bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of three cDNAs coding for Rab proteins from the albumen gland of the mollusc Lymnaea stagnalis
Authors: AGTERBERG Marja , VAN DIE Irma , YANG Hui , ANDRIESSEN J. Aino , VAN TETERING Angelique , VAN DEN EIJNDEN Dirk H. , PLOEGH Hidde L.
Published: March 2005

Three cDNA clones encoding small GTP‐binding proteins, LS‐Rab1, LS‐Rab2 and LS‐Rab18a were isolated from a cDNA library from the albumen gland of the pulmonate snail Lymnaea stagnalis. Comparison of the deduced amino acid sequences with available sequences from the EMBL/Data Bank revealed that LS‐Rab1 and LS‐Rab2 show a sequence identity of 89–90% to the mammalian Rab1 and Rab2 proteins, and can therefore be regarded as the L. stagnalis homologs. LS‐Rab18a may be considered a new member of the Rab subfamily, closely related to mouse Rab18 (74% amino acid identity). Interestingly, LS‐Rab1 and LS‐Rab2 share a very high sequence conservation with their mammalian homologs (95–97%) over the first 178–191 N‐terminal amino acids, whereas the C‐terminal part is almost completely divergent, except for their extreme ends (2–4 amino acids). The implications of these observations for the understanding of Rab‐targeting signals are discussed.

The LS‐rab cDNAs were expressed in COS‐7M6 cells. The resulting 22‐kDa products were shown to bind GTP. In the albumen gland mRNA, levels of LS‐rab1 appeared to be much higher than those of LS‐rab2 and LS‐rab18a, suggesting an important role for the LS‐Rab1 protein in the albumen gland.
Third isoform of the prostaglandin‐E‐receptor EP3 subtype with different C‐terminal tail coupling to both stimulation and inhibition of adenylate cyclase
Authors: IRIE Atsushi , SUGIMOTO Yukihiko , NAMBA Tsunehisa , HARAZONO Akira , HONDA Akiko , WATABE Akiko , NEGISHI Manabu , NARUMIYA Shuh , ICHIKAWA Atsushi
Published: March 2005

A functional cDNA clone for a third isoform of the mouse prostaglandin‐E‐receptor EP3 subtype, derived by alternative RNA splicing, named the EP3γ receptor, was obtained in addition to those for the two other isoforms, EP3α and EP3β. The three isoforms are only different in the amino acid sequence of the putative cytoplasmic carboxy‐terminal tail. When expressed, EP3γ shows identical ligand‐binding properties to these of the other isoforms. The EP3‐selective agonist, M&B 28767, increased the basal cAMP level and inhibited the forskolin‐induced increase in the cAMP level in EP3γ, while it decreased both the basal and forskolin‐elevated cAMP levels in EP3α and EP3β. The M&B 28767‐stimulated GTPase activity consisted of pertussis‐toxin‐sensitive and cholera‐toxin‐sensitive portions in the EP3γ‐expressing cell membrane, suggested that EP3γ is coupled to both guanine nucleotide‐binding inhibitory and stimulatory proteins. These results indicate that EP3γ is coupled to both stimulation and inhibition of adenylate cyclase, but that EP3α and EP3β are exclusively coupled to inhibition of adenylate cyclase. Thus, alternative splicing produces a third isoform with a different carboxy‐terminal tail, which differs from the other two isoforms in the specificity of coupling to a signal‐transduction pathway.
Amidase coupled with low‐molecular‐mass nitrile hydratase from Rhodococcus rhodochrous J1
Authors: KOBAYASHI Michihiko , KOMEDA Hidenobu , NAGASAWA Toru , NISHIYAMA Makoto , HORINOUCHI Sueharu , BEPPU Teruhiko , YAMADA Hideaki , SHIMIZU Sakayu
Published: March 2005

The cloned 9.4‐kb insert of plasmid pNHJ20L containing low‐molecular‐mass nitrile hydratase (L‐NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23–33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96‐kb EcoRI–SphI region located 1.9‐kb downstream of the L‐NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N‐774 and Pseudomonas chlororaphis B23. The nucleotide‐deter‐mined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N‐774 and P. chlororaphis B23 and to indole‐3‐acetamide hydrolase from Pseudomonas savastanoi.

The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell‐free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S‐enantiomer of 2‐phenylpropionamide, but could not recognize the configuration of 2‐chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.
Glycine decarboxylase complex from higher plants
Authors: BOURGUIGNON Jacques , VAUCLARE Pierre , MERAND Véronique , FOREST Eric , NEUBURGER Michel , DOUCE Roland
Published: March 2005

