bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of an insulin‐sensitive cyclic AMP phosphodiesterase from rat liver
Authors: BOYES Stewart , LOTEN Ernest G. 
Published: March 2005

A low‐Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola‐cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE‐substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 μM and a Vmax of 6.2 μmol mg−1 min−l with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 μM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 μM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size‐exclusion chromatography, suggesting that it exists as a dimer.A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin‐activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin‐sensitive phosphodiesterase.
Human aldolase A gene
Authors: IZZO Paola , COSTANZO Paola , LUPO Angelo , RIPPA Emilia , PAOLELLA Giovanni , SALVATORE Francesco 
Published: March 2005

The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned gene sequence, spanning 7530 bp, includes twelve exons and occurs as a single copy per haploid human genome. The structural organization of the gene is quite complex: eight exons containing the coding sequence are common to all mRNAs extracted from human and other mammalian sources; four additional exons are present in the 5′ untranslated region, of these one is contained in the ubiquitous type of mRNA, the second is in the muscle‐specific type of mRNA and the third and fourth are in a minor species of mRNA found in human liver tissue. Furthermore, the determined sequence includes 1000 nucleotides upstream from the first exon (exon I) in the 5′ flanking region, and 400 nucleotides, which include the polyadenylation signal, downstream from the termination codon.S1‐nuclease‐protection analysis of the 5′ end of mRNA extracted from human cultured fibroblasts, muscle and hepatoma cell lines indicates the existence of four different transcription‐initiation sites. The latter are also supported by the presence of conventional sequences for eukaryotic promoters. Therefore, the four promoters on the same gene generate different tissue‐specific transcripts, which share the translated sequence, but each has a unique 5′ untranslated region as a result of differential mRNA processing.The nucleotide homology at the coding region and the intron‐exon organization of the three human and mammalian aldolase A, B and C genes confirm that they arose from a common ancestral gene, and that aldolase B diverged first.
Maitotoxin triggers the cortical reaction and phosphatidylinositol‐4,5‐bisphosphate breakdown in amphibian oocytes
Authors: BERNARD Véronique , LAURENT Anne , DERANCOURT Jean , CLÉMENT‐DURAND Monique , PICARD André , Le PEUCH Christian , BERTA Philippe , DORÉE Marcel 
Published: March 2005

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5‐trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave‐like propagation of contraction in the animal hemisphere.All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage‐dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase‐II‐arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.
New insights into maitotoxin action
Authors: SLADECZEK Fritz , SCHMIDT Bernhard Hubert , ALONSO Richard , VIAN Laurence , TEP Aline , YASUMOTO Takeshi , CORY Robert Nassim , BOCKAERT Joël 
Published: March 2005

Maitotoxin (3 ng/mol) induced a massive uptake of 45Ca2+ into BC3H1 cells. This effect exhibits a lag phase of 3 min. Inositol diphosphate formation occurred concomittantly with the 45Ca2+ uptake but inositol monophosphate formation was found only after a 5‐min delay following toxin addition. Maitotoxin‐induced 45Ca2+ influxes could not be blocked by either 1 μM verapamil, 1 μM nifedipine or 1 mM La3+ but was blocked by Zn2+ (IC50= 41 μM). In addition to inositol phosphate formation and 45Ca2+ uptake, maitotoxin stimulated a large uptake of Na+ and a great loss of K+ in BC3H1 cells. In the absence of Ca2+ (1 mM EGTA) none of the four maitotoxin effects could be detected. After restoration of Ca2+ the maitotoxin effects reappeared even when the toxin itself was no longer present. The divalent cation, Co2+ (1 mM), inhibited ion movements induced by maitotoxin and also digitonin (8.1 μM). The toxin action showed a very pronounced pH dependence. At low pH, maitotoxin was inactive. The dose‐response curves for H+ ion inhibition of maitotoxin‐induced Ca2+ uptake showed a shift to the right when determined in the absence of HCO3‐ and HCO3‐/Cl‐ ions.It was concluded that the primary action of maitotoxin in BC3H1 cells was a pore‐forming or channel‐forming activity of a non‐classical type. Some properties of maitotoxin resemble those of α‐latrotoxin, others those of pore‐forming agents such as melittin or α‐toxin of Staphylococcus aureus.
Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase
Authors: GADHER Suresh J. , EYRE David R. , DUANCE Victor C. , WOTTON Sandra F. , HECK Louis W. , SCHMID Thomas M. , WOOLLEY David E. 
Published: March 2005

