bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antagonistic properties are shifted back to agonistic properties by further N‐terminal shortening of pituitary adenylate‐cyclase‐activating peptides in human neuroblastoma NB‐OK‐1 cell membranes
Authors: VANDERMEERS André , VANDENBORRE Stéphane , HOU Xue , DE NEEF Philippe , ROBBERECHT Patrick , VANDERMEERS‐PIRET Marie‐Claire , CHRISTOPHE Jean 
Published: March 2005

N‐terminally shortened analogs of the 27‐amino‐acid and 38‐amino‐acid forms of the pituitaryadenylate‐cyclase‐activating neuropeptide, PACAP(1–27) and PACAP(1–38), were synthesized by a solid‐phase method. Systematic deletion of the first 13 amino acids of both PACAP was tested by evaluating their ability to occupy the specific and selective PACAP receptor of human neuroblastoma NB‐OK‐1 cell membranes and to stimulate adenylate cyclase or, when inactive per se, to inhibit PACAP‐stimulated adenylate cyclase activity. For each peptide, the Kact (concentration required for half‐maximal adenylate cyclase activation) or Ki [concentration required to shift the dose/response curve of PACAP(1–27) twofold to the right] was in good agreement with the corresponding IC50 [concentration inhibiting 50% of 125I‐[AcHis1]PACAP(1–27) binding to membranes], suggesting interaction with the same homogeneous class of adenylate cyclase‐coupled receptors.The deletion of the two first amino acids (His1 and Ser2) sufficed to decrease the affinity for receptors and to suppress the capacity to activate adenylate cyclase. The shorter fragments 3–27 and 3–38, 4–27 and 4–38, 5–27 and 5–38, 6–27 and 6–38, 7–27 and 7–38, 8–27 and 8–38, and 9–27 and 9–38 were all competitive antagonists of PACAP(1–27)‐stimulated activity with the N‐terminally shortened PACAP(1–38) derivatives being 4–30‐fold more potent than the equivalent PACAP(1–27) derivatives. In this group PACAP(6–38) was the most potent antagonist (Ki 1.5 nM).Surprisingly, the N‐terminally shorter fragments 10–27 and 10–38, 11–27 and 11–38, 12–27 and 12–38, 13–27 and 13–38, and 14–27 and 14–38 were again able to stimulate adenylate cyclase, the smallest fragments, PACAP(14–27) and PACAP(14–38), being the most potent and efficient (Kact 2 μM and 0.1 μM, respectively). In this group of agonists, PACAP(1–38) derivatives deleted at the N‐terminus were also more potent than the equivalent PACAP(1–27) derivatives.
Relationship between the major protein kinase C substrates acidic 80‐kDa protein‐kinase‐C substrate (80K) and myristoylated alanine‐rich C‐kinase substrate (MARCKS)
Authors: HERGET Thomas , BROOKS Susan F. , BROAD Simon , ROZENGURT Enrique 
Published: March 2005

Two major protein‐kinase‐C (PKC) substrates have been described in the literature; an 87‐kDa bovine and human PKC substrate, called MARCKS, and an acidic 80‐kDa PKC substrate, isolated from rat brain and Swiss 3T3 cells, termed 80K. Since there is only 66–74% sequence similarity between MARCKS and 80K, we have further investigated their relationship in this study.Southern‐blot experiments with gene‐specific probes demonstrated the presence of the 80K, but not MARCKS, gene in the mouse genome. Furthermore, polymerase‐chain‐reaction (PCR) analyses using three pairs of primers that specifically recognise either 80K, MARCKS or conserved sequences of both genes, revealed the presence of only the 80K gene in the mouse and rat genomes and only the MARCKS gene in the bovine and human genomes with mRNA expression in the corresponding brain tissues. Northern‐blot analysis of a variety of tissues indicated that both 80K and MARCKS have similar patterns of expression. Most components of signal‐transduction pathways are present in multiple molecular isoforms as members of a gene family. In contrast, the findings presented in this study indicate that rodent 80K and bovine and human MARCKS are not distinct members of a gene family, but represent the equivalent substrates in different species.
Short‐chain dehydrogenases
Authors: KROOK Maria , PROZOROVSKI Vladimir , ATRIAN Silvia , GONZÀLEZ‐DUARTE Roser , JÖRNVALL Hans 
Published: March 2005

