bluuebunny/crossref_metadata_2025 huggingface dataset:
The mechanism by which rat liver glucokinase is inhibited by the regulatory protein
Authors: VANDERCAMMEN Annick , van SCHAFTINGEN Emile
Published: March 2005

The fructose‐6‐phosphate‐sensitive and fructose‐1‐phosphate‐sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179–184] was purified close to homogeneity by a procedure involving poly(ethyleneglycol) precipitation, chromatography on anion‐exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel‐filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer.

The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat‐stable or a trypsin‐resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1–2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low‐molecular‐mass inhibitor.

Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5‐fold increase in the concentration of regulator required to half‐maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6‐phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6‐phosphate and the regulatory protein, although not when fructose 1‐phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6‐phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6‐phosphate, and that fructose 1‐phosphate antagonises this inhibition by preventing the formation of the complex.
Apparently irreversible GTP hydrolysis attends tubulin self‐assembly
Authors: ANGELASTRO James M. , PURICH Daniel L.
Published: March 2005

The pathway of GTP hydrolysis associated with microtubule polymerization was investigated using an assay of intermediate 18O‐exchange reactions. Under a variety of conditions influencing tubulin self‐assembly, GTP was hydrolyzed without any evidence of multiple reversals characteristic of reversible phosphoanhydride‐bond cleavage. These results also accord with published findings that ATP hydrolysis during actin polymerization fails to display intermediate exchange reactions [Carlier, M. F., Pantaloni, D., Evans, J. A., Lambooy, P. K., Korn, E. D. and Webb, M. R. (1988) FEBS Lett. 235, 211–214].
Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement
Authors: SHIMIZU Takashi , OKUNO Makoto , MARCHESE‐RAGONA Silvio P. , JOHNSON Kenneth A.
Published: March 2005

The phosphorothioate analog of ATP has a sulfur atom replacing a non‐bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymena cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule‐dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility.

It has been shown that the microtubule–22S‐dynein complex is dissociated by the binding of ATP to the dynein enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5′‐[α‐thio]triphosphate (ATP[αS]), Sp or Rp, by light‐scattering stopped‐flow methods. The dissociation by (Sp)ATP[αS] was rapid and the rate of the light‐scattering change by (Sp)ATP[αS] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two‐step process. On the other hand, (Rp)ATP[αS] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s−1). The adenosine 5′‐[β‐thio]triphosphate (ATP[βS]) isomers did not induce dissociation.

The hydrolytic activity of the outer arm dynein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of Vmax (nmol · mg protein−1· min−1)/apparent Km (μM) of this dynein were 400–720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[αS] (Sp or Rp), ATP[βS] (Sp or Rp), respectively, in the presence of Mg2+ as the supporting cation. This dynein exhibited the same stereospecificity at β phosphate as the 22S dynein or myosin.

The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (Sp)ATP[αS] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (Sp)ATP[αS] was one‐quarter to one‐half of that produced by ATP reactivation.
Molecular cloning of a cDNA for the smallest nuclear‐encoded subunit of sweet potato cytochrome c oxidase
Authors: NAKAGAWA Tsuyoshi , MAESHIMA Masayoshi , NAKAMURA Kenzo , ASAHI Tadashi
Published: March 2005

A cDNA clone for the subunit Vc, the smallest nuclear‐encoded subunit, of sweet potato cytochrome c oxidase was isolated. Nucleotide sequence analysis showed that the subunit is synthesized as a precursor from which only the amino‐terminal amino acid, methionine, residue is removed to form the mature subunit. The mature subunit consists of 63 amino acid residues which are arranged so as to form one hydrophobic cluster lying between two hydrophilic clusters; such an arrangement is also seen with some nuclear‐encoded small subunits of the enzymes from other eukaryotes. The labelled precursor protein was synthesized with the cDNA by in vitro transcription (SP6 system) and translation (wheat germ system). This precursor was imported into isolated sweet potato tuberous root mitochondria without any significant processing, the import being dependent on membrane potential.
Phorbol esters induce differentiation of U‐937 human promonocytic cells in the absence of LFA‐1/ICAM‐1‐mediated intercellular adhesion
Authors: CABAÑAS Carlos , SANCHEZ‐MADRID Francisco , ALLER Patricio , YAGUE Enriqueta , BERNABEU Carmelo
Published: March 2005