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25°C, collagenase attacked type II and type X (45‐kDa pepsin‐solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35°C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin‐solubilized type M) and collagen type XI were resistant to collagenase attack at both 25°C and 35°C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25°C.Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25°C or 35°C. At 25°C collagen types IX (pepsin‐solubilized type M) and XI were also resistant to elastase, but at 35°C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45‐kDa pepsin‐solubilized) was susceptible to elastase attack at 25°C and 35°C as judged by the production of specific products that corresponded closely with those produced by collagenase.Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35°C by conditioned culture medium from IL1‐activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte‐derived collagenase(s) has yet to be resolved.
Nucleotide and thioredoxin specificity of the manganese ribonucleotide reductase from Brevibucterium ammoniagenes
Authors: WILLING Andreas , FOLLMANN Hartmut , AULING Georg 
Published: March 2005

The manganese‐containing ribonucleotide reductase previously identified in gram‐positive bacteria has been purified and its nucleotide specificity and other requirements were determined. The enzyme isolated from Brevibacterium ammoniagenes is a ribonucleoside‐diphosphate reductase which, in the presence of allosteric effectors, reduces all four common substrates at comparable rates; very little activity is observed in the absence of effector nucleotides. Ribonucleoside triphosphates are reduced at 20% the rate of the diphosphates. Cytidine and uridine nucleotide reduction is specifically stimulated by ATP and dATP, adenylate reduction by dGTP, and guanosine nucleotide reduction by dTTP. Unlike the iron‐containing ribonucleotide reductase systems, high concentrations of dATP do not inhibit substrate reduction. The new bacterial enzyme tolerates high salt concentrations (up to 250 mM ionic strength) and does not require divalent metal ions for activity in vitro.The presence of thioredoxin has been demonstrated in heat‐ and acid‐treated protein extracts of B. ammoniagenes and the protein was purified to homogeneity. It is very similar to the thioredoxins isolated from other organisms in relative molecular mass (12000), isoelectric point (4.3) and enzyme‐activating properties. In the presence of 0.3 mM dithiothreitol, the bacterial thioredoxin can serve as hydrogen donor for B. ammoniagenes ribonucleotide reductase in vitro, indicating the presence of a functional ribonucleotide reductase‐thioredoxin system in these bacteria.The properties described in this and in our preceding paper in this journal [Eur. J. Biochem. 170, 603–611 (1988)] suggest that the B. ammoniagenes ribonucleotide reductase is intermediate in structure and specificity between the deoxyadenosylcobalamin‐dependent and the iron‐containing enzyme classes and that it is adapted to the specific requirements of deoxyribonucleotide synthesis in this organism.
Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver
Authors: PÜSCHEL Gerhard P. , JUNGERMANN Kurt 
Published: March 2005

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by α‐receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied.

In cell‐suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose‐dependent and time‐dependent increase of inositol mono, bis and trisphosphate.
In the perfused rat liver 1 μM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time‐dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions.
In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate.

These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.
Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus
Authors: YAMADA Akihiro , TSUKAGOSHI Norihiro , UDAKA Shigezo , SASAKI Takuji , MAKINO Shio , NAKAMURA Shin , LITTLE Clive , TOMITA Masahiro , IKEZAWA Hiroh 
Published: March 2005

