bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of hepatic mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase during the foetal/neonatal transition, suckling and weaning in the rat
Authors: QUANT Patti A. , ROBIN Danielle , ROBIN Pierre , FERRE Pascal , BRAND Martin D. , GIRARD Jean
Published: March 2005

We assayed active and total (i.e. active plus succinylated) 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) synthase in mitochondria isolated from foetal, neonatal, suckling or weaned rats.

HMG‐CoA synthase was substantially succinylated and inactivated in mitochondria isolated from termfoetal, (1‐h‐old, 6‐h‐old, 1‐day‐old) neonatal, suckling and high carbohydrate/low‐fat (hc)‐weaned rats. Succinylation of HMG‐CoA synthase was very low in mitochondria isolated from the livers of foetal, 30‐min‐old neonatal and high‐fat/carbohydrate‐free (hf)‐weaned rats.

There was a negative correlation between active HMG‐CoA synthase and succinyl‐CoA content in mitochondria isolated from term‐foetal, suckling and hc‐weaned rats.

Differences in active enzyme could not be entirely accounted for by differences in succinylation and inactivation of the synthase. Immunoassay confirmed that the absolute amounts of mitochondrial HMG‐CoA synthase increased during the foetal/neonatal transition and decreased with hc weaning. The levels remained elevated with hf weaning.

From these data we propose that mitochondrial HMG‐CoA synthase is controlled by two different mechanisms in young rats. Regulation by succinylation provides a mechanism for rapid modification of existing enzyme in response to changing metabolic states. Changes in the absolute amounts of HMG‐CoA synthase provide a more long‐term control in response to nutritional changes.
Highly conserved structure of proliferating cell nuclear antigen (DNA polymerase δ auxiliary protein) gene in plants
Authors: SUZUKA Iwao , HATA Shingo , MATSUOKA Makoto , KOSUGI Shunichi , HASHIMOTO Junji
Published: March 2005

In a previous paper [Suzuka, I., Daidoji, H., Matsuoka, M., Kadowaki, K., Takasaki, Y., Nakane, P. K. & Moriuchi, T. (1989) Proc. Natl Acad. Sci. USA 86, 3189–3193], we demonstrated the presence of a rice genomic‐DNA fragment which hybridized with a rat proliferating‐cell nuclear antigen (PCNA) cDNA probe, and proposed that plants possess the homolog of the PCNA gene which plays an essential role in DNA replication in mammals. Here we show that the sequence of the rice PCNA gene supports this proposal. The rice gene consists of four exons. The 5′‐flanking region contains a variety of sequences similar to several consensus sequences believed to be important for the regulation of gene expression in eukaryotes. Determination of the transcription‐start site and the structure of the 3′‐flanking region of the gene indicated that the coded mRNA is 1073 bases. The rice PCNA genomic and cDNA clones are composed of 789 nucleotides; they encode a putative protein of 263 amino acids having a molecular mass of 29 275 Da. Comparison of the coding regions of rice PCNA and rat PCNA revealed about 64% similarity at the nucleotide level and about 62% similarity at the amino acid level. Furthermore, comparison of the predicted amino acid sequence of the soybean PCNA with the rice PCNA sequence indicated a high degree of similarity (88%). The structural similarity between PCNA from plants and animals, despite the distant evolutionary relationship, indicates a strong selection pressure for conservation of this structure and suggests the presence of conserved DNA‐replication mechanisms throughout the eukaryotes.
Secondary structure of the 5′ external transcribed spacer of vertebrate pre‐rRNA
Authors: MICHOT Bernard , BACHELLERIE Jean‐Pierre
Published: March 2005