Intercellular adhesions which occur during the mononuclear phagocyte differentiation are predominantly mediated by the lymphocyte‐function‐associated antigen‐1 (LFA‐1) family and the intercellular‐adhesion molecule‐1 (ICAM‐1) which is a ligand for LFA‐1. Thus, differentiation of U‐937 promonocytic cells induced by phorbol esters occurs concomitantly with intercellular LFA‐1/ICAM‐1‐dependent cluster formation. Since these homotypic adhesions can be inhibited by monoclonal antibodies (mAb) directed to either LFA‐1 or ICAM‐1, we have analyzed whether the lack of cell–cell adhesions impairs the differentiation process. Treatment of U‐937 cells with the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate in the presence of mAb to LFA‐1 or ICAM‐1 antigens yielded cells free from homotypic adhesions but differentiated as evidenced by their decreased proliferation and enhanced capacity for generation of superoxide anion. In addition, expression of the CD11c antigen was increased, whereas the transferrin receptor disappeared from the cell surface. Vimentin gene transcription was also greatly augmented as opposed to a clear diminution in the levels of c‐myc and ornithine decarboxylase transcripts. These results clearly demonstrate that phorbol esters can induce differentiation of monocytic cells independently of cell–cell adhesion.
Characterisation of tissue‐specific trans‐acting factor binding to a proximal element in the rat growth hormone gene promoter
Authors: LARKIN Sarah , TAIT Sarah , TREACY Maurice , MARTIN Finian
Published: March 2005

Using an exonuclease III protection assay, tissue‐specific binding of rat pituitary tumour cell (GH3 cell) nuclear factors to a proximal region (−68 to −138) of the rat growth hormone gene promoter has been detected. The binding is particularly strong between the borders −68 to −102. The binding is eliminated in the presence of excess unlabelled rat growth hormone gene promoter sequences but also by proximal (−423 to +38) or distal (−1960 to −1260) rat prolactin gene promoter sequences and simian virus 40 enhancer/promoter sequences. Extracts of rat pituitaries showed identical binding characteristics. Methylation interference analysis indicated that the contact points between the pituitary‐specific factor and the proximal rat growth hormone gene promoter‐binding element (−65 to −95) are over a conserved sequence which occurs twice in the rat growth hormone gene promoter and at least eight times in the rat prolactin gene 5′‐flanking sequences. This sequence has previously been proposed to constitute the binding site for the somatotroph/lactotroph tissue‐specific transcription factor.

Gel‐retardation and exonuclease III competition analysis showed that three of the rat prolactin gene promoter elements (−46 to −71, −156 to −180 and −174 to −204) share the ability to bind the pituitary‐specific factor. The binding to the most proximal rat prolactin gene promoter element (−46 to −71) was clearly more avid than to the rat growth hormone gene promoter (−65 to −95) proximal element. However, both these elements displayed the formation of two gel‐retarded complexes while the more distal rat prolactin gene binding elements (−156 to −180 and −174 to −204) formed only the smaller of the two complexes.

Finally, we demonstrated by co‐transfection competition analysis that plasmids containing the most proximal rat prolactin gene promoter binding element completely inhibited transcription from the rat growth hormone gene promoter while rat growth hormone gene promoter sequences only partially inhibited transcription from the rat prolactin gene promoter. This suggests that the higher affinity for factor binding displayed by the proximal rat prolactin gene promoter binding site in vitro is reflected in factor binding activity in vivo.
Conformational changes in intact and papain‐modified α1‐proteinase inhibitor induced by guanidinium chloride
Authors: HERVÉ Mireille , GHÉLIS Charis
Published: March 2005

Equilibrium unfolding–refolding processes of active and proteolytically modified α1‐proteinase inhibitor induced by guanidinium chloride were studied. Spectroscopic methods of ultraviolet absorption, fluorescence emission and circular dichroism were used. The functional inhibitor unfolds following a multistate process: a first transition (midpoint at 0.6 M guanidinium chloride) was observed whatever the method used and was attributed to a limited conformational modification of the region including the two tryptophan residues. At higher denaturant concentrations, two other transitions were observed, one in fluorescence (midpoint at 1.7 M guanidinium chloride), attributed to the unfolding of the polypeptide chain in the same region and the other one, observed in circular dichroism and in ultraviolet absorption (midpoint at 2.3 M guanidinium chloride), leading to the totally unfolded protein. Evidence for several intermediates was also obtained with the proteolytically modified inhibitor. If total unfolding is considered, the modified inhibitor was found to be more stable towards the denaturant than the functional form (obtained at 5.5 M and 3.5 M guanidinium chloride, respectively). The unfolding irreversibility observed was attributed to the C‐terminal fragment Ser359–Lys394 associated with the main chain of the cleaved inhibitor.
Function of oligosaccharide modification in glucocerebrosidase, a membrane‐associated lysosomal hydrolase
Authors: VAN WEELY Sonja , AERTS Johannes M. F. G. , VAN LEEUWEN Marinella B. , HEIKOOP Judith C. , DONKER‐KOOPMAN Wilma E. , BARRANGER John A. , TAGER Joseph M. , SCHRAM André W.
Published: March 2005

The nature and function of oligosaccharide modification in glucocerebrosidase, a membrane‐associated lysosomal hydrolase, have been investigated in cultured human skin fibroblasts.

