bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein kinase C stimulates adenylate cyclase activity in prolactin‐secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP‐binding protein Gi
Authors: GORDELADZE Jan O. , BJÖRO Trine , TORJESEN Peter A. , ØSTBERG BjöRN C. , HAUG Egil , GAUTVIK Kaare M. 
Published: March 2005

The phorbol ester 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5′‐[β,γ‐imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin‐enhanced, 2.2 mM) for free Mg2+. The TPA‐mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half‐maximal activation) was 60 μM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA‐stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4α‐phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberinsensitive adenylate cyclase remained unaffected.Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin‐stimulated enzyme activities, while absolute TPA‐enhanced adenylate cyclase was maintained. Association of extracted GTP‐binding inhibitory protein, Gi, from S49 cyc− murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone‐stimulated adenylate cyclase activities, while net inhibition of the cyclase by somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone‐elicited adenylate cyclase activities in the Gi‐enriched membranes. Finally, TPA completely abolished the somatostatin‐induced inhibition of adenylate cyclase in both hybrid and non‐hybrid membranes.These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the nαi subunit of the inhibitory GTP‐binding protein, leaving the nβ subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP‐binding protein, Gs.
Characterization of distinct mRNAs coding for putative isozymes of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase
Authors: CREPIN Karine M. , DARVILLE Martine I. , HUE Louis , ROUSSEAU Guy G. 
Published: March 2005

Three distinct clones encoding full‐length 6‐phosphofructo‐2‐kinase (PFK‐2)/fructose‐2,6‐bisphosphatase (FBPase‐2) were characterized from a rat liver cDNA library. Clone 22c was 1859 bp long and coded for the 470 amino acids of the bifunctional subunit of the liver homodimer. This polypeptide is phosphorylated on serine 32 by cyclic‐AMP‐dependent protein kinase. Clone 4c (2681 bp) had a coding region identical to that of clone 22c but it included a putative intron of 959 bp. In clone 5c (1750 bp), the sequence upstream from amino acid 33 differed from that in clone 22c and coded for a unique N‐terminal portion of 10 amino acids.Poly(A)‐rich RNA from rat tissues was hybridized with cDNA probes corresponding to the unique N‐terminal portions of clones 22c and 5c. Dot and Northern blots showed signals indicative of three distinct PFK‐2/FBPase‐2 mRNAs. There were a 6.8‐kb mRNA typical of cardiac tissue, a 2.1‐kb mRNA typical of liver, corresponding to clone 22c, and a 1.9‐kb mRNA typical of skeletal muscle, corresponding to clone 5c. Primer extension analysis showed that clones 22c and 5c were nearly complete since their respective 5′‐untranslated sequences were at most 96/97 bp and 44 bp shorter than the corresponding mRNAs. These data provide a molecular basis for the existence of PFK‐2/FBPase‐2 isozymes.
Streptomyces griseus aminopeptidase is a calcium‐activated zinc metalloprotein
Authors: SPUNGIN Anya , BLUMBERG Shmaryahu 
Published: March 2005

A heat‐stable aminopeptidase with an N‐terminal Ala‐Pro‐Asp‐Ile‐Pro‐Leu sequence has been purified from Streptomyces griseus by heat treatment followed by gel‐exclusion and anion‐exchange chromatographic procedures. The enzyme is a monomeric zinc metalloenzyme showing an apparent molecular mass of 33 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 21 kDa by gel filtration on Superose 12.Calcium ions bind to the enzyme, pKCa 4.5, and activate it about sixfold when the substrate is leucine‐4‐nitroanilide (0.4 mM in 50 mM Tris/HCl pH 8.0, 25°C). Binding of Ca2+ also contributes to the thermal stability of the protein. This aminopeptidase may be useful for two‐stage assays of bacterial and mammalian metalloendopeptidases; it may also serve in studies of proteolytic enzyme activation by calcium ions.
Differential induction of peroxisomal and microsomal fatty‐acid‐oxidising enzymes by peroxisome proliferators in rat liver and kidney
Authors: SHARMA Rajesh K. , LAKE Brian G. , MAKOWSKI Richard , BRADSHAW Tony , EARNSHAW Dave , DALE Jeremy W. , GIBSON G. Gordon 
Published: March 2005

