofructo‐1‐kinase could also not be demonstrated. Nevertheless, our results indicate a weak interaction between phosphofructo‐1‐kinase and phosphofructo‐2‐kinase.
Regulation of Olfactory Signalling via cGMP‐Dependent Protein Kinase
Authors: Kroner Christine , Boekhoff Ingrid , Lohmann Suzanne M. , Genieser Hans‐Gottfried , Breer Heinz
Published: August 2004

Strong odor stimuli elicit a slow and sustained increase of the cGMP concentration in isolated rat olfactory cilia. Elevated cGMP levels appear to attenuate the primary response to odorant stimulation. Incubating cilia with membrane‐permeable cGMP derivates caused a significantly reduced cAMP signal in response to odorant stimulation. This inhibitory effect was mimicked by 8‐(4‐chlorophenlythio)‐cGMP, a selective activator of cGMP‐activated protein kinases; in contrast, a selective inhibitor, [8‐(4‐chlorophenylthio)‐guanosine‐3′,5′‐cyclic monophosphorothioate] of cGMP kinases enhanced the reactivity to odorant stimulation. The data suggest that the responsiveness of olfactory sensory cells is governed by a cGMP‐dependent protein kinase. Western‐blot analysis using subtype‐specific antibodies indicated that cytosolic type‐I cGMP kinase, but not the membrane‐associated type‐II cGMP kinase, is expressed in olfactory sensory neurons.
Methylcobalamin:Coenzyme M Methyltransferase Isoenzymes MtaA and MtbA from Methanosarcina barkeri
Authors: Harms Ulrike , Thauer Rudolf K.
Published: August 2004

Methanosarcina barkeri is known to contain two methyltransferase isoenzymes, here designated MtaA and MtbA, which catalyze the formation of methyl‐coenzyme M from methylcobalamin and coenzyme M. The genes encoding the two soluble 34‐kDa proteins have been cloned and sequenced. mtaA and mtbA were found to be located in different parts of the genome, each forming a monocystronic transcription unit. Northern blot analysis revealed that mtaA is preferentially transcribed when M. barkeri is grown on methanol and the mtbA gene when the organism is grown on H2/CO2 or trimethylamine. Comparison of the deduced amino acid sequences revealed the sequences of the two isoenzymes to be 37% identical. Both isoenzymes showed sequence similarity to uroporphyrinogen III decarboxylase from Escherichia coli. The mtaA gene was tagged with a sequence encoding six His placed six bp before the mtaA start codon, and was functionally overexpressed in E. coli. 25% of the E. coli protein was found to be active methyltransferase which could be, purified in two steps to apparent homogenity with a 70% yield.
Regulation of Glutamate Dehydrogenase Expression in the Developing Rat Liver
Authors: Das Atze T. , Salvadó Josepa , Boon Louis , Biharie Gaitric , Moorman Antoon F. M. , Lamers Wouter H.
Published: August 2004

To study the regulation of the expression of glutamate dehydrogenase (Glu‐DH) in rat liver during development, the Glu‐DH mRNA concentration in the liver of rats ranging in age from 14 days prenatal development to 3 months after birth was determined. This concentration increased up to two days before birth, decreased rapidly between two days before and one day after birth and increased again in the second and third postnatal week. The ratio of Glu‐DH mRNA/protein decreased more than 10‐fold in the prenatal period, whereas it did not change significantly after birth. Thus, whereas the ratio between the Glu‐DH monomer protein molecules and Glu‐DH mRNA molecules is found to be approximately 1400 at 14 days of prenatal development, it is approximately 17000 four weeks after birth. We argue that an increase in the translational efficiency after birth is the most likely cause of the observed developmental changes in Glu‐DH mRNA/protein ratio. Our results suggest that the expression after birth is predominantly regulated at the pretranslational level, whereas the prenatal Glu‐DH expression is regulated both at the translational level and at the pretranslational level.