Eucaryotic pre‐rRNA spacers are evolutionarily highly variable in sequence and size, and are markedly expanded in vertebrates, particularly in mammals. The longest mammalian spacer by far is the 3.5–4‐kb 5′ external transcribed spacer (5′‐ETS), which is excised in two steps. We present a folding model for the entire mammalian 5′‐ETS, derived from comparative analyses and thermodynamic predictions for mouse, rat and human sequences, which should prove helpful in identifying cis‐acting processing signals, particularly those involved in its early internal cleavage, for which U3 RNA is an essential factor. Although the rodent and primate sequences have extensively diverged, a series of relatively conserved sequence tracts can nevertheless be identified: they participate in base‐pairing, preserved through the occurrence of compensatory base changes, which delineate four independent domains of secondary structure. The first domain is located entirely upstream from the site of internal cleavage. The second domain, immediately downstream from this cleavage site, encompasses most of the region required for faithful and efficient in vitro processing at this site. Phylogenetically supported conserved structures also define two other independent domains, encompassing most of the 5′‐ETS length, with the presence of giant hairpins (extending from the conserved core elements) which exhibit both some analogous features and substantial differences between man and mouse. The comparative analysis was extended to the two other vertebrate sequences available so far, amphibians Xenopus laevis and Xenopus borealis. The amphibian folding model, supported by comparative evidence between these two species, displays some features in common with the mammalian model, with a similar organization into four separate domains of secondary structure, suggesting the functional relevance of these structures in the process of ribosome formation.
Cuttlefish sperm protamines
Authors: SCHINDLER Patrick , BITSCH Francis , KLARSKOV Klaus , ROEPSTORFF Peter , BRIAND Gilbert , WOUTERS‐TYROU Danièle , SAUTIÈRE Pierre , VAN DORSSELAER Alain
Published: March 2005

The sequence of very basic proteins such as protamines (more than 50% arginines) and related peptides has been determined using mass spectrometry in conjunction with Edman degradation. The capabilities of three mass spectrometric (MS) techniques [fast‐atom‐bombardment (FAB), 252Cf plasma desorption (252CFPD) and electrospray (ES)] have been evaluated on stallion protamine 1, cuttlefish protamine, and the corresponding cleavage peptides. In contrast to FAB‐MS and 252Cf PD‐MS, ES‐MS made possible an easy determination of the molecular mass of the intact protamines (∼8 kDa). With ES‐MS about 0.2 nmol was sufficient to yield a mass measurement with an accuracy of 0.05%. On peptides smaller than 3500 Da, both FAB‐MS and 252Cf PD‐MS allowed mass measurements with an accuracy of 0.1%. 252Cf PD‐MS appeared more sensitive than FAB‐MS by about a factor of 10. FAB‐MS is nevertheless particularly interesting since in most cases it produced spectra with intense A‐type fragmentation ions which provided reliable primary structure information.
Two structural domains as a general fold of the toxic fragment of the Bacillus thuringiensisδ‐endotoxins
Authors: CONVENTS Daniel , CHERLET Marc , VAN DAMME José , LASTERS Ignace , LAUWEREYS Marc
Published: March 2005

The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two‐step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265, 1369–1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease‐susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N‐terminal half of both toxins.
Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in 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

Treatment of rabbit pancreatic acini with the phorbol ester, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), resulted in a time‐ and dose‐dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA‐induced inactivation of the enzyme. The biologically inactive phorbol ester, 4α‐phorbol 12,13‐didecanoate, did not induce such a translocation of protein kinase C. The half‐maximal concentration for TPA‐induced translocation of protein kinase C was 40 nM, and was equal to that for TPA‐induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA‐induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium‐mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6‐{2‐[(4‐fluorophenyl)phenylmethylene]‐1‐piperidinyl} ethyl‐7‐methyl‐5H‐thiazolo[3,2‐a]pyrimidin‐5‐one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid.
ADP is a substrate for the AAUAAA‐directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts
Authors: LAKOTA Jan , NELSON B. Dean
Published: March 2005

The specific poly(A) addition reaction catalyzed by crude nuclear extracts from HeLa cells can use ADP as efficiently as ATP as the donor of AMP residues. Both the ADP‐ and ATP‐supported reactions require an intact upstream polyadenylation signal sequence element (AAUAAA). The mutated signal sequence (AACAAA) supports neither reaction. The ADP‐supported poly(A) addition reaction can be resolved by glycerol gradient centrifugation of the crude nuclear extract into two components which are active when recombined but are inactive individually. The ATP‐supported poly(A) addition is reconstituted by recombining the same gradient fractions, but the activity is lower than that supported by ADP, suggesting that an ATP‐specific factor has been removed. A 150 mM KCl fraction DEAE‐Sepharose of the nuclear extract, also devoid of the ATP‐supported poly(A) addition reaction, retains a normal ADP‐supported reaction. Together, these data show that ADP is a substrate for polyadenylation, and suggest that different factors might be required to induce ADP‐ or ATP‐specificity in the poly(A) addition reaction.
A tetranectin‐related protein is produced and deposited in extracellular matrix by human embryonal fibroblasts
Authors: CLEMMENSEN Inge , LUND Leif R. , CHRISTENSEN Lise , ANDREASEN Peter A.
Published: March 2005

Tetranectin is a tetrameric human plasma protein that binds to plasminogen kringle 4. Its amino acid sequence is homologous with the C‐terminal parts of asialoglycoprotein receptors and proteoglycan core proteins. In the present study, we have demonstrated that the human embryonal fibroblast cell line WI‐38 produce a tetranectin‐related molecule, which might, by several criteria, be similar to tetranectin from plasma. These criteria include immunoblotting analysis of conditioned cell medium revealing a protein band with Mr 17,000, indistinguishable from the Mr of plasma tetranectin. A preparation obtained by purification of conditioned medium by affinity chromatography on an anti‐(plasma tetranectin) IgG column also contained the Mr 17000 protein. This protein (partly purified from the conditioned medium) was shown by crossed immunoelectrophoresis to bind to heparin, CaCl2 and plasminogen kringle 4, as previously described for tetranectin in plasma. Importantly, this tetranectin‐related protein is not only present in conditioned culture medium, but the Mr 17000 protein reacting with anti‐(plasma tetranectin) IgG was also present in the extracellular material, remaining after removal of WI‐38 cells from the culture dishes, as demonstrated by immunoblotting analysis and immunocytochemical staining. We conclude that WI‐38 cells produce a tetranectin‐related protein and secrete it into the extracellular matrix.
Dependence of lysozyme‐catalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O‐acetylation
Authors: DUPONT Claude , CLARKE Anthony John
Published: March 2005

The degree of peptidoglycan O‐acetylation in 14 strains of Proteus mirabilis has been accurately determined by a procedure which employs the quantitation of mild‐base‐released acetic acid by HPLC, and the estimation of peptidoglycan concentration by cation‐exchange amino acid analysis. The β‐D‐N,6‐O‐diacetylmuramyl content of all isolated and purified peptidoglycans was ranged 20 – 52.8%, relative to the total muramic acid concentration. Each of the O‐acetylated peptidoglycans was found to be resistant to solubilization by both human and hen egg‐white lysozymes and for hen egg‐white lysozyme, the extent of this resistance was dependent upon the degree of O‐acetylation. The steady‐state parameters, Km and V, for the hen‐egg‐white‐lysozyme‐catalysed solubilization of various peptidoglycan preparations were determined at pH 6.61 and 25°C. Values of Km for the different peptidoglycan samples were found to increase with increasing O‐acetylation, whereas with V no such relationship appeared to exist. An increase in the overall change in the standard Gibbs free energy of activation [Δ(ΔG±)], a consequence of increasing O‐acetylation, was observed, and is shown to result from the weaker affinity of the enzyme for the modified substrates.
Blocking of the receptor‐mediated invasion of erythrocytes by Plasmodium knowlesi malaria with sulfated polysaccharides and glycosaminoglycans
Authors: DALTON John P. , HUDSON Diana , ADAMS John H. , MILLER Louis H.
Published: March 2005

Invasion of human erythrocytes by Plasmodium knowlesi requires the Duffy blood group antigen. P. knowlesi merozoites synthesize a 135‐kDa polypeptide which binds to the Duffy antigen with receptor‐like specificity. In this study, we show that the sulfated polysaccharide fucoidan and the glycosaminoglycan dextran sulfate inhibit the binding of the 135‐kDa polypeptide to human Duffy‐positive and rhesus erythrocytes while the chondroitin sulfates do not. Fucoidan and dextran sulphate also blocked the in vitro invasion of human Duffy b and rhesus erythrocytes cells by P. knowlesi merozoites. These inhibitors were more effective at blocking the binding of the 135‐kDa polypeptide to human Duffy b erythrocytes than to rhesus erythrocytes, which correlated with them having