The induction of renal fatty‐acid‐oxidising enzymes has been investigated following short‐term exposure to a group of structurally diverse peroxisome proliferators and compared to the more extensively documented hepatic responses in the rat. There was a marked compound dependence on induction of both cytochrome P‐450‐IVA1‐dependent ω‐hydroxylation of lauric acid and enzymes of the peroxisomal fatty acid β‐oxidation pathway (measured as cyanide‐insensitive palmitoyl‐CoA oxidation and enoyl‐CoA hydratase).Cytochrome P‐450 IVA1 (or a very closely related isoenzyme in the same gene family) was a major constitutive haemoprotein in rat kidney microsomes and actively supported the ω‐hydroxylation of lauric acid. This activity was induced 2–3‐fold by peroxisome proliferators such as clofibrate, di‐(2‐ethylhexyl)phthalate, bezafibrate and nafenopin. By using a cDNA probe to the cytochrome P‐450 IVA1 gene in Northern blot analysis, we have shown that increased renal and hepatic ω‐hydroxylation of lauric acid, after treatment with peroxisome proliferators is a consequences of a substantial increase in the mRNA coding for this haemoprotein. In addition, programming of an in vitro rabbit reticulocyte translation system with both renal and hepatic RNA resulted in the synthesis of similar (if not identical) cytochrome‐P‐450‐IVA1‐related polypeptides. Furthermore, we have provided Western blot evidence that both rat liver and kidney microsomes contain two closely related cytochrome P‐450 IVA1 polypeptides, the major one characterised by a monomeric molecular mass of 51.5 kDa (identical to authentic, purified hepatic cytochrome P‐450 IVA1) and a minor one of 52 kDa. The kidney‐supported fatty acid ω‐hydroxylase activity was refractory to inhibition by a polyclonal antibody to liver cytochrome P‐450 IVA1, which may be related to the existence of two closely related (but immunochemically distinct) fatty acid hydroxylases in this tissue.Our studies have also demonstrated that certain of the compounds tested (including clofibrate, bezafibrate and nafenopin) induced renal fatty acid β‐oxidation, mirroring the increased ω‐hydroxylase activity in the endoplasmic reticulum. Our studies have also indicated that the kidney was more refractory to induction of the endoplasmic reticulum and peroxisomal fatty‐acid‐oxidising enzymes than the liver. Taken collectively, our data is strongly suggestive of a possible linkage of the renal fatty acid oxidative enzymes in these two organelles, a situation that also occurs in the liver. In addition, our studies have provided a possible conceptual framework that may rationalise the decreased susceptibility of the kidney to the toxicity of peroxisome proliferators.
Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non‐activated carboxylic acids to aldehydes
Authors: WHITE Hiltrud , STROBL Gerd , FEICHT Richard , SIMON Helmut 
Published: March 2005

An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non‐activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 μM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum.Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red‐Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity.The very labile enzyme has been enriched by a factor of about 145 by binding to octyl‐Sepharose and further chromatographic separation by red‐Sepharose and FPLC using Mono‐Q and phenyl‐Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels.
Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi.V) human erythrocytes
Authors: VIGNAL Alain , RAHUEL Cécile , EL MALIKI Bouchra , LONDON Jacqueline , LE VAN KIM Caroline , BLANCHARD Dominique , ANDRE Catherine , d'AURIOL Luc , GALIBERT Francis , BLAJCHMAN Morris A. , CARTRON Jean‐Pierre 
Published: March 2005

In the Miltenberger class V (Mi.V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing‐over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti‐GPA antibodies specific for discrete domains of GPA, it was found that the Mi.V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N‐terminus.As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in λEMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A‐1–A‐7) and five exons (B‐1–B‐5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi.V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi.V glycoprotein gene was composed of exon B‐1 of the GPB gene followed by exons A‐2 and A‐3 of the GPA gene and the exons B‐3, B‐4 and B‐5 of the GPB gene. Exon B‐1 can be distinguished from exon A‐1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N‐terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A‐3 and exon B‐3 was confirmed by amplification of the DNA region where the putative crossing‐over has occurred and it was deduced that the Mi.V glycoprotein is a hybrid molecule composed of amino acid residues 1–58 from GPA fused to amino acid residues 27–72 of GPB. In addition, the finding that part of the signal peptide and the 5′‐untranslated region are derived from GPB suggests that the genetic background of the Mi.V variant is rather complex and may involve a cascade of recombination or gene conversion events.
Presence of fucosylated triantennary, tetraantennary and pentaantennary glycans in transferrin synthesized by the human hepatocarcinoma cell line Hep G2
Authors: CAMPION Bruno , LÉGER Didier , WIERUSZESKI Jean‐Michel , MONTREUIL Jean , SPIK Geneviève 
Published: March 2005

