bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and characterization of a novel β‐agarase from Vibrio sp. AP‐2
Authors: AOKI Takahiko , ARAKI Toshiyoshi , KITAMIKADO Manabu 
Published: March 2005

β‐Agarase was purified from the culture fluid of a porphyran‐decomposing marine bacterium (strain AP‐2) by ammonium sulfate precipitation, successive column chromatography and DNase and RNase treatment. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 20 kDa, a pH optimum of 5.5, and was stable in the pH region 4.0–9.0 and at temperatures below 45°C.The β‐agarase was a novel endo‐type enzyme which hydrolyzed neoagarotetraose, larger neoagarooligosaccharides and agar to give neoagarobiose [3,6‐anhydro‐α‐L‐galactopyranosyl‐(1→3)‐D‐galactose] as the predominant product. The enzyme did not act on K‐carrageenan.According to the criteria of Bergey's Manual of Systematic Bacteriology, the strain was assigned to the genus Vibrio.
δ‐Haemolysin from Staphylococcus aureus and model membranes
Authors: DUFOURC Erick J. , DUFOURCQ Jean , BIRKBECK Thomas H. , FREER John H. 
Published: March 2005

Solid‐state 2H NMR and 31P NMR of 2H‐enriched chains and polar head groups, respectively, of dipalmitoylglycerophosphatidylcholine/water dispersions were undertaken to investigate the action of δ‐haemolysin from Staphylococcus aureus on biomembranes. When the lipid/toxin molar ratio, Ri is 10, the gelphase 2H powder patterns and the temperature of the gel–fluid phase transition, tc, are unchanged by the presence of the toxin whereas the 31P powder spectra of polar head groups are perturbed. At t > tc, a detailed analysis of methylene ordering indicates that δ‐haemolysin orders the lipid chains near tc and disorders them for t≫tc. These findings are interpreted in terms of peptide location with regard to the membrane and suggest that the position of the toxin depends on the temperature relative to tc.Experiments carried out at Ri= 4 exhibit sharp, isotropic 2H‐NMR lines, at t > tc, indicating that δ‐haemolysin promotes the appearance of very small objects undergoing fast isotropic reorientation which average to zero the quadrupolar interaction. Below tc, one observes gel‐phase powder patterns which indicate that the bacterial toxin is unable to form such small structures with ordered dipalmitoylglycerophosphocholine phospholipids. From comparison of the action of δ‐haemolysin with that of melittin on same lipids [Dufourc et al. (1986) Biochemistry 25, 6448–6455] it results that both toxins perturb similarly fluid‐phase lipids at elevated temperature, but they behave differently with gel‐phase lipids, the former toxin being less efficient in membrane restructuring than the latter.
Functional and immunological evidence for stable association of solubilized vasoactive‐intestinal‐peptide receptor and stimulatory guanine‐nucleotide‐binding protein from rat liver
Authors: COUVINEAU Alain , ROUYER‐FESSARD Christiane , VOISIN Thierry , LABURTHE Marc 
Published: March 2005

We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP‐sensitive form of Mr 150 000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14 482–14 489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide‐binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48 000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I‐VIP‐receptor complexes solubilized in Triton X‐100 were adsorbed on WGA‐Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I‐VIP and (b) αs (Mr 42 000) and β (Mr 35 000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA‐Sepharose. When Triton‐solubilized 125I‐VIP‐receptor complexes were adsorbed on WGA‐Sepharose, then retained material was specifically eluted with 0.3 M N‐acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I‐VIP‐receptor complexes of Mr 150 000 contained in the eluate indicating that 125I‐VIP‐receptor‐G protein complexes had been adsorbed to the WGA column. (b) The Mr‐42 000 αs subunit can be specifically ADP‐ribosylated by cholera toxin. (c) Immunoblotting using antisera against the αs and β subunits of Gs protein, reveals Mr‐42 000 and Mr‐35 000 components corresponding to αs and β subunits, respectively. (d) Affinity cross‐linking using dithiobis(succinimidyl‐propionate) of 125I‐VIP‐receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150 000. Immunoblotting using antisera against the β‐subunit shows the presence of the β subunit (Mr 35 000) in this Mr‐150 000 component.In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP‐receptor complexes with αs and β subunits of Gs protein.
Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin
Authors: KARIEGLSTEIN Kerstin , HENSCHEN Agnes , WELLER Ulrich , HABERMANN Ernst 
Published: March 2005