Prokaryotic 3α/20β‐hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu‐C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp‐N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu‐C and Asp‐N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s).Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10‐fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short‐chain dehydrogenase subunits, one mid‐chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C‐terminus.
Lithium, an inhibitor of cAMP‐induced inositol 1,4,5‐trisphosphate accumulation in Dictyostelium discoideum, inhibits activation of guanine‐nucleotide‐binding regulatory proteins, reduces activation of adenylylcyclase, but potentiates activation of guanylyl cyclase by cAMP
Authors: PETERS Dorien J. M. , SNAAR‐JAGALSKA B. Ewa , VAN HAASTERT Peter J. M. , SCHAAP Pauline 
Published: March 2005

Li+ drastically alters pattern formation in Dictyostelium by inhibiting cAMP‐induced presporegene expression and promoting cAMP‐induced prestalk‐gene expression. We reported previously that Li+ inhibits inositol monophosphatases in this organism and strongly reduces basal and cAMP‐stimulated inositol 1,4,5‐trisphosphate levels. We show here that Li+ also reduces cAMP‐induced accumulation of cAMP. but promotes cAMP‐induced accumulation of cGMP. This effect is not due to inhibition of cGMP hydrolysis or inhibition of adaptation and may therefore reflect stimulation of guanylyl‐cyclase activation. Li+ does not affect the binding of cAMP to surface receptors but interferes with the interaction between receptors and guanine‐nucleotide‐binding regulatory (G) proteins. These effects are complex; in the absence of Mg2+, Li+ increases guanosine 5′‐[γ‐thio]triphosphate(GTP[S])‐binding activity to similar levels as 1 mM Mg2+. However, while Mg2+ potentiates cAMP‐induced stimulation of GTP[S]‐binding activity, Li+ effectively inhibits stimulation. Li+ also inhibits cAMP‐stimulated, but not basal high‐affinity GTP‐ase activity, indicating an inhibitory effect on cAMP‐induced activation of G‐proteins.Our data suggest that in addition to inositolphosphate metabolism, the activation of G‐proteins may be a second biochemical target for Li+ effects on pattern formation and signal transduction in Dictyostelium.
Purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum
Authors: RICHTER Oliver‐Matthias H. , SCHÄFER Günter 
Published: March 2005

Cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme was purified to electrophoretic homogeneity by combining ion‐exchange and affinity‐chromatographic separations. This soluble pyrophosphatase probably consists of six identical subunits, since SDS/PAGE gave an estimate of about 22 kDa for a single subunit and size‐exclusion chromatography under non‐denaturing conditions indicates a molecular mass of 110 ± 5 kDa.The two most prominent catalytic features of this enzyme are the absolute requirement for divalent cations for catalytic action, Mg2+ conferring the highest activity, and the pronounced specificity for PPi. The catalytic behavior apparently follows simple Michaelis‐Menten kinetics with a Km of about 7 μM for PPi and a specific acitivity of about 1200 U/mg at 56°C. Surprisingly, maximum activity could be observed at 85°C which is more than 20°C above the temperature for optimal growth.Several cytoplasmic extracts of eubacteria and archaebacteria have been probed with a polyclonal antiserum raised against the purified archaebacterial protein. The only noticeable cross‐reactivity could be detected with an extract from the methanogen Methanosarcina barkeri although this probably does not reflect the inferred phylogenetic relationship between methanogens and Thermoplasma acidophilum.
Cloning and sequence of the gene for the cytoplasmic inogranic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum
Authors: RICHTER Oliver‐Matthias H. , SCHÄFER Günter 
Published: March 2005