Transferrin synthesized by a human hepatocellular carcinoma cell line Hep G2 (called Hep G2 transferrin) was purified from culture media by immunoaffinity chromatography on a rabbit anti‐(human serotransferrin) IgG column. The eluted transferrin was then resolved into five peaks on a cation‐exchange column using the fast protein liquid chromatography system. The major fraction, named Hep G2 transferrin fraction C, having a molecular mass of 82.5 kDa was found to be homogeneous in polyacrylamide gel electrophoresis and in concanavalin‐A‐affinity crossed immunoelectrophoresis.A comparative analysis of the molar carbohydrate composition of normal human serotransferrin and of Hep G2 transferrin fraction C shows an increase in the latter in the number of galactose and N‐acetylglucosamine residues and in the presence of fucose, which is absent in normal transferrin.By a combination of methylation analysis and NMR spectroscopy, the primary structure of the oligosaccharide alditols released from Hep G2 transferrin fraction C by reductive alkaline cleavage has been established as triantennary, tetraantennary and pentaantennary N‐acetyllactosaminic structures with fucose residues (α1–3)‐linked to peripheral N‐acetylglucosamine residues.These results indicate that the increase in the number of antennae in transferrin glycans synthesized by the hepatocarcinoma cell line is much more pronounced than in liver diseases such as alcoholic cirrhosis and that in addition, the malignant transformation of human liver induces the presence of fucose.
Conformational consequences of the incorporation of arabinofuranosylcytidine in DNA
Authors: PIETERS Jane M. L. , de VROOM Erik , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

The self‐complementary octamers d(CGCTAGCG) and d(CGaCTAGCG) (aC, arabinofuranosylcytidine) were studied by means of NMR spectroscopy. It is shown that d(CGaCTAGCG), under suitable conditions of oligonucleotide concentration, ionic strength and temperature, exclusively adopts a hairpin structure. However, under the same experimental conditions (5 mM DNA, no added salt, 295 K) d(CGCTAGCG) mainly adopts a B‐DNA‐type duplex. At lower temperatures (290 K) the hairpin form of d(CGaCTAGCG) occurs in slow exchange with an intact B‐DNA‐type duplex. When the DNA concentration of d(CGCTAGCG) is dramatically reduced (0.5 mM) the hairpin form becomes highly favoured at the expense of the dimer. Moreover, proton‐chemical‐shift considerations indicate that the structural features of the hairpin structure of d(CGCTAGCG) mimic, in part, those of the modified octamer d(CGaCTAGCG), i.e. a loop comprising only the two central residues with the thymine located into the minor groove (Pieters, J. M. L., de Vroom, E., van der Marel, G. A., van Boom, J. H., Koning, T. M. G., Kaptein, R. and Altona, C. unpublished results).Thermodynamic analysis of d(CGCTAGCG) yields an average Tmd value of 342 K (1 M DNA) and a ΔHd° value of −266 kJ/mol for the dimer/coil transition and an average Tmh value of 321 K and ΔHh°−102 kJ/mol for the hairpin/coil equilibrium. For the duplex/coil equilibrium of d(CGaCTAGCG) an average Tmd value of 336 K (1 M DNA) and ΔHd° value of −253 kJ/mol are deduced. The hairpin/coil transition of d(CGaCTAGCG) is characterized by a ΔHh° value of −104 kJ/mol and an average Tmh value of 331 K. It is concluded that incorporation of an arabinofuranosylcytidine in the octamer d(CGaCTAGCG) results in stabilization of the hairpin form, whereas the dimer is destabilized by two aC · dG base pairs.
Glucosephosphate isomerase from Trypanosoma brucei
Authors: MARCHAND Martine , KOOYSTRA Ulco , WIERENGA Rik K. , LAMBEIR Anne‐Marie , Van BEEUMEN Jos , OPPERDOES Fred R. , MICHELS Paul A. M. 
Published: March 2005

In Trypanosoma brucei the enzyme glucose‐6‐phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody‐like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54–58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose‐6‐phosphate isomerases the trypanosomal enzyme contains an additional 38–49 amino acids in its N‐terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5‐kDa‐larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose‐6‐phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure.Comparison of the kinetic properties of the T. brucei glucose‐6‐phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6‐phosphate (Km= 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6‐phosphate (Ki= 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose‐6‐phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki= 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose‐6‐phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.
Isolation of similar rolipram‐inhibitable cyclic‐AMP‐specific phosphodiesterases from rat brain and heart
Authors: NÉMOZ Georges , MOUEQQIT Mohammed , BRIGENT Annie‐France , PACHECO Henri 
Published: March 2005