Tetanus toxin is a 151‐kDa protein. The complete amino acid sequence is known. The mature toxin is made up of the two peptide chains and contains 10 half‐cystine residues. Treatment with 4‐vinylpyridine in the presence of 6 M guanidine converted six of them into S‐pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half‐cystine residues were recovered in the S‐pyridyl‐ethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds.The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4‐vinylpyridein or with 4‐dimethylaminoazobenzene‐4′‐iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed in reversed‐phase HPLC compared. The chromatography components were identified by N‐terminal amino acid sequence and amino acid composition. In the chromatography of the non‐mercaptolysed, DABIA‐treated sample four chromophore‐carrying components were detected which could be demonstrated by N‐terminal sequence analysis to correspond to six half‐cystine‐containing cyanogen bromide fragments. In the mercaptolysed, DABIA‐treated sample three additional chromophore‐carrying components were present, corresponding to two previously disulfide‐linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA‐labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine‐labelled fragments.It was established that the half‐cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide‐bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide‐bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein‐chemical methods.
The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin‐induced enzyme secretion from rabbit pancreatic acini
Authors: EDERVEEN Antoine G. H. , van EMST‐DE VRIES Sjenet E. , DE PONT Jan Joep H. H. M. , WILLEMS Peter H. G. M. 
Published: March 2005

The putative inhibitor of diacylglycerol kinase activity, 6‐{2‐[(4‐fluorophenyl)phenylmethylene]‐1‐piperidinyl}‐ethyl‐7‐methyl‐5H‐thiazolo[3,2‐a]pyrimidin‐5‐one (R59022), markedly potentiated cholecystokinin‐C‐terminal‐octapeptide(CCK‐8‐)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5–10 μM R59022. Potentiation depended both on the concentration of R59022 and CCK‐8. No potentiation was observed when acini were half‐maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50–60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12‐O‐tetradecanoylphorbol 13‐acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor‐mediated activation of the inositol phospholipid pathway is required for R59022‐induced potentiation. R59022‐inhibited the CCK‐8‐stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK‐8‐stimulated hydrolysis of 32P‐labelled phosphatidylinositol 4,5‐bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12‐O‐tetradecanoylphorbol 13‐acetate added simultaneously with CCK‐8.Therefore, it is concluded that in the presence of 5–10 μM R59022 the receptor‐mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK‐8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol protein kinase C branch of the signal‐transduction route is rate‐limiting.
Molecular analysis of the structural gene for yeast transaldolase
Authors: SCHAAFF Ine , HOHMANN Stefan , ZIMMERMANN Friedrich K. 
Published: March 2005

We have cloned the structural gene for yeast transaldolase. Transformants carrying the TAL1 gene on a multicopy plasmid over‐produced transaldolase. A deletion mutant which was constructed using the cloned gene did not show any detectable transaldolase activity in vitro. Furthermore, both transaldolase isoenzymes which were detected in wild‐type crude extracts by immunoblotting were missing in the deletion mutants. Thus, TAL1 is the only transaldolase structural gene in yeast. TAL1 is not an essential gene. Deletion of the transaldolase gene did not affect growth on complete media with different carbon sources or on synthetic media. However, the transaldolase‐deficient strains accumulated sedoheptulose 7‐phosphate, an intermediate of the pentose‐phosphate pathway. Mutants lacking both transaldolase and phosphoglucose isomerase grew more slowly than the single mutants. They accumulated more sedoheptulose 7‐phosphate on medium containing fructose than on glucose medium. This shows that fructose 6‐phosphate and glyceraldehyde 3‐phosphate, metabolites of glycolysis, can enter the nonoxidative part of the pentose‐phosphate pathway.
Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5
Authors: HOHMANN Stefan , CEDERBERG Håkan 
Published: March 2005