Glucocerebrosidase is synthesised as a 62.5‐kDa precursor with high‐mannose‐type oligosaccharide chains and an apparent native isoelectric point of 6.0–7.0. Subsequent processing of the oligosaccharide moieties to sialylated complex‐type structures results in formation of 65–68‐kDa forms of the enzyme with apparent native isoelectric points of 4.3–5.0. These forms are transported to lysosomes and subsequently modified by the sequential action of lysosomal exoglycosidases, finally resulting in a 59‐kDa form with an isoelectric point near neutrality. The existence of oligosaccharide modification of the enzyme in the lysosomes is illustrated by the accumulation of different intermediate forms of glucocerebrosidase in mutant cell lines deficient in lysosomal exoglycosidases. The enzyme does not undergo proteolytic modification during maturation.

The possible physiological relevance of the oligosaccharide modification of glucocerebrosidase in the lysosomes was investigated by studying the properties of the enzyme in fibroblasts deficient in lysosomal exoglycosidases, and also the properties of homogeneous pure glucocerebrosidase from placenta, modified in the oligosaccharide moieties by digestion in vitro with glycosidases. Modification of the oligosaccharide moieties of glucocerebrosidase had no significant effect on the catalytic activity of the enzyme as measured with either artificial or natural substrates in the presence of artificial or natural activators. There was also no effect of modification of the oligosaccharide chains on the intracellular stability of the enzyme or on its apparent hydrophobicity. We conclude that oligosaccharide modification of glucocerebrosidase in the lysosomes simply reflects further maturation of the enzyme in the lysosome and is of no importance to its function.
Kinetic misinterpretation of a coupled enzyme reaction can lead to the assumption of an enzyme—enzyme interaction
Authors: VAS Mária , BATKE József
Published: March 2005

The time course of the conversion of 3‐phospho‐D‐glycerate (GriP) to glyceraldehyde‐3‐phosphate (GraP) catalyzed by 3‐phospho‐D‐glycerate kinase (GriP kinase) and glyceraldehyde‐3‐phosphate dehydrogenase (GraPDH) couple has been reinvestigated. The dependence of the steady‐state rate on the dehydrogenase concentration is fully compatible with the consecutive nature of the reaction and therefore is not necessarily related to a complex formation of the two enzymes. To derive a Kd value of a bienzyme complex, as was done by Sukhodolets et al. [Sukhodolets, M. V., Muronetz, V. I. & Nagradova, N. K. (1987) Biochem. Int. 15, 373–379], is basically erroneous. In contrast with some previous reports, the maximal activity of GriP kinase is not influenced by the auxiliary enzyme present in the coupled assay system. Thus, no special accelerating effect can be attributed to GraPDH. 1,3‐Bisphospho‐D‐glycerate (GriP2) bound to GriP kinase does not seem to be a substrate for GraPDH, providing evidence against channelling of GriP2 between the two enzymes.
Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462
Authors: OHSHIMA Toshihisa , SAKANE Masaru , YAMAZAKI Takashi , SODA Kenji
Published: March 2005

Alanine dehydrogenase (L‐alanine:NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation. red‐Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 °C for at least 60 min and with incubation in pH 5.5–9.5 at 75 °C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (– 20 °C, at pH 7.2) for over 5 months. The optimum pH for the L‐alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L‐alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2‐oxobutyrate and 3‐fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L‐alanine then NAD+. A dead‐end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruyate was included in the reaction. A possible role of the dead‐end inhibition is to prevent the enzyme from functioning in the L‐alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM: ammonia. 38.0 mM; L‐alanine, 10.5 mM and NAD+, 0.26 mM.
Phylogenetic and biochemical evidence for a secondary structure model of a small cytoplasmic RNA from Bacilli
Authors: STRUCK Joachim C. R. , ERDMANN Volker A.
Published: March 2005

Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant, stable RNA identified in the Gram‐positive eubacterium Bacillus subtilis. Several findings suggest an important role of scRNA in protein biosynthesis: shares structural and biochemical features with the Escherichia coli 4.5s RNA (1 14 nucleotides), a molecule known to be involved in this process, and it can complement the essential function of 4.5s RNA in vivo. The common apical hairpin motif of scRNA and 4.5s RNA also exists in eukaryotic 7SL RNA, the RNA component of the signal recognition particle. To elucidate the higher‐order structure of scRNA, we have combined a phylogenetic approach with a biochemical one. The sequence of scRNA from a thermophilic relative of B. subtilis, Bacillus stearotherrnophilus, was determined and compared with the B. subtilis scRNA. In addition, the solution structure of B. stearotherrnophilus scRNA was probed with single‐ and double‐strand‐specific nucleases. Both types analysis support a secondary structure model for scRNA that strongly resembles 4.5s RNA and respective parts of 7SL RNA. The results Drovide further evidence for the suggestion of a functional relationship between these RNAs.
Isolation of a gene for a regulatory 15‐kDa subunit of mitochondrial F1F0‐ATPase and construction of mutant yeast lacking the protein
Authors: YOSHIDA Yukuo , SATO Takeya , HASHIMOTO Tadao , ICHIKAWA Naoki , NAKAI Sumiko , YOSHIKAWA Hiroshi , IMAMOTO Fumio , TAGAWA Kunio
Published: March 2005

A gene coding for yeast 15‐kDa protein, a regulatory factor of mitochondrial F1F0‐ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15‐kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP‐synthesizing activity of the mutant mitochondria was the same as that of wild‐type cells, suggesting that the 15‐kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP‐hydrolyzing activity of F1F0‐ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild‐type cells. Thus, it is inferred that the 15‐kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP‐hydrolyzing activity of the enzyme upon de‐energization of mitochondrial membranes.
Enhanced phosphorylation of a nucleolar 110‐kDa protein in rat liver by dietary manipulation
Authors: MASUMI Atsuko , SUZUKI Toshikazu , IIJIMA Shigeyuki , TSUKADA Kinji
Published: March 2005

RNA polymerase I activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein‐free diet. Phosphorylation in vitro of a 110‐kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein‐free diet. In nuclear extracts the 110‐kDa protein in heat‐treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein‐free diet. Nuclear extracts from control rat liver and livers of rats fed a protein‐free diet were fractionated by DEAE‐cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110‐kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein‐free diet, but not from control liver, yielded fractions which eluted at 0.21–0.25 M NaCl and predominantly phosphorylated the 110‐kDa protein. The phosphorylation of 110‐kDa protein was not appreciably affected by a heparin concentration of 5 μg/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110‐kDa protein in liver nucleoli from rats fed a protein‐free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110‐kDa protein in livers from rats fed a protein‐free diet is due to the induction of a 110‐kDa protein kinase distinct from casein kinase II.
Cycloheximide induces stromelysin mRNA in cultured human fibroblasts
Authors: OTANI Yoshihide , QUINONES Susan , SAUS Juan , KURKINEN Markku , HARRIS Edward D.
Published: March 2005

Stromelysin is a metalloproteinase that degrades extracellular matrix macromolecules including fibronectin, laminin, collagen IV and proteoglycans. We now report that cycloheximide, an inhibitor of protein synthesis, induces human stromelysin mRNA in fibroblast cultures in a time‐ and dose‐dependent fashion. As determined by Northern hybridization, a 24‐h treatment with cycloheximide increased stromelysin mRNA about 20‐fold over the control level. In vitro translation or translation in cells after removal of cycloheximide resulted in increased levels of immunoprecipitable stromelysin suggesting that the cycloheximide‐induced stromelysin mRNA was functional. Analysis of mRNA stability suggested that the cycloheximide effect is in part due to the increased activation of the stromelysin gene. In contrast to these results, cycloheximide did not induce collagenase mRNA but, rather, prevented its induction by interleukin‐1β. These data provide evidence for discoordinate regulation of collagenase and stromelysin genes and suggest that a short‐lived repressor protein may play a role in the stromelysin gene expression.
Mitosis‐specific histone H3 phosphorylation in vitro in nucleosome structures
Authors: SHIBATA Kiyotaka , INAGAKI Masaki , AJIRO Kozo
Published: March 2005

A mechanism of mitosis‐specific enhancement of histone H3 phosphorylation was analyzed in vitro in terms of nucleosome structure. The incorporation of [32P]phosphate into DNA‐bound H3 was approximately 5–7 times higher than in DNA‐free H3 using the catalytic subunit of cAMP‐dependent protein kinase. The two major N‐terminal serine sites, including the mitosis‐specific site (Ser10) and Ser28, were extensively phosphorylated in the DNA‐bound forms. These phosphorylation patterns were identical to those of nucleosomal H3. In contrast, the H3 in DNA‐free octamers was very slightly phosphorylated. The major site of H3 phosphorylation in DNA‐free H3 was Thr118 in the C‐terminus. Results indicate that DNA‐binding is essential for the high level of mitosis‐specific H3 phosphorylation, and that the nucleosome structure promotes H3 N‐terminal phosphorylation in vitro It also suggests the possibility that H1 prevents H3 phosphorylation during interphase of the cell cycle.
Chronic low‐frequency stimulation of rabbit fast‐twitch muscle induces partial inactivation of the sarcoplasmic reticulum Ca2+‐ATPase and changes in its tryptic cleavage
Authors: DUX László , GREEN Howard J. , PETTE Dirk
Published: March 2005