A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 μM, while hydrolysis of cyclic GMP displayed anomalous, negatively cooperative kinetics. At micromolar concentrations, this enzyme form hydrolyzed highly specifically cyclic AMP (50‐fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 μM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were observable by electrophoresis on a polyacrylamide gradient gel, compatible with an equilibrium between dimeric and monomeric forms. Isoelectric focusing of the preparation gave rise to two activity peaks of pI 4.8 and 6.7, with identical properties. probably representing two charge isomers of the same protein. An enzyme prepared from rat heart cytosol by the same techniques as for brain phosphodiesterase isolation shared numerous characteristics with the enzyme of cerebral origin, suggesting identity of the rolipramsensitive form between the two tissues.Since the rolipram‐sensitive form detected in crude brain preparations markedly differs from the above‐described isolated enzyme, both by its molecular mass in exclusion chromatography and by its pI, it is suggested that an alteration of the native protein, due to dissociation of putative subunits, occurs during the purification procedure.
Studies of the cellulolytic system of Trichoderma reesei QM 9414
Authors: VAN TILBEURGH Herman , LOONTIENS Frank G. , ENGELBORGS Yves , CLAEYSSENS Marc 
Published: March 2005

Binding onto cellobiohydrolase II from Trichoderma reesei of glucose, cellobiose, cellotriose, derivatized and analogous compounds, is monitored by protein‐difference‐absorption spectroscopy and by titration of ligand fluorescence, either at equilibrium or by the stopped‐flow technique. The data complete earlier results [van Tilbeurgh, H., Pettersson, L. G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Eur. J. Biochem. 148, 329–334] indicating an extended active center, with putative subsites ABCD.Subsite A specifically complexes with β‐d‐glucosides and d‐glucose; at 25°C the latter influences the concomitant binding of other ligands at neighbouring sites. For several ligands this cooperative effect for binding (at 0.33 M glucose and temperature range 4–37°C) was characterized by a substantial increase of the enthalpic term (ΔΔH°=–35 kJ mol–1). Glucose (0.33 M) decreases the association and dissociation rate parameters of 4‐methylumbelliferyl β‐d‐cellobioside by one order of magnitude: k+= (3.6 ± 0.5)×10–5 M–1 s–1 versus (5.1 ± 0.1)×10–6 M–1 s–1 (in the absence of glucose) and k−= (1.3 ± 0.1) s−1 versus (14.0 ± 0.3) s–1.As deduced from substrate‐specificity studies and inhibition experiments, subsite B interacts with terminal non‐reducing glucopyranosyl residues of oligomeric ligands and substrates, whereas catalytic (hydrolytic) cleavage occurs between C and D.Association constants 10–100 times higher than those for cellobiose or its glycosides were observed for d‐glucopyranosyl‐(1 → 4)‐β‐d‐xylopyranose and cellobionolactone derivatives, suggesting ‘transition‐state’‐type binding for these ligands at subsite C.Although subsite D can accomodate a bulky chromophoric group (MeUmb) its preference for a glucosyl residue is reflected in the lower binding enthalpy of cellotriose (–34 kJ mol–1) as compared to cellobiose (–28.3 kJ mol–1) and MeUmb(Glc)2 (–11.6 kJ mol–1).This model indicates that oligomeric ligands (substrates) interact through cooperativity of their subunits at the extended binding site of cellobiohydrolase II.
Site‐directed mutagenesis of the spinach acyl carrier protein‐I prosthetic group attachment site
Authors: JAWORSKI Jan G. , POST‐BEITTENMILLER Martha A. , OHLROGGE John B. 
Published: March 2005

Site‐directed mutagenesis was used to change the phosphopantetheine attachment site (Ser38) of spinach acyl carrier protein I (ACP‐I) from a serine to a threonine or cysteine residue.

Although the native ACP‐I is fully phosphopantethenylated when expressed in Escherichia coli, the THACP‐I and CY‐ACP‐I mutants were found to be completely devoid of the phosphopantetheine group. Therefore, the E. coli holoACP synthase requires serine for in vivo phosphopantetheine addition to spinach ACP‐I.
Spinach holoACP‐synthase was completely inactive in vitro with either the TH‐ACP‐I or CY‐ACP‐I mutants. In addition, TH‐ACP‐I and CY‐ACP‐I were strong inhibitors of spinach holoACP synthase.
The mutant ACPs were weak or ineffective as inhibitors of spinach fatty acid synthesis and spinach oleoyl‐ACP hydrolase.
Compared to holoACP‐I, the mutant apoACP‐I analogs had: (a) altered mobility in SDS and native gel electrophoresis, (b) altered binding to anti‐(spinach ACP‐I) antibodies and (c) altered isoelectric points. The combined physical, immunological and enzyme inhibition data indicate that attachment of the phosphopantheine prosthetic group alters ACP conformation.