Recently we deleted the pyruvate decarboxylase structural gene PDC1 from the genome of the yeast Saccharomyces cerevisiae. The pdc1 deletion mutants had pyruvate decarboxylase activity due to the presence of a second structural gene [Schaaff, I., Green, J. B. A., Gozalbo, D. & Hohmann, S. (1989) Curr. Genet. 15, 75–81]. We cloned and sequenced this gene which we call PDC5. The predicted amino acid sequences of PDC1 and PDC5 are 88% identical.Deletion of PDC5 did not cause any decrease in the specific pyruvate decarboxylase activity while pdc1 deletion mutants had 80% of the wild‐type activity. Deletion mutants lacking both PDC1 and PDC5 did not show any detectable pyruvate decarboxylase activity in vitro and were unable to ferment glucose. This indicates that PDC1 and PDC5 are the only structural genes for pyruvate decarboxylase in yeast.The PDC5 isoenzyme showed a slightly higher Km value for its substrate pyruvate than the PDC1 product (PDC5: Km= 8 mM; PDC1: Km= 5 mM), as measured in crude extract of pdc1 and pdc5 deletion mutants, respectively. PDC5 is only expressed in pdc1 deletion mutants. No mRNA transcribed from PDC5 could be detected in wild‐type cells. Thus, in addition to the control by glucose induction, pyruvate decarboxylase activity seems to be subject to autoregulation. Similar phenomena have been described previously for tubulin, histones and a ribosomal protein but not for metabolic enzymes.
The active centers of adenylylsulfate reductase from Desulfovibrio gigas
Authors: LAMPREIA Jorge , MOURA Isabel , TEIXEIRA Miguel , PECK Harry D. , LEGALL Jean , HUYNH Boi H. , MOURA José J. G. 
Published: March 2005

In order to utilize sulfate as the terminal electron acceptor, sulfate‐reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non‐identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (∼ 400 kDa). It is an iron‐sulfur, flavincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa.Low‐temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g= 2.02 and only detectable below 20 K. This signal represented a minor species (0.10–0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe‐grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe‐S center I) observable below 30 K and a concomitant decrease in intensity of the g= 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase‐reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g= 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin–spin‐coupled nature (observable below 20 K), indicating the presence of at least two iron‐sulfur centers, (centers I and II).Mössbauer studies on 57Fe‐enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin‐type [4Fe‐4S] cluster. Center I exhibits spectra with atypical Mössbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin‐type [4Fe‐4S] cluster, suggesting a different structure and/or a distinct cluster‐ligand environment.
Purification, characterization and inhibition of d‐3‐aminoisobutyrate aminotransferase from the rat liver
Authors: TAMAKI Nanaya , KANEKO Masae , MIZOTA Chizuru , KIKUGAWA Mariko , FUJIMOTO Sigeko 
Published: March 2005

d‐3‐Aminoisobutyrate–pyruvate aminotransferase was purified 2000‐fold from rat liver extract using heat treatment, ammonium sulfate fractionation, carboxylmethyl‐Sepharose CL‐6B, DEAE‐Sepharose CL‐6B, hydroxyapatite, Sephacryl S‐200 and electrofocusing chromatographies. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 220 kDa and the subunit molecular mass was 52 kDa. The enzyme exhibited absorption maxima at 280 nm and 412 nm with a shoulder at 330 nm at neutral pH. The pH optimum for enzyme activity was 9.5 and the Km values for β‐alanine and pyruvic acid were calculated to be 0.81 mM and 0.45 mM, respectively. The purified enzyme catalyzed the transamination of ω‐amino acids; β‐alanine and d‐3‐aminoisobutyric acid served as good amino donors, and pyruvic acid, glyoxylic acid and oxaloacetic acid were favorable amino acceptors.6‐Azauracil and 6‐azathymine were found to be potent inhibitors of purified rat liver d‐3‐aminoisobutyrate–pyruvate aminotransferase. 6‐Azauracil acted as a competitive inhibitor with respect to β‐alanine, and was an uncompetitive inhibitor with respect to pyruvic acid with a Ki of approximately 8.9 mM.
Escherichia coli DNA helicase I
Authors: BENZ Inga , MÜLLER Holger 
Published: March 2005

Gene traI of the Escherichia coli F sex factor which encodes DNA/helicase I was subcloned in a λ pL‐based plasmid vector and expressed in a background of pL non‐repressing cells. Neither the non‐repressed pL promoter nor the production of a high level of functional helicase I are toxic. Enzyme purified from this source was studied in the electron microscope. The results show that helicase I binds cooperatively to single‐stranded DNA. DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth‐contoured filament with a diameter of 12.5 ± 0.4 nm and an axial periodicity of 7.0 ± 0.2 nm. In unfixed specimens, discrete particles with axes of 12.7 ± 0.5 nm and 7.2 ± 0.5 nm are visible. They are consistent in size with helicase I monomers (Mr 180000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient. Helicase I free of DNA appears as aggregates.For comparison, a truncated traI. lacking coding for the amino‐terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment. The chimeric gene product (named helicase I del29) retains strand‐separating activity although it fails to show cooperative DNA binding behavior. Judged from the length of the helicase‐I‐specific sequence of this polypeptide. tral is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map. The revised location of traI has implications for understanding distal functions of the transfer operon.
Purification and properties of carnitine acetyltransferase from human liver
Authors: BLOISI Wilma , COLOMBO Irma , GARAVAGLIA Barbara , GIARDINI Roberto , FINOCCHIARO Gaetano , DIDONATO Stefano 
Published: March 2005