Persistently increased contractile activity as induced by low‐frequency stimulation in fast‐twitch rabbit muscle elicits a partial inactivation of the sarcoplasmic reticulum Ca2+‐ATPase function with regard to Ca2+ transport and ATP hydrolysis. Electron microscopy showed no differences in the frequency and structure of the two‐dimensional Ca2+‐ATPase crystals between microsomal fractions from normal and stimulated muscles. However, differences existed between the tryptic digestion of the Ca2+‐ATPase in both the membrane‐bound and solubilized enzyme at the first tryptic cleavage site, named T1 (Arg505). This followed from a delayed appearance of the A and B fragments of the Ca2+‐ATPase in the electrostimulated muscle. No differences existed with regard to the second tryptic cleavage site, named T2 (Arg198). Confirming previous results, fluorescein isothiocyanate (FITC) binding to the enzyme of the chronically stimulated muscle was markedly reduced. The FITC‐labeled fraction of the enzyme from both the normal and the stimulated muscle followed similar time courses of tryptic cleavage. The fraction of Ca2+‐ATPase that did not bind FITC was identified by immunoblot analysis as the trypsinresistant form. In view of the vicinity of T1, the FITC‐ and the ATP‐binding sites, these results point to a modification of the enzyme in that region leading to an inactivation of about 50% of the sarcoplasmic reticulum Ca2+‐ATPase molecules.
Characterization of matteuccin, the 2.2S storage protein of the ostrich fern
Authors: RÖDIN Joakim , RASK Lars
Published: March 2005

The 2.2S spore storage protein (matteuccin) of the ostrich fern, Matteuccia struthiopteris, has been isolated and characterized. It is a small basic protein consisting of two disulfide‐linked polypeptides with approximate molecular masses of 3.0 kDa and 8.0 kDa. At least four different isoforms exist where two of the forms differ from the other by having a slightly smaller heavy chain. Amino acid analysis reveals that the 2.2S protein is rich in arginine. Almost complete amino acid sequence information was obtained for the light chain and a partial sequence for the heavy chain. Amino acid sequence comparison reveals that this protein shows a high similarity to seed storage proteins in different angiosperm species in spite of the fact that the common ancestor of ferns and angiosperms lived more than 300 million years ago.
A guanyloribonuclease of mouse liver cytosol
Authors: PANTAZAKI Anastasia , GEORGATSOS John G.
Published: March 2005

The acid RNase activity of mouse liver cytosol has been resolved into two different enzymes named acid RNase I and acid RNase II respectively. Acid RNase I is a typical pancreatic‐type enzyme hydrolyzing CpN and UpN bonds. Acid RNase II, however, hydrolyzes GpN bonds in non‐hydrogen‐bonded regions of the substrate.
Expression of the chemically synthesized coding region for the cytotoxin α‐sarcin in Escherichia coli using a secretion cloning vector
Authors: HENZE Peter‐Paul C. , HAHN Ulrich , ERDMANN Volker A. , ULBRICH Norbert
Published: March 2005

The coding region for the cytotoxin α‐sarcin from Aspergillus giganteus has been chemically synthesized by the ligation of 19 overlapping oligodeoxyribonucleotides. An Escherichia coli clone producing the cytotoxin was constructed by inserting the synthesized gene directly downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN‐III‐OmpA2. the enzyme encoded by the chemically synthesized gene expressed in E. coli displayed properties identical to those of native α‐sarcin isolated from A. giganteus with respect to its chemistry, antigenicity and ribonucleolytic activity in qualitative assays.
Effect of Mn(II) on reactions catalyzed by lignin peroxidase from Phanerochaete chrysosporium
Authors: BONO Jean‐Jacques , GOULAS Philippe , BOE Jean‐François , PORTET Nadine , SERIS Jean‐Louis
Published: March 2005

The effect of manganese on lignin peroxidase activity was studied. The enzyme was produced with a new process using an air‐lift‐type reactor. The experiments were performed with veratryl alcohol and a dimeric lignin model compound. It was shown that when Mn(II) · lactate complex was present the amount of veratraldehyde formed and the uptake of oxygen were significantly enhanced during the aerobic oxidation of varatryl alcohol. A similar effect can be obtained with superoxide dismutase. These results strongly suggest that the superoxide anion can occur during the reaction; its scavenging by Mn(II) or superoxide dismutase generates H2O2. In contrast, no evidence for the formation of superoxide anion was found during oxidation of the lignin model, compound veratrylglycerol‐β‐guaiacyl ether.
Homogeneous preparation of human thymidine‐5′‐triphosphatase by electroelution from SDS/PAGE with subsequent renaturation
Authors: SCHULTES Birgit C. , DAHLMANN Nicolaus
Published: March 2005