The gene (ppa) from the thermoacidophilic archaebacterium Thermoplasma acidophilum, encoding the cytoplasmic pyrophosphatase, has been cloned. Two degenerate oligonucleotide probes, synthesized according to the N‐terminal amino acid sequence of the isolated protein, were used to screen subgenomic libraries.The DNA‐derived amino acid sequence of the archaebacterial enzyme allows, for the first time, comparative studies of cytoplasmic pyrophosphatases to be extended to all three urkingdoms. The archaebacterial pyrophosphatase more closely resembles the eubacterial enzymes on the basis of sequence similarity and subunit size. The majority of amino acid residues considered to be essential for hydrolysis of pyrophosphate seem to have been conserved throughout evolution, as inferred from the results of an alignment of sequences from all three urkingdoms.
Effect of anisotonic cell‐volume modulation on glutathione‐S‐conjugate release, t‐butylhydroperoxide metabolism and the pentose‐phosphate shunt in perfused rat liver
Authors: SAHA Nirmalendu , STOLL Barbara , LANG Florian , HÄUSSINGER Dieter 
Published: March 2005

1. Addition of 1‐chloro‐2,4‐dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione‐S‐conjugate [S‐(2,4‐dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Ahisotonic perfusion did not affect total S‐conjugate formation, but release of the S‐conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S‐conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume‐regulatory net K+ uptake.2. Basal steady‐state release of oxidized glutathione (GSSG) into bile was 1.30 ± 0.12 nmol · g−1· min−1 (n= 18) during normotonic (305 mOsmol/I) perfusion and was 3.8±0.3 nmol · g−1· min−1 in the presence of t‐butylhydroperoxide (50 μmol/I). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t‐butylhydroperoxide(50 μmol/I)‐stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/I) increased. Anisotonic exposure was without effect on t‐butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver‐cell swelling due to addition of glutamine plus glycine (2 mmol/I, each).3. Hypotonic exposure led to a persistent stimulation 14CO2 production from [1‐14C]glucose by about 80%, whereas 14CO2 production from [6‐14C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited 14CO2 production from [1‐14C]glucose by about 40%, whereas 14CO2 production from [6‐14C]glucose was unaffected. The effect of anisotonicity on 14CO2 production from [1‐14C]glucose was also observed in presence of t‐butylhydroperoxide (50 μmol/1), which increased 14CO2 production from [1‐14C]glucose by about 40%.4. t‐Butylhydroperoxide (50 μmol/1) was without significant effect on volume‐regulatory K+ fluxes following exposure to hypotonic (225 mOsmol/1) or hypertonic (385 mOsmol/1) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t‐butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions.5. The data suggest that hypotonic cell swelling stimulates flux through the pentose‐phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide‐induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione‐S‐conjugates into the sinusoidal circulation at the expense of biliary secretion.
The mechanism of stimulation of MgATPase activity of chloroplast F1‐ATPase by non‐catalytic adeninenucleotide binding
Authors: MURATALIEV Marat B. , BOYER Paul D. 
Published: March 2005

The presence of ATP at non‐catalytic sites of the chloroplast F1‐ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551–11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non‐catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non‐catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non‐catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non‐catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non‐catalytic sites, theKm remains constant, at about 50 γM for MgATP and 400 γM for MgGTP.The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1· ADP · Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non‐catalytic sites promotes the ATP‐dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non‐catalytic sites results from increasing the portion of CF1 in an active state during steady‐state ATP hydrolysis.
Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli
Authors: CIONI Patrizia , ONUFFER James Joseph , STRAMBINI Giovanni Battista 
Published: March 2005

The Trp phosphorescence spectrum, intensity and decay kinetics of apo‐aspartate aminotransferase, pyridoxamine‐5P‐aspartate‐aminotransferase and pyridoxal‐5P‐aspartate aminotransferase were measured over a temperature range 160–273 K. The fine structure of the phosphorescence spectra in low‐temperature glasses, with 0–0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine‐5P‐enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal‐5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0–0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp‐sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer.All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal‐5P enzyme shows a decrease in lifetime‐normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal‐5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.
Cloning and specific polymerised‐chain‐reaction amplification of a third charge‐separable human metallothionein isoform
Authors: SOUMILLION Ann , Van DAMME Jo , De LEY Marc 
Published: March 2005