Bacillus cereus secretes phospholipases C, which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. A 7.5‐kb HindIII fragment of B. cereus DNA cloned into Escherichia coli, with pUC18 as a vector, directed the synthesis of the sphingomyelin‐hydrolyzing phospholipase C, sphingomyelinase. Nucleotide sequence analysis of the subfragment revealed that it contained two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy‐terminal portion of the phosphatidylcholine‐hydrolyzing phospholipase C, and the downstream open reading frame to the entire translational portion of the sphingomyelinase. The two phospholipase C genes form a gene cluster.As inferred from the DNA sequence, the B. cereus sphingomyelinase has a signal peptide of 27 amino acid residues and the mature enzyme comprises 306 amino acid residues, with a molecular mass of 34233 Da. The signal peptide of the enzyme was found to be functional in protein transport across the membrane of E. coli.The enzymatic properties of the sphingomyelinase synthesized in E. coli resemble those of the donor strain sphingomyelinase. The enzymatic activity toward sphingomyelin was enhanced 20–30‐fold in the presence of MgCl2, and the adsorption of the enzyme onto erythrocyte membranes was accelerated in the presence of CaCl2
Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli
Authors: FLACHMANN Ralf , KUNZ Norbert , SEIFERT Jochen , GÜTLICH Markus , WIENTJES Franz‐Josef , LÄUFER Albrecht , GASSEN Hans Günter 
Published: March 2005

The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine‐2,3‐dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD.Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840‐bp open reading frame coding for a 31555‐Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the l‐aspartate oxidase activity of its gene product. Sequence analysis showed a 1620‐bp open reading frame coding for a 60306‐Da protein.For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, l‐aspartate oxidase, was purified to homogeneity and the N‐terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for l‐aspartate.High‐copy‐number vectors, carrying either gene nadA, nadB or nadA+nadB, increased quinolinate production 1.5‐fold, 2.0‐fold and 15‐fold respectively. Both gene products seem to be equally rate‐limiting in quinolinate synthesis.
Demonstration of a novel ecto‐enzyme on human erythrocytes, capable of degrading ADP and of inhibiting ADP‐induced platelet aggregation
Authors: LÜTHJE Jürgen , SCHOMBURG Axel , OGILVIE Adaling 
Published: March 2005

The role of ADP as an important inducer of platelet aggregation is generally accepted. Therefore it has been postulated by many authors that the enzymatic removal of extracellular ADP from the circulation is essential to avoid platelet aggregation and thrombus formation. Here we show that erythrocytes essentially contribute to the clearance of ADP. The removal of ADP from suspensions of washed human erythrocytes was due to at least two different activities. One activity, which had already been observed by earlier workers, was identified as adenylate kinase, on the basis of the reaction products and the inhibition by adenosine(5′)pentaphospho(5′)adenosine (Ap5A). This enzyme was not associated with the cells and was always detectable in cell‐free supernatants, indicating that the enzyme had leaked from the cells into the extracellular medium.In contrast, the second activity, which is described here for the first time, was tightly bound to the cells. The activity was not inhibited by Ap5A. The main product of the reaction was AMP, and enzyme activity depended on the presence of divalent cations. The Michaelis constant was about 28 μmol/1. This activity seemed to be an ecto‐ADPase. Studies with various inhibitors revealed that degradation of ADP was not due to a non‐specific phosphatase.Besides the well known ADPase on the endothelium, the ecto‐activity on erythrocytes may play an important part in destroying pro‐aggregatory ADP.
Phorbol 12,13‐dibutyrate and mitogens increase fructose 2,6‐bisphosphate in lymphocytes
Authors: BOSCA Lisardo , MOJENA Marina , DIAZ‐GUERRA M. José M. , MARQUEZ Carlos 
Published: March 2005

The influence of tumour promoters and growth factors on glycolysis and on fructose‐2,6‐bisphosphate concentration was studied in isolated mouse spleen lymphocytes and in purified B‐cells. The intracellular concentration of fructose 2,6‐bisphosphate and the rate of lactate release were increased 2–3‐fold in spleen lymphocytes exposed to active phorbol esters, mitogenic lectins, interleukin 4 or lipopolysaccharide. The maximal effect was observed after 1 h of exposure. In these cells hexose 6‐phosphates increased 2‐fold and 6‐phosphofructo‐2‐kinase activity remained unchanged after treatment with phorbol 12,13‐dibutyrate or with lectins. Exposure of B‐cells to phorbol 12,13‐dibutyrate, interleukin 4 or lipopolysaccharide increased the glycolytic flux and the concentration of fructose 2,6‐bisphosphate without relation to their mitogenic activity.Lymphocytes and rat liver 6‐phosphofructo‐2‐kinase were partially purified using the same procedure. The lymphocyte enzyme was not inhibited by sn‐glycerol 3‐phosphate in contrast to the potent inhibition observed in liver. Treatment of both enzymes with the catalytic subunit of the cyclic‐AMP‐dependent protein kinase failed to inactivate 6‐phosphofructo‐2‐kinase from lymphocytes. These differences suggest that lymphocytes and liver contain different forms of this enzyme.
Control of the tRNA‐tufB operon in Escherichia coli
Authors: VAN DELFT Joost H. M. , VERBEEK Hans M. , DE JONG Paul J. , SCHMIDT Denise S. , TALENS Anneke , BOSCH Leendert 
Published: March 2005