This paper describes a rapid and inexpensive method for homogeneous enzyme preparation from SDS/ polyacrylamide gels with subsequent renaturation. The method was optimized for an enzyme of pyrimidine metabolism, thymidine‐5′‐triphosphatase (dTTPase), present in human serum in small amounts. After gel electrophoresis, the enzyme was eluted from gel pieces in an elution chamber based on a tube gel electrophoresis system. Renaturation conditions were optimized in preliminary tests. The best results were obtained with an initial acetone precipitation to remove sodium dodecyl sulfate. The precipitate was then dissolved in 8 M guanidine hydrochloride and diluted 50‐fold for renaturation. Adding 1.5 mg/ml lauryl maltoside to the renaturation buffer, followed by subsequent dialysis of the renaturating samples, improved the renaturation yield up to 95%. This method was used to purify dTTPase to homogeneity from a partially purified sample, and to determine the molecular mass of the subunits. The procedure can also be applied to other enzymes and could give rise to a general strategy for enzyme purification.
Simian immunodeficiency virus reverse transcriptase
Authors: KRAUS Günter , BEHR Elke , BAIER Michael , KÖNIG Herbert , KURTH Reinhard
Published: March 2005

Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA‐dependent and DNA‐dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA) · oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC) · oligo(dG). Other homopolymeric substrates, including poly(rC) · oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis‐Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non‐competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.
The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae)
Authors: BALSHÜSEMANN Dirk , JAENICKE Lothar
Published: March 2005

The sexuality‐inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS‐22 and separated by reverse‐phase HPLC into several isoforms which differ in the degree of O‐glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N‐glycans which are clustered in the middle of the polypeptide chain. The N‐glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gas‐chromatographic sugar analysis and two‐dimensional HPLC separation to possess the following definite structures:

image

Two of the three N‐glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N‐glycosylation site. The O‐glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.
The use of the Ca2+‐sensitive intramitochondrial dehydrogenases and entrapped fura‐2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria
Authors: McCORMACK James G. , OSBALDESTON Nicola J.
Published: March 2005

In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2+‐sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2‐oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1–100 μM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2–3‐fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2‐oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2‐oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100‐fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura‐2 or indo‐1 in a similar manner to Ca2+, but with higher kd values. In fura‐2‐loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated.
Carbon‐13 nuclear magnetic resonance analysis of [1‐13C]glucose metabolism in Trypanosoma cruzi
Authors: FRYDMAN Benjamin , de los SANTOS Carlos , CANNATA Joaquin J. B. , CAZZULO Juan J.
Published: March 2005

The non‐invasive technique of 13C‐nuclear magnetic resonance was applied to study glucose metabolism in vivo in Trypanosoma cruzi, the causative agent of American trypanosomiasis (Chagas' disease). It was found that under anaerobic conditions [1‐13C]glucose undergoes a glycolytic pathway whose main metabolic products were identified as [3‐13C]alanine, [2‐13C]succinate and phosphoryl[1‐13C]choline; [2‐13C]alanine was also a minor metabolite. The addition of 70%2H2O to the incubation mixture led to the formation of [3‐13C, 3‐2H]alanine derived from the prior incorporation of 2H+ into pyruvate. The existence of a [3‐13C, 3‐2H]pyruvate precursor, although not isolated, could be inferred from the formation of [2‐13C, 2‐2H]succinate in the same experiment. The latter derives from the CO2 fixation reaction on pyruvate or phosphoenolpyruvate to give malate, which is then converted to succinate through the fumarate intermediate step. The presence of [2‐13C]alanine must be traced to a randomization of label at the malate – fumarate stage. Both [3‐13C, 3‐2H]alanine and [2‐13C, 2‐2H]succinate were excreted from the cells into the supernatant. When the cell pellet was lysed with perchloric acid it released [3‐13C]alanine which was devoid of 2H+. Hence, T. cruzi has two alanine pools: one which incorporates 2H+ from the 2H2O present in the medium and excretes alanine into the latter, and another which is impervious to 2H+ exchange. The fixation of CO2 on a C3 precursor was confirmed by incubation of the T. cruzi cells with [1‐13C]glucose and sodium [13C]bicarbonate which led to the formation of [1,2‐13C2]succinate (Jcc– 51.8 Hz). Incubation with sodium [13C]bicarbonate and [13C]glucose led to the formation of [1‐13C]succinate (182.5 ppm) derived from the 13CO2 fixation on the C3 precursor, and of phosphoryl[1‐13C]choline (59.39 ppm) which revealed the presence in T. cruzi of a reductive pathway of CO2 which is independent of the CO2 fixation reaction. The formation of phosphoryl[1‐13C]choline from [1‐13C]glucose should be attributed to 13CO2 liberated from the former by glucose‐6‐phosphate dehydrogenase.
Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa
Authors: BOURDENET Sylviane , VACHERON Marie‐Jeanne , GUINAND Micheline , MICHEL Georges , ARMINJON François
Published: March 2005