cDNA clones encoding the precursor of the T protein of the glycine decarboxylase complex have been isolated from a pea leaf cDNA library in λ gt11. The longest cDNA insert of 1430 bp encodes a polypeptide of 408 amino acid residues of which 30 residues constitute an N‐terminal cleavable presequence and 378 residues make up the mature protein. Several results confirmed the identity of the cDNA and the exactness of the predicted primary structure. Firstly, we purified the T protein to homogeneity and its mass was measured by mass spectrometry. The mass obtained (40966 ± 5 Da) was the value predicted from the cDNA (40961 Da). Secondly, the purified T protein was chemically cleaved with cyanogen bromide and the peptide fragments were analysed by high‐performance liquid chromatography/electrospray ionization mass spectrometry and/or fast‐atom‐bombardment mass spectrometry. The mass values of all the peptides generated by chemical cleavage and measured by these techniques were very close to the values calculated from the predicted primary structure. Thirdly, microsequencing of some of these peptides, which represent 35% of the total protein, fits perfectly with the primary structure deduced from the cDNA. In the present HPLC/electrospray ionization MS studies we never detected the presence of covalently bound tetrahydropteroylpolyglutamate (H4PteGlun), either in the native T protein or in the different peptide fragments generated by the chemical cleavage. The absence of H4PteGlun bound to the T protein in our experimental conditions demonstrates that H4PteGlun is not covalently linked to the T protein. Northern blot analysis showed that the steady‐state level of the mRNA corresponding to the T protein was high in green leaves compared to the level in etiolated leaves (∼ 8 – 10‐fold higher). Surprisingly, a non‐negligible amount of mRNA corresponding to the T protein was present in roots whereas the mRNA encoding the H protein was not detectable. Western blot analysis showed that the P, L and T proteins of the glycine decarboxylase complex were present in roots whereas the H protein was not detectable. Southern hybridization to pea genomic DNA indicated the presence of a single gene encoding the T protein of the glycine decarboxylase complex in the haploid genome.
Roulette mutagenesis of the FMN‐binding site of Klebsiella pneumoniae flavodoxin
Authors: DRUMMOND Martin , HUFF Susan , GREEN Andrew
Published: March 2005

A method of randomising specific regions of coding sequences has been devised which utilises the Lac phenotype to identify mutants. Intact genes can be mutagenised, making it unnecessary to reclone the mutations before examining mutant phenotypes. The method has been applied to three residues around the N‐terminus of the first α helix of the Klebsiella pneumoniae nitrogenase flavodoxin, which are predicted to form part of the phosphate‐binding subsite. Surprisingly, most substitutions at Gly12, a highly conserved residue in the chain reversal preceding the α helix, appeared to be fairly stable in vivo and were found to retain some function. Substitutions at Lys13, a surface residue which contributes to a patch of positive charge characteristic of the nitrogenase flavodoxins, had no major effect on stability or function. However, most substitutions at Thr14, which is predicted to hydrogen bond to the phosphate of the prosthetic group FMN, were much more destabilising and grossly reduced function. The exceptions were Ala, Cys, Ser and Val, which suggests that the bulk of the residue at this position is critical.
The amino acid sequence of human ribonuclease 4, a highly conserved ribonuclease that cleaves specifically on the 3′ side of uridine
Authors: ZHOU Hai‐Meng , STRYDOM Daniel J.
Published: March 2005

A ribonuclease (RNase) that cleaves specifically on the 3′ side of uridine [Shapiro, R., Fett, J. W., Strydom, D. J. & Vallee, B. L. (1986a) Biochemistry 25, 7255–7264] was purified from human plasma and its amino acid sequence was determined. This protein is a 119‐residue single‐chain polypeptide cross‐linked by four disulfide bonds and has an amino‐terminal pyroglutaminyl residue. No post‐translational modifications were observed during extensive sequence studies on peptide fragments, except for the amino‐terminal pyroglutamic acid and a possible deamidation of Asn66. The protein is homologous to the pancreatic ribonucleases and angiogenin, but differs substantially from both of these proteins; the protein sequence has 43% identity with human pancreatic ribonuclease and 39% identity with human angiogenin, as compared to 35% identity between human angiogenin and pancreatic ribonuclease. It is referred to as RNase 4, based on the nomenclature currently used for the genes of pancreatic RNase (RNase 1) and the eosinophil‐derived RNases (RNase 2 and RNase 3).

Virtually all of the RNase active‐site components, including the catalytic residues His12, His119 and Lys41, are preserved. However, some invariant residues of RNase 1 are replaced, e.g. Lys7 by arginine, Asp14 by histidine, and Pro42 by arginine. RNase 4 contains a unique two‐residue deletion at the position corresponding to amino acids 77 and 78 of pancreatic RNase, and its carboxy‐terminal sequence is truncated at position 122. The deletion in angiogenin at position 21 is also found in RNase 4.

RNase 4 is very similar to two RNases isolated from bovine and porcine liver, and together they form a new family in the RNase superfamily. The degree of inter‐species similarity (90%) is much greater than within the pancreatic RNase and angiogenin families, which suggests that this ribonuclease could possess a physiologically important function other than general RNA catabolism.
Catalytic and binding properties of restriction endonuclease Cfr9I
Authors: SIKSNYS Virginius , PLECKAITYTE Milda
Published: March 2005