Metallothionein (MT) fractions isolated from human adult liver tissue are readily separated by anion‐exchange chromatography in two isoforms, MT‐1 and MT‐2, which differ from each other in the nature of the amino acid residue at position 11. In fetal liver tissue, the presence of a third charge‐separable MT isoform has been previously reported. We determined its partial amino acid sequence and the sequence of a cDNA clone encoding this MT form. This confirmed the existence of another human MT isoform, hereafter named MT‐0, which is characterized by the presence of a negatively charged amino acid at position 8, and by a Glu23 to Lys substitution in a strictly conserved region of the protein. Taking into account these substitutions, we are able to classify human MT isoforms into three instead of two charge‐separable groups, based on the nature of three amino acid residues.The unique presence of Glu8 in MT‐0 enabled us to develop an MT‐0‐specific amplification by the polymerase chain reaction, which revealed the presence of MT‐0 mRNA in adult liver RNA samples, in spite of the total absence of this isoform at the protein level. This suggests the involvement of post‐transcriptional regulatory mechanisms in the expression of this fetal MT form.
Amino acid and DNA sequences of an extracellular basic protease of Dichelobacter nodosus show that it is a member of the subtilisin family of proteases
Authors: LILLEY Glenn G. , STEWART David J. , KORTT Alexander A. 
Published: March 2005

A DNA fragment encoding an extracellular basic protease (pI ∼ 9.5) from Dichelobacter nodosus, a Gram‐negative obligate anaerobe and the causative agent of ovine footrot, has been cloned and expressed in Escherichia coli and sequenced. E. coli harbouring a plasmid with a 3‐kb DNA fragment containing the D. nodosus basic‐protease gene exhibited proteolytic activity when tested on skim‐milk plates. The sequence of the native basic protease isolated from D. nodosus was also determined by direct amino acid sequencing. Comparison of the deduced sequence of the primary translation product (603 residues) and that of the native protease (344 residues) indicates that the protease is synthesized as a precursor molecule, containing a signal peptide (21 residues), a 111 amino acid pro‐peptide and a 127 residue C‐terminal extension which is subsequently processed to the mature active form.Comparison of the D. nodosus basic protease sequence with that of other serine proteases showed that it is related to the subtilisin family of proteases with strong conservation of sequence identity around the catalytic site residues. A remarkable similarity in structure was found to the serine protease of Xanthomonas campestris, a plant pathogen, with respect to the length of the precursor segments, conservation of disulfide bridges and approximately 50% sequence identity of the mature proteases.
Substrate‐analogue‐induced changes in the nickel‐EPR spectrum of active methyl‐coenzyme‐M reductase from Methanobacterium thermoautotrophicum
Authors: ROSPERT Sabine , VOGES Markus , BERKESSEL Albrecht , ALBRACHT Simon P. J. , THAUER Rudolf K. 
Published: March 2005

Methyl‐coenzyme‐M reductase (MCR) catalyzes the formation of methane from methyl‐coenzyme M [2‐(methylthio)ethanesulfonate] and 7‐mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR‐red1 and MCR‐red2, probably derived from Ni(I). In the presence of the substrate methyl‐coenzyme M, the rhombic MCR‐red2 signal is quantitatively converted to the axial MCR‐red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectum of the enzyme. 3‐Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki= 0.05 μM), converted the EPR signals MCR‐red1 and MCR‐red2 to a novel axial Ni(I) signal designated MCR‐BrPrSO3.3‐Fluoropropanesulfonate (apparent Ki < 50 μM) and 3‐iodopropanesulfonate (apparent Ki < 1 μM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I= 1/2, I= 3/2 and I= 5/2, respectively. This finding suggests that MCR‐BrPrSO3 is not the result of a close halogen‐Ni(I) interaction.7‐Bromoheptanoylthreonine phosphate (BrHpo ThrP) (apparent Ki= 5 μM), which is an inhibitory substrate analogue of 7‐mercaptoheptanoylthreonine phosphate, converted the signals MCR‐red1 and MCR‐red2 to a novel axial Ni(I) signal, MCR‐BrHpoThrP, similar but not identical to MCR‐BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to differnt enzyme/substrate and enzyme/inhibitor complexes.
Calorimetric measurements of thermal denaturation of stefins A and B
Authors: ŽEROVNIK Eva , LOHNER Karl , JERALA Roman , LAGGNER Peter , TURK Vito 
Published: March 2005

Thermal denaturation of two homologous proteins, low‐Mr cysteine‐proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65°C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half‐denaturation at 50.2°C and 90.8°C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively.Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three‐dimensional structure has been determined. The calculated enthalpy, heat‐capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data.