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N‐terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365–404). As a result, the polypeptides contained a large portion (13–34 amino acids) of domain Ib linked to the adjacent C‐terminal domain III (amino acids 405–613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S‐fragment; residues 392–613; 24201 Da; pI 4.7) possessed the same level of ADP‐ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37‐fold higher NAD‐glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP‐ribosyltransferase activity of both exotoxin A and the S‐fragment, but not the NAD‐glycohydrolase activity of the S‐fragment. Antibodies against the S‐fragment neutralized the ADP‐ribosyltransferase activity of exotoxin A.

These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392–404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF‐2) and show that antibodies recognize the EF‐2‐binding site but not the NAD+‐binding site.
Amino acid sequences of four isoforms of amphioxus sarcoplasmic calcium‐binding proteins
Authors: TAKAGI Takashi , COX Jos A.
Published: March 2005

The protochordate amphioxus (Branchiostoma lanceolatum) contains different isoforms of sarcoplasmic, high‐affinity Ca2+‐binding proteins (SCP). The amino acid sequences of the two major isoforms SCP I and II, reported previously [Takagi, T., Konishi, K. & Cox, J. A. (1986) Biochemistry 25, 3585–3592], have been corrected and differ from each other by seven amino acid substitutions in a 17‐residue‐long segment (positions 20–36). We also report on the isolation and amino acid sequence determination of two minor isoforms, i.e. amphioxus SCP III and IV. Although they behave very differently from the major forms with respect to net charge, they differ from SCP I by only one amino acid: SCP III has Met at position 20 (Tyr in SCP I) and SCP IV has Asn at position 23 (Asp in SCP I). Together the sequence data on amphioxus SCP suggest that, in contrast with SCP of other invertebrate phyla, the isoforms are generated by alternative splicing of the primary RNA transcript with a mutually exclusive pattern.
Purification and properties of a NADH‐dependent 5,10‐methylenetetrahydrofolate reductase from Peptostreptococcus productus
Authors: WOHLFARTH Gert , GEERLIGS Gabriele , DIEKERT Gabriele
Published: March 2005

The methylenetetrahydrofolate reductase from the carbon‐monoxide‐utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 μmol · min−1 mg protein−1 (37°C; pH 5.5). The apparent Km for NADH was near 10 μM. The apparent molecular mass of the enzyme was determined by gel filtration to be ∼250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylene‐tetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyl‐tetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used as substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin‐dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP+‐dependent reductase of eucaryotes investigated so far.
Structure determination of five sialylated trisaccharides with core types 1, 3 or 5 isolated from bovine submaxillary mucin
Authors: SAVAGE Angela V. , DONOGHUE Carmel M. , D'ARCY Sinead M. , KOELEMAN Carolien A. M. , van den EIJNDEN Dirk H.
Published: March 2005

In this study we have investigated the structures of five sialylated trisaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high‐performance liquid chromatography. Three of the trisaccharides contained NeuAc while two contained NeuGc. One oligosaccharide contained core‐type 1, two contained core‐type 3 and two contained core‐type 5. The structures, determined by a combination of one‐ and two‐dimensional 1H‐NMR spectroscopy at 270 MHz and methylation analysis involving gas‐liquid chromatography/mass spectrometry, were as follows: A4b, GalNAcα(1 → 3)[NeuAcα(2 → 6)]GalNAcol; A4c, GlcNAcβ(1 → 3)[NeuAcα(2 → 6)]GalNAcol; A4d, Galβ(1 → 3)[NeuAcα(2 → 6)]GalNAcol; A4e, GalNAcα(1 → 3)‐[NeuGcα(2 → 6)]GalNAcol; A4f, GlcNAcβ(1 → 3)[NeuGcα(2 → 6)]GalNAcol. The oligosaccharides occurred in the approximate molar ratios 1.0:12.0:0.3:0.2:2.0. This is the first report of oligosaccharides containing core‐type 5 and of the occurrence of oligosaccharides A4b, A4e, and A4f in bovine submaxillary mucin. 1H‐NMR data for structure A4e, which is a novel structure, are presented for the first time.
Inhibition of inositol 1,4,5‐trisphosphate metabolism in permeabilised SH‐SY5Y human neuroblastoma cells by a phosphorothioate‐containing analogue of inositol 1,4,5‐trisphosphate
Authors: WOJCIKIEWICZ Richard J. H. , COOKE Allan M. , POTTER Barry V. L. , NAHORSKI Stefan R.
Published: March 2005

Electrically permeabilised [3H]inositol‐labelled SH‐SY5Y human neuroblastoma cells were employed to examine the effects of two synthetic, phosphatase‐resistant analogues of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] on the metabolism of cell membrane‐derived [3H]Ins(1,4,5)P3 or exogenous [5‐32P]Ins(1,4,5)P3.

Incubation of permeabilised SH‐SY5Y cells for 5 min at 37°C with carbachol and guanosine 5′‐[γ‐thio]triphosphate caused a decrease in [3H]phosphoinositol phospholipid levels and an increase in [3H]inositol phosphate accumulation with inositol 4‐phosphate, inositol 1,4‐bisphosphate, Ins(1,4,5)P3 and inositol 1,3,4,5‐tetrakisphosphate comprising approximately 79%, 16%, 3% and 2%, respectively, of the increase. Inositol 1‐phosphate levels did not increase upon stimulation, nor was inositol 4‐phosphate converted rapidly to inositol.

In parallel incubations, the analogues, DL‐inositol 1,4,5‐trisphosphorothioate (DL‐InsP3S3) and DL‐inositol 1,4‐bisphosphate 5‐phosphorothioate (DL‐InsP3S), and synthetic racemic Ins(1,4,5)P3 (DL‐InsP3), altered the profile of the [3H]inositol phosphates recovered and led, at millimolar concentrations, to a 10–15‐fold increase in [3H]Ins(1,4,5)P3. The extent of inhibition of [3H]Ins(1,4,5)P3 metabolism was, however, greatest in the presence of synthetic D‐Ins(1,4,5)P3 (5 mM), when [3H]Ins(1,4,5)P3 comprised approximately 50% of the increase in total [3H]inositol phosphates. Thus, under these conditions, at least 50% of [3H]inositol phosphates were derived from [3H]phosphatidylinositol 4,5‐bisphosphate. [32P]Pi release from exogenous [5‐32P]Ins(1,4,5)P3 was also inhibited by DL‐InsP3S3, DL‐InsP3S and DL‐InsP3, with half‐maximal inhibition at approximately 50 μM, 160 μM and 240 μM respectively. These actions were approximately ten times more potent than the effects of these compounds on [3H]inositol phosphate accumulation, indicating that homogenous mixing of exogenous and cellmembrane‐derived Ins(1,4,5)P3 does not occur.

These findings indicate that DL‐InsP3S3 and DL‐InsP3S inhibit Ins(1,4,5)P3 5‐phosphatase. In contrast. the effects of synthetic DL‐InsP3 and d‐Ins(1,4,5)P3 are due to isotopic dilution. Whilst DL‐InsP3S3 was the most potent inhibitor of dephosphorylation of exogenous or cell‐membrane‐derived Ins(1,4,5)P3, it was the weakest inhibitor of 3‐kinase‐catalysed Ins(1,4,5)P3 phosphorylation. Similarly, although approximately 50 times less potent than DL‐InsP3S3, 2,3‐diphosphoglycerate inhibited Ins(1,4,5)P3 5‐phosphatase activity and was apparently without effect of Ins(1,4,5)P3 3‐kinase activity.

These data show that both DL‐InsP3S3 and DL‐InsP3S are potent inhibitors of Ins(1,4,5)P3 5‐phosphatase. In contrast, phosphorylation of Ins(1,4,5)P3 was inhibited potently only by DL‐InsP3S.
Plasma membrane Fe2‐transferrin reductase and iron uptake in K562 cells are not directly related
Authors: GOLDENBERG Hans , DODEL Beate , SEIDL Dagmar
Published: March 2005

Receptor‐mediated endocytosis and recycling of transferrin is partly inhibited by the ferrous iron chelator bipyridine, which almost completely blocks iron uptake. Bipyridine causes iron release at the cell surface, but inhibition of iron uptake is also due to a blockage of its passage through the endosomal membrane. The rate of release of iron to bipyridine is decreased by the competing electron acceptor ferricyanide and by amiloride, but not by iron uptake inhibiting acidotropic amines.

Transferrin reduction at the plasma membrane may be artificially induced by presence of a ferrous chelator and caused by low‐affinity transmembrane NAD(P)H oxidoreductase.