The Cfr9I restriction endonuclease recognizes and cleaves duplex DNA sequence C°CCGGG. The binding of restriction endonuclease Cfr9I to DNA was examined in the absence of Mg2+ using gel‐mobility‐shift and nitrocellulose‐filter‐binding assays. It was shown that restriction endonuclease Cfr9I bound DNA fragments either containing or lacking the canonical recognition sequence with equal affinity. These results suggest that the specificity of restriction endonuclease Cfr9I is expressed during the catalytic step. The cleavage of supercoiled pUC18 DNA by restriction endonuclease Cfr9I showed that at low concentrations of MgCl2, only with open‐circular DNA, nicks appeared in one strand at the recognition sequence, while the cleavage of the second strand was very slow. At higher concentrations of MgCl2 the enzyme cleaves either one or both strands of the DNA. Under these conditions the supercoiled DNA was converted to open‐circular and linear forms simultaneously rather than consecutively. It was shown that open‐circular DNA was a poor substrate for restriction endonuclease Cfr9I. These results suggested that both Mg2+ and intact recognition sequence are required to drive the enzyme into correct conformation to ensure DNA cleavage.
Cloning, expression and mutational analysis of the urocanase gene (hutU) fromPseudomonas putida
Authors: LENZ Martin , RÉTEY János
Published: March 2005

The histidine‐utilizinghutUgene was isolated from a λ‐EMBL3 phage of a genomic library fromPseudomonas putidanicII and subcloned into the expression vector pT7‐7.Escherichia coliBL21 cells were transformed with the recombinant plasmid and produced a catalytically active protein, amounting to approximately 30% of the total protein in the crude cell‐free extract. The addition of NAD+to the growth medium ensured the full occupation of active sites by the cofactor. This requires a mechanism for the transport of NAD+intoE. colicells.

Using the overproducing mutant a new, fast and efficient isolation procedure is described which yields electrophoretically homogeneous urocanase within two days. The yield of pure enzyme, based on the culture volume, has been improved 50–80‐fold compared with the traditional method.

To investigate the possible role of cysteine residues in the catalysis or in the tight binding of the cofactor NAD+, six different mutants were prepared. In each mutant protein, one conserved cysteine was exchanged for alanine. The resulting clones were tested for the expression of urocanase with catalytic activity; theKmandVmaxvalues were determined. Only Cys410 was essential for catalysis. There was no detectable reconstitution or increase of activity after the addition of NAD+, either in the essential Cys/Ala mutant or the other mutant proteins. Electrospray‐mass spectroscopy of the wild‐type enzyme revealed that the coenzyme is not covalently bound to the protein and computational analysis showed no typical sequence for a mononucleotide‐binding domain like the Rossman fold.

To obtain urocanase apoenzyme,P. putidanicII was transformed with pGP1‐2 and pTET7‐U and grown in nicotinate‐depleted medium. Like the mutant proteins, no activation of the apoform occurred after the addition of NAD+. These observations led us to postulate a new model for the non‐covalent but tight binding of NAD+to the enzyme by ‘trapping’ the cofactor while folding the nascent protein.
Metabolic flux determination in C6 glioma cells using carbon‐13 distribution upon [1‐13C]glucose incubation
Authors: PORTAIS Jean‐Charles , SCHUSTER Ronny , MERLE Michel , CANIONI Paul
Published: March 2005

A mathematical model of mammalian cell intermediary metabolism is presented. It describes the distribution of the carbon‐13 isotope (13C) at the different carbon positions of metabolites in cells fed with 13C‐enriched substrates. The model allows the determination of fluxes through different metabolic pathways from 13C‐ and 1H‐NMR spectroscopy and mass spectrometry data. The considered metabolic network includes glycolysis, gluconeogenesis, the citric acid cycle and a number of reactions corresponding to protein or fatty acid metabolism. The model was used for calculating metabolic fluxes in a rat tumor cell line, the C6 glioma, incubated with [1‐13C]glucose. After evolution to metabolic and isotopic steady states, the intracellular metabolites were extracted with perchloric acid. The specific enrichments of glutamate, aspartate and alanine carbons were determined from 13C‐, 1H‐NMR spectroscopy, or mass spectrometry data. Taking into account the rate of glucose consumption and of lactate formation, determined from the evolution of glucose and lactate contents in the cell medium, and knowing the activity of the hexose monophosphate shunt, it was possible to estimate the absolute values of all the considered fluxes. From the analysis the following results were obtained. (a) Glucose accounts for about 78% of the pyruvate and 57% of the CoASAc. (b) A metabolic channelling occurs at the citric acid cycle level; it favours the conversion of carbons 2, 3, 4, and 5 of 2‐oxoglutarate into carbons 1, 2, 3, and 4 of oxaloacetate, respectively. The percentage of channelled metabolites amounts to 39%. (c) The pyruvate carboxylase activity and the efflux from the citric acid cycle are estimated to be very low, suggesting a lack of glutamine production in C6 cells. The results emphasize different metabolic characteristics of C6 cells when compared to astrocytes, their normal counterpart.
The role of the NAD‐dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant
Authors: BOLES Eckhard , LEHNERT Wolfgang , ZIMMERMANN Friedrich K.
Published: March 2005

Phosphoglucose isomerase pgi1‐deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over‐expression on a multi‐copy plasmid of the structural gene GDH2 coding for the NAD‐dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD–glutamate dehydrogenase. Suppression of the pgi1 deletion by over‐expression of GDH2 was abolished in strains with a deletion of the glucose‐6‐phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH‐dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over‐expression of the NAD‐dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2‐oxoglutarate which can be converted back to glutamate by the NADPH‐dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over‐expression of the NAD‐dependent glutamate dehydrogenase causes a substrate cycling between 2‐oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.
TKL2, a second transketolase gene of Saccharomyces cerevisiae
Authors: SCHAAFF‐GERSTENSCHLÄGER Ine , MANNHAUPT Gertrud , VETTER Irene , ZIMMERMANN Friedrich K. , FELDMANN Horst
Published: March 2005

Transketolase activity is indispensable for the generation of erythrose 4‐phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4‐phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identitity with TKL1 [Sundström, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi‐copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti‐transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.
Interactions of calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae
Authors: URIBE Salvador , RANGEL Pablo , PARDO Juan Pablo , PEREIRA‐DA‐SILVA Lucia
Published: March 2005

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd= 1.9 μM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd= 50 μM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.
Increased levels of alpha‐class and pi‐class glutathione S‐transferases in cell lines resistant to 1‐chloro‐2,4‐dinitrobenzene
Authors: WAREING Clare J. , BLACK Stephen M. , HAYES John D. , WOLF C. Roland
Published: March 2005

Glutathione S‐transferase (GST) enzymes are often over‐expressed in tumor cells made resistant to cytotoxic drugs but it is unclear whether GST over‐expression is directly linked to the resistance mechanism. We have made a human lung tumor cell line resistant to 1‐chloro‐2,4‐dinitrobenzene (CDNB) in order to establish whether selection for resistance with a model GST substrate results in selection of a cell line with higher GST levels. The resistant line (CDNBr), although only twofold more resistant to this compound, exhibited a marked (15‐fold) increase in GST activity compared to the wild‐type cell line (28 ± 10 versus 357 ± 30 nmol CDNB conjugated · min−1· mg−1 protein, respectively). Resistance to CDNB was associated with a marked increase in the level of both alphaclass and pi‐class GST. Resolution of the GST by reverse‐phase HPLC demonstrated that the increase in the expression of the alpha‐class enzymes was due to elevated levels of both the B1 and B2 subunits. The increased levels of alpha‐class and pi‐class GST in the CDNBr cells was not due to either gene amplification or increased mRNA levels and appears to involve either altered mRNA utilization or protein stabilization. In addition to being resistant to CDNB, the CDNBr cell line also showed a 2.5‐fold resistance to cumene hydroperoxide but was not cross‐resistant to the anticancer drug chlorambucil. To demonstrate that the increased GST level was part of the resistance mechanism the alpha‐class GST B1 cDNA under control of the β‐actin promoter was stably expressed in the breast tumor cell line MCF‐7. The cell lines generated were twofold more resistant to CDNB relative to the parental line.
Repression of histone gene transcription in quiescent 3T6 fibroblasts
Authors: ZAHRADKA Peter , ELLIOT Tracy , HOVLAND Kenneth , LARSON Dawn E. , SAWARD Laura
Published: March 2005

Maintaining murine 3T6 fibroblasts in serum‐depleted medium for a period of three days results in a resting cell population that does not synthesize DNA. Histone mRNA levels, closely tied to the cell‐proliferation rate, are low due to a reduced rate of synthesis. A comparison of histone gene transcription in vitro by nuclear extracts of quiescent or proliferative 3T6 cells showed that a 200‐bp segment of the promoter was responsible for repressing gene activity when cells were in a G0 state. In the absence of the distal promoter region (–200 to –400), gene transcription remained high in quiescent cells, indicating the proximal promoter region (+1 to –200) was responsible for basal gene activity. Alterations in protein binding to the dista! promoter region correlated with histone H4 gene activity, suggesting that repression of histone gene transcription is linked to the attachment of a specific nuclear protein. During G1, the histone H4 gene was efficiently transcribed in vitro, but an inability to process the histone pre‐mRNA limited the cellular content of mature histone mRNA. This distinction between transcriptional (in G0) and post‐transcriptional (in G1) mechanisms for modulating histone mRNA levels suggests that gene‐regulatory factors are specifically activated in quiescent cells to reduce expression of non‐essential genes.
Intramolecular distances within the Ca2+‐ATPase from sarcoplasmic reticulum as estimated through fluorescence energy transfer between probes
Authors: CORBALAN‐GARCIA Senena , TERUEL José A. , GOMEZ‐FERNANDEZ Juan C.
Published: March 2005

Fluorescence energy transfer measurements have been carried out to estimate intramolecular distances between probes bound to Ca2+‐transporting ATPase (Ca2+‐ATPase) as well as distances between these probes and the phospholipid headgroup. The nucleotide binding site was monitored by using 1,N6‐ethenoadenosine 5′‐triphosphate, a fluorescent analogue of ATP, and also by labelling Lys515 with fluorescein 5′‐isothiocyanate. Three different cysteine residues were individually labelled using the following probes: 5‐[(2‐iodoacetyl)aminoethyl]amino‐naphthalene‐1‐sulfonic acid (I‐AEDANS), 7‐chloro‐4‐nitro‐2,1,3‐benzoxadiazole (NBD‐Cl) and fluorescent maleimides. The surface of the membrane was labelled by reconstitution with fluorescent phospholipids (fluorescein and rhodamine derivatives). We found a distance of 4.1 nm from the nucleotide binding site to NBD (at Cys344), and the same distance to fluorescent maleimides (at Cys364). The AEDANS label (at Cys670,672) was found separated 3.5 nm from NBD, 4.4 nm from fluorescent maleimides, and 3.9 nm from the lipid matrix. The NBD label was 3.2 nm apart from fluorescent maleimides and 2.2 nm from the lipid matrix. Finally, fluorescent maleimides were found to be located 4.2 nm above the membrane surface. All these distances agree with a molecular model in which NBD is located in the stalk portion of the Ca2+‐ATPase, near the surface of the membrane, and the rest of the probes are above it, in the globular domain of the protein.
Steady‐state kinetics of substrate hydrolysis by vacuolar H+‐pyrophosphatase
Authors: BAYKOV Alexander A. , BAKULEVA Natalia P. , REA Philip A.
Published: March 2005

The results of analyses of the steady‐state kinetics of the vacuolar H+‐translocating pyrophosphatase (V‐PPase) of native tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata (mung bean) and purified enzyme from the same source under a wide range of Mg2+, pyrophosphate (PPi) and K+ concentrations are consistent with a minimal reaction scheme in which dimagnesium pyrophosphate is the active substrate species and catalysis is mediated by preformed enzyme‐Mg2+ complex. When account is taken of the sensitivity of the V‐PPase to ionic strength, additional kinetic interactions are not required to describe the behavior of the enzyme. N‐Ethylmaleimide‐protection assays show that the dissociation constant for Mg2+ binding in the absence of PPi is an order of magnitude smaller than that estimated from the steady‐state kinetics of PPi hydrolysis. Two distinct Mg2+‐binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cation and Mg2+ and K+ do not compete during substrate hydrolysis, divalent and monovalent cations are concluded to bind at separate sites. The pH dependencies of the kinetic parameters are consistent with the participation of groups of pKa 5.7 and 8.6 in substrate binding and groups of pKa 6.1 and 9.0 in the substrate‐conversion step, indicating that at least four ionizable groups are essential for catalysis. These findings are discussed with respect to the reaction mechanism of the V‐PPase and the potential regulatory significance of cytosolic free Mg2+ and K+in vivo.
Higher plant mitochondria encode an homologue of the nuclear‐encoded 30‐kDa subunit of bovine mitochondrial complex I
Authors: LAMATTINA Lorenzo , GONZALEZ Daniel , GUALBERTO José , GRIENENBERGER Jean‐Michel
Published: March 2005

We describe the structure and expression of a wheat mitochondrial gene, which codes for a subunit of mitochondrial NADH dehydrogenase. The deduced protein sequence has 70% similarity to the 30‐kDa subunit of bovine mitochondrial complex I and 65% similarity to the 31‐kDa subunit of Neurospora crassa complex I, components of the iron‐sulfur‐protein fraction, both nuclear‐encoded proteins. We named this wheat mitochondrial gene as nad9. The wheat nad9 gene is transcribed in a single mRNA of 0.9 kb that is edited (C‐to‐U conversions) in 14 positions. Transcript mapping revealed that the first ATG codon is just 20 nucleotides downstream of the mRNA 5′ end and that the 3′ end is just 23 nucleotides downstream of the nad9 stop codon

The expression of the nad9 gene in plant mitochondria was studied. Polyclonal antibodies prepared against a wheat NAD9 fusion protein specifically recognise the 30‐kDa subunit of bovine mitochondrial complex I and a 27.5‐kDa protein in the membrane fractions of wheat, maize and common bean mitochondria, whereas the same serum recognizes a 30‐kDa protein in the mitochondria of pea, chickpea and lentil.
Sites of nidogen cleavage by proteases involved in tissue homeostasis and remodelling
Authors: MAYER Ulrike , MANN Karlheinz , TIMPL Rupert , MURPHY Gillian
Published: March 2005

The cleavage of recombinant mouse nidogen in its native form was examined with granulestored proteases (leucocyte elastase, mast‐cell chymase), blood proteases (thrombin, plasmin, kallikrein), matrix metalloproteinases (stromelysin, matrilysin, collagenases) and, for comparison, with trypsin and the endoproteinase Glu‐C. More than 50 major cleavage sites were identified by Edman degradation of several large fragments and smaller peptides. The data show an almost exclusive localization of protease‐sensitive sites to the flexible segment, connecting the N‐terminal globular domains G1 and G2, and within the C‐terminal, laminin‐binding domain G3. Domains G1, G2 and the rod‐like segment were much more stable against proteolysis. Kinetic analysis indicated a fast cleavage of several different sites in the link region followed by destruction of G3 but this was to some extent variable depending on the particular protease. Leucocyte elastase was identified as the most active protease in the cleavage of nidogen whilst stromelysin, matrilysin, plasmin and kallikrein were of distinctly lower activity. No cleavage could be detected with interstitial collagenase and gelatinase A. The peptide analyses also allowed the location of two disulfide bridges within the G3 domain. Complex formation between nidogen and laminin fragments caused some protection against cleavage by thrombin, leucocyte elastase and stromelysin particularly in domain G3. The data indicate a relatively uniform cleavage pattern of nidogen which may be relevant in the context of protein/ligand‐binding activities associated with domains G2 and G3. The proteolytic processes involved in remodelling and the cellular penetration of basement membranes could therefore be essential for the modulation of the mediator function of nidogen.
Structural and functional properties of a multi‐enzyme complex from spinach chloroplasts
Authors: RAULT Magali , GIUDICI‐ORTICONI Marie‐Thérèse , GONTERO Brigitte , RICARD Jacques
Published: March 2005

Antibodies have been raised specifically against chloroplast phosphoribulokinase, glyceraldehyde‐3‐phosphate dehydrogenase and ribulose 1,5‐bisphosphate carboxylase‐oxygenase. Each of these antibodies recognizes the same macromolecular entity isolated and purified from chloroplasts. This entity is a multi‐enzyme complex, previously isolated and made up of ribose‐phosphate isomerase, phosphoribulokinase, ribulose 1,5‐bisphosphate carboxylase‐oxygenase, phosphoglycerate kinase and glyceraldehyde‐3‐phosphate dehydrogenase.

Under denaturing conditions the multi‐enzyme complex contains two polypeptides of 54 kDa and 15 kDa corresponding to the large and the small subunits of ribulose 1,5‐bisphosphate carboxylase‐oxygenase, the two polypeptides of the glyceraldehyde‐3‐phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa very likely pertaining to ribose‐phosphate isomerase.

The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corresponding isolated enzymes. Ribulose 1,5‐bisphosphate carboxylase‐oxygenase of higher plants consists of eight large and eight small subunits. Glyceraldehyde‐3‐phosphate dehydrogenase is made up of two types of polypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two identical subunits. Therefore, for the three isolated enzymes, the stoichiometry of the polypeptide chains is always 1:1. Within this multi‐enzyme complex, there are two subunits of phosphoribulokinase, two A and B subunits of glyceraldehyde‐3‐phosphate dehydrogenase and two large and four small subunits of ribulose 1,5‐bisphosphate carboxylase‐oxygenase. Therefore the number and the stoichiometry of the polypeptide chains of phosphoribulokinase and glyceraldehyde‐3‐phosphate dehydrogenase are the same in the multi‐enzyme complex and in the free enzymes, but those of ribulose 1,5‐bisphosphate carboxylase‐oxygenase are completely different.

This conclusion that the multi‐enzyme complex contains two active sites for ribulose 1,5‐bisphosphate may be confirmed independently by kinetic inhibition studies using 6‐phosphogluconate.
Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen
Authors: GUDMUNDSDÓTTIR Ágüsta , GUDMUNDSDÓTTIR Elín , ÓSKARSSON Skarphédinn , BJARNASON Jón B. , EAKIN Ann K. , CRAIK Charles S.
Published: March 2005

The cDNAs encoding two different anionic forms of Atlantic cod trypsinogen have been isolated and sequenced. The nucleotide sequences include the 5′‐noncoding and 3′‐noncoding regions in addition to preproenzymes of 241 amino acids. These consist of hydrophobic signal peptides, activation hexapeptides and trypsins of 222 amino acid residues. The cod trypsins contain all the major structural features common to trypsins such as the catalytic triad His57, Asp102 and Ser195. Furthermore, the obligatory Asp189 and the six disulphide bonds are conserved. Eight amino acid residues are different between the isozymes, leading to a difference of four charges. Both cod trypsins are one‐amino‐acid‐residue shorter than most mammalian trypsins as a result of deletion of proline at position 152, and have a high methionine content. In addition, the cod preproenzyme signal and activation peptides differ markedly from their mammalian analogues. The amino acid identity between the cod and bovine trypsins is approximately 60%.
Sequence and expression of the gene encoding the respiratory nitrous‐oxide reductase from Paracoccus denitrificans
Authors: HOEREN Frank U. , BERKS Ben C. , FERGUSON Stuart J. , McCARTHY John E. G.
Published: March 2005

The structural gene for the respiratory nitrous‐oxide reductase from Paracoccus denitrificans has been cloned using a probe derived from the structural gene, nosZ, for this enzyme from Pseudomonas stutzeri. The cloned gene could be expressed surprisingly well (presumably yielding an apo‐protein) using an expression vector in Escherichia coli.

Sequencing the nosZ gene from P. denitrificans has shown that the periplasmic nitrous‐oxide reductase of this organism is highly similar in sequence to previously derived primary sequences for the enzyme from three other organisms. As with the other reductases, an unusually long signal sequence is deduced and a common motif of GXXRRXXLG near the beginning of this sequence is present. The results of N‐terminal sequencing of the mature nitrous‐oxide reductase from the closely related organism Thiosphaera pantotropha indicate that processing of the P. denitrificans precursor occurs between amino acids at positions 57 and 58. The predicted signal peptide is therefore of the same length and of similar overall structure to that previously described for the P. denitrificans methylamine dehydrogenase small subunit (MauA). The P. denitrificans sequence for the mature nitrous‐oxide reductase reduces from 14 to 11 and 6 to 4, respectively, the number of conserved histidine and methionine residues compared to previous sequences. Three cysteine and four tryptophan residues, previously identified as conserved amongst nitrous‐oxide reductases, are found in the Paracoccus enzyme. A comparison of the sequence of the C‐terminal region of the nitrous‐oxide‐reductase sequence with that for the CuA region of subunit II of the cytochrome aa3 from P. denitrificans reveals considerable sequence similarities.

Upstream of the structural gene for nosZ are sequences TTGAAGCTTAACCAG (centred at position –21 with respect to the start codon) and CCCGGTGGTCATCAAG (centred at position –126). Although both could be FNR (ANR) boxes, the latter is far more probable to have this role because only it is likely to be upstream of a promoter site. This is the first indication at the DNA sequence level for the existence of this regulatory system in P. denitrificans.

Analysis of the flanking DNA sequences revealed reading frames upstream and downstream of the nosZ gene showing similarity to the nosR and nosD genes, respectively, of Pseudomonas species.

An S30 in vitro transcription/translation system was developed for P. denitrificans which permitted the expression of the cloned gene for nitrous‐oxide reductase and which will be of general value in other studies of this organism.
Permeability properties of peroxisomes in digitonin‐permeabilized rat hepatocytes
Authors: VERLEUR Nicolette , WANDERS Ronald J. A.
Published: March 2005

In order to investigate the permeability properties of rat‐liver peroxisomes in situ, we selectively permeabilized hepatocytes with digitonin in a medium mimicking the cytosol. This system permitted us to study the latency of peroxisomal oxidases by means of measurement of their activities in permeabilized compared to disrupted hepatocytes. The activity of peroxisomal oxidases was studied using three different methods: (1) measurement of the oxidase‐mediated production of H2O2 in a system containing homovanillic acid, horseradish peroxidase and azide; (2) measurement of the rate of substrate utilization or product formation; (3) measurement of the production of H2O2 via the peroxidative action of catalase in the presence of an excess of methanol. The results obtained depended on which system was used to measure the activity of the different oxidases. Our observations lead us to conclude that method 1 cannot be used for latency studies, whereas methods 2 and 3 are suitable under defined circumstances. Based on the results of methods 2 and 3, we conclude that urate oxidase, L‐α‐hydroxyacid oxidase A and D‐amino acid oxidase show no structure‐linked latency in digitonin‐permeabilized hepatocytes, suggesting that the substrates for these enzymes permeate freely through the peroxisomal membrane.
Determinants of the brain‐specific expression of the rat aldolase C gene: ex vivo and in vivo analysis
Authors: THOMAS Muriel , MAKEH Iman , BRIAND Pascale , KAHN Axel , SKALA Henriette
Published: March 2005

A 115‐bp promoter fragment of the aldolase C gene is sufficient for conferring neural cell specificity on a reporter gene, in cultured PC12 cells and in transgenic mice. In vitro DNase I protection experiments detected two footprints on the promoter, termed boxes A/A', and B. The 5′ A/A' box contains overlapping Sp1 and Krox20/Krox24 binding sites; it binds Sp1 in fibroblasts (box A') and a different complex in brain (box A). Any deletion or mutation of this box that impairs protein recognition also suppresses promoter activity. The replacement of box A/A' by a Sp1 consensus binding site results in the loss of the brain specificity of expression in transgenic mice. Further 3′, box B is composed of a 5′ direct repeat and a 3′ GC box consisting of overlapping Sp1 and Krox20/Krox24 binding sites. Mutation of the direct repeat subregion appears to be more deleterious for the promoter activity than mutation of the G+C‐rich subregion.
Isolation and characterization of the human inter‐α‐trypsin inhibitor heavy‐chain H1 gene
Authors: BOST Frédéric , BOURGUIGNON Jeannette , MARTIN Jean‐Pierre , SESBOÜÉ Richard , THIBERVILLE Luc , DIARRA‐MEHRPOUR Maryam
Published: March 2005

The human inter‐α‐trypsin inhibitor heavy chain H1 (ITI heavy chain H1) gene was isolated from two overlapping clones. It spans 14 kbp and is composed of 22 exons from 15 bp to 281 bp in size and has consensus splice sites. Intron sizes range from 80 bp to 2000 bp. It codes for the precursor of HC1 that is part of the serum ITI form of 220 kDa. Two major transcriptional initiation sites were identified in the 5′‐flanking region, which contained putative promoter elements, but no typical TATA and CAAT boxes. mRNA for the ITI heavy chain H1 was found only in liver. The tissue‐specific transcription of the gene might be due to the presence of binding sites for the hepatocyte nuclear factor HNF‐5 and to the octameric motifs. A previous overlapping of cDNA clones indicated the absence of 29 bp in one of these clones. The present study shows that the 29 bp is located within the gene at the end of exon 21. A reverse‐transcriptase polymerase chain reaction mapping analysis of liver mRNA identified the two types of the mRNA for ITI heavy chain H1. Accordingly, the data demonstrate that there is alternative splicing of at least one exon of the ITI heavy chain H1 gene.
Expression in yeast of three allelic cDNAs coding for human liver P‐450 3A4
Authors: PEYRONNEAU Marie‐Anne , RENAUD Jean‐Paul , JAOUEN Maryse , URBAN Philippe , CULLIN Christophe , POMPON Denis , MANSUY Daniel
Published: March 2005

Three natural allelic cDNAs coding for P‐450 3A4, the major form in human liver, namely NF25, NF10 and hPCN1, have been expressed in Saccharomyces cerevisiae. NF25 and hPCN1 were functionally expressed in yeast microsomes, yielding proteins with an absorption maximum at 448 nm in the CO‐reduced difference spectrum. Some catalytic activities and substrate binding properties of P‐450 NF25 and P‐450 hPCN1 in yeast microsomes have been compared; no striking difference was found, showing that the two point substitutions between their amino‐acid sequences (Trp392 and Thr431 in P‐450 NF25 are replaced by Val392 and Ile431 in P‐450 hPCN1) have no significant effect on the functional properties of these two variants. By contrast, P‐450 NF10, which differs from P‐450 NF25 by a one‐amino‐acid deletion (Ile224 replacing Thr224‐Val225), was produced as a denatured form, as revealed by an absorption maximum at 420 nm, and was not catalytically active. This suggests that the deletion prevents the correct folding of the protein. The results of this study show that P‐450 NF25 and P‐450 hPCN1 are two roughly equivalent, functionally active variants of P‐450 3A4, but that P‐450 NF10 is a defective, unstable gene product that could arise from an alternative mRNA splicing. This could contribute to the large variations reported for nifedipine oxidation, a typical P‐450 3A4 activity, in human liver.
Kinetic studies of pea carbonic anhydrase
Authors: JOHANSSON Inga‐Maj , FORSMAN Cecilia
Published: March 2005

Chloroplast carbonic anhydrase from Pisum sativum has been isolated. The kinetic properties of the enzyme have been studied and comparisons to the well characterised human carbonic anhydrase II made. Pea carbonic anhydrase was found to be dependent on a reducing agent in order to retain the catalytic activity. Oxidised, inactive, enzyme could be activated by the addition of a SH‐agent. However, such activation gave only 60% of the activity of an enzyme kept in a reduced state all the time. The kinetics of CO2 hydration show an increase in Kcat as well as in kcat/Km with pH, but the pH profile does not follow a simple titration curve. The pH dependence is more complicated and it seems as if there are several titratable groups affecting the activity. At pH 9 we obtain a turnover number of 4x105 s−1 and a Kcat/Km value of 1.8x108 M−1 s−1 with reference to the subunit. We also find that the enzyme needs high concentrations of buffer to work at a maximal rate. Apparent Km values with respect to the total buffer concentration are found between 52–185 mM at neutral and high pH. At low pH the situation is complex with deviations from Michaelis‐Menten kinetics.

Chloroplast carbonic anhydrase from higher plants have been reported to have primary structures that are completely different from the enzyme from animals. In addition, we find the circular dichroic spectrum of pea carbonic anhydrase to be well distinguished from that of human carbonic anhydrase II. Despite those structural differences the kinetic parameters indicate that pea carbonic anhydrase is equally efficient as human carbonic anhydrase II in catalysing the hydration of CO2. However, the mechanism for proton transfer from the active site to the surrounding medium seems to differ between the two enzymes.
The actin monomers in the ternary gelsolin: 2 actin complex are in an antiparallel orientation
Authors: HESTERKAMP Thomas , WEEDS Alan G. , MANNHERZ Hans Georg
Published: March 2005

Gelsolin forms ternary complexes with two actin monomers in the presence of Ca2+, which nucleate actin polymerization and cap the barbed ends of filaments. It has therefore been assumed that the two actins are oriented in a similar manner to the terminal subunits in the genetic helix of F‐actin. We have tested this using chemical cross‐linking with N,N′‐1,4‐phenylenedimaleimide. For all conditions tested, we identified as the only cross‐linked dimeric species an actin dimer indistinguishable from the lower actin dimer of 86 kDa. This lower dimer was previously identified in the initial phase of actin polymerization and also when actin paracrystals are chemicallv cross‐linked [Millonig, R., Salvo, H. & Aebi, U. (1988) J. Cell Biol. 106, 785–796]. It probably defines a contact between adjacent monomers oriented in an antiparallel orientation. In contrast, when F‐actin is cross‐linked by the same reagent, an upper dimer of apparent molecular mass 115 kDa is formed, which corresponds to adjacent monomers in the genetic helix. The formation of this upper dimer was specifically inhibited by addition of gelsolin to F‐actin. Evidence is presented for a Cys374–Cys374 cross‐link in the lower dimer. Isolated lower dimer binds to gelsolin in a 1:1 stoichiometry, but it inhibits nucleation of polymerization by gelsolin. Other gelsolin constructs that bind two actin subunits (e.g. the N‐terminal half of the molecule, which has severing and capping but no nucleating activity) also form only lower dimer when cross‐linked with N,N′‐1,4‐phenylenedimaleimide. Only segment 2–6 (gelsolin fragment devoid of the N‐terminal segment 1) induces an upper dimer orientation of the two actins under nucleating conditions. Our evidence suggests that the two actins associated with gelsolin are not fixed in the orientation of adjacent subunits in F‐actin; instead they have a flexible orientation with respect to each other, which permits cross‐linking into a stable anti‐parallel form that does not correspond to the presumed nucleating conformation.