‘Ribosome feedback’ effects on the expression of the genes specifying tRNA and EF‐Tu in E. coli have been studied at increased rrnB doses (rRNA gene doses). We confirm previous observations that the introduction into the cell of a multicopy plasmid carrying the rrnB operon reduces the cellular content of most tRNAs, including those encoded by the tRNA‐tufB operon, but leaves the 5S rRNA content unaffected. Increase of the dosage of intact, but not of deleted rRNA genes, causes a slight drop in total EF‐Tu that can be fully accounted for by a decrease in EF‐TuB level. The drop in EF‐TuB content (approx. 25%) is much smaller than that in tRNA content (approx. 80%). The synthesis rate of total EF‐Tu is hardly affected, indicating that the turnover of EF‐Tu has not changed. The ratio of tRNA over tuf RNA synthesis rates remains the same after elevation of rrnB dosage. Considering the large decrease in tRNA content this means that both RNA synthesis rates decrease to approximately the same extent. The relatively small drop in EF‐Tu synthesis must be due, therefore, to an enhancement of the number of EF‐Tu molecules synthesized per mRNA molecule. Apparently a post‐trans‐criptional mechanism, regulating EF‐Tu synthesis, becomes operative under these conditions.Growth‐rate‐dependent regulation of the tRNA‐tufB operon has been studied using lysogens carrying tRNA': lacZ and tRNA‐tufB': lacZ operon fusions and a tufB': lacz' gene fusion. These experiments show that the cellular contents of tRNA, tufB RNA and EF‐TUB vary in direct proportion to the growth rate. This indicates that growth rate control of tRNA‐tufB operon transcription resembles that of stable RNA operons and not of r‐protein operons, and that the read‐through of the terminator at the end of the tRNA gene cluster remains unaltered.
Porphyrin cytochrome c
Authors: KORNBLATT Jack A. , LABERGE Monique 
Published: March 2005

Porphyrin cytochrome c, the iron‐free derivative of cytochrome c, has been used extensively as a fluorescent analog of cytochrome c. It appears as though its fluorescence intensity but not its relative quantum yield is affected by pH in the physiological range; an apparent pK of about 6.2 is found suggesting a histidine close to the porphyrin.
The fluorescence intensity of the porphyrin cytochrome c in the presence of cytochrome c oxidase is independent of pH; this suggests that the oxidase has the capacity to control the pK of whichever group is responsible for the pH sensitivity of the free porphyrin cytochrome c. The most likely candidate for this pH‐sensitive group is histidine‐18. The N‐3 nitrogen of this residue forms one of the axial ligands to the iron in the intact cytochrome c but it is uncoordinated in the iron‐free derivative.
Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast‐atom‐bombardment mass spectrometry
Authors: POULTER Linda , ANG Siau‐Gek , GIBSON Bradford W. , WILLIAMS Dudley H. , HOLMES Charles F. B. , CAUDWELL F. Barry , PITCHER Julie , COHEN Philip 
Published: March 2005

The in vivo phosphorylation state of glycogen synthase was re‐examined by fast‐atom‐bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S‐ethylcysteine. In animals injected with the β‐adrenergic antagonist propranolol, the phosphorylation sites in the N‐terminal (N) and C‐terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28‐C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28‐C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin.Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic‐AMP‐dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28–C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed.Residue N3, a major site phosphorylated by casein kinase‐I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase‐I is not a glycogen synthase kinase in vivo.