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit · mg−1 with acetyl‐CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl‐CoA and a pH optimum of 8.7. Apparent Km values for acetyl‐CoA are three times lower than for decanoyl‐CoA. Km values for L‐carnitine in the presence of acetyl‐CoA are six times lower than in the presence of decanoyl‐CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine.The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5‐kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver.Immunoprecipitation and SDS/PAGE analysis of 35S‐labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa.We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet‐extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.
Metabolism and actions of 2‐deoxy‐2‐fluoro‐d‐galactose in vivo
Authors: GRÜN Bernhard R. , BERGER Ulrike , OBERDORFER Franz , HULL William E. , OSTERTAG Hermann , FRIEDRICH Eckhard , LEHMANN Jochen , KEPPLER Dietrich 
Published: March 2005

The synthetic d‐galactose analog 2‐deoxy‐2‐fluoro‐d‐galactose (dGalF) offers unique advantages for studies of the d‐galactose pathway by non‐invasive techniques using 19F‐NMR spectroscopy or positron emission from the 18F‐labeled compound. The metabolism of 2‐deoxy‐2‐fluoro‐d‐galactose was studied in rodents using the unlabeled, the 18F‐labeled, and the 14C‐labeled d‐galactose analog. Analyses for the metabolites of 2‐deoxy‐2‐fluoro‐d‐galactose were performed by HPLC, enzymatic methods, and 19F‐NMR spectroscopy in vivo and in vitro. The metabolism of 2‐deoxy‐2‐fluoro‐d‐galactose was most active in the liver which took up the major part of the administered dose of the 14C‐labeled d‐galactose analog, but renal excretion was also pronounced. This was confirmed by in vivo scanning of the rat using the 18F‐labeled sugar (1.5 μCi/g; 25 nmol/g) and examination by positron‐emission tomography and gamma camera.The dose dependence of the levels of the hepatic metabolites of 2‐deoxy‐2‐fluoro‐d‐galactose was investigated for doses between 25 nmol/g body mass and 1 μmol/g body mass. After 1 h, the major part of the acid‐soluble uracil nucleotides consisted of UDP‐2‐deoxy‐2‐fluoro‐d‐hexoses when the dose was at least 0.1 μmol/g. With higher doses, 2‐deoxy‐2‐fluoro‐d‐galactose 1‐phosphate became the predominant initial metabolite. After a dose of 1 μmol/g 2‐deoxy‐2‐fluoro‐d‐galactose 1‐phosphate accumulated rapidly (5.3±0.4 μmol/g liver after 30 min) followed by the formation of UDP‐2‐deoxy‐2‐fluoro‐d‐galactose and UDP‐2‐deoxy‐2‐fluoro‐d‐glucose (0.7±0.1 μmol/g and 1.8±0.1 μmol/g, respectively, after 5 h).The diversion of uridylate, due to the accumulation of UDP‐2‐deoxy‐2‐fluoro‐d‐hexoses, was associated with a rapid depletion of hepatic UTP, UDP‐glucose, and UDP‐galactose. The UTP content was decreased to 11±6% of normal within 15 min after administration of 2‐deoxy‐2‐fluoro‐d‐galactose at a dose of 1 μmol/g. The UTP‐depleting action was minimal, however, at a dose of 25 nmol/g or less, indicating that interference in uridylate metabolism would be negligible at the doses required for positron‐emission tomography of the liver using the 18F‐labeled compound. At higher doses, the UTP deficiency induced by 2‐deoxy‐2‐fluoro‐d‐galactose could be useful in the chemotherapy of d‐galactose‐metabolizing tumors such as hepatocellular carcinoma. At moderate doses of this d‐galactose analog, 19F‐NMR spectroscopy in vivo or in vitro could pinpoint defects of enzymes in galactosemia, e.g. of galactokinase, uridylyltransferase, or UDP‐glucose 4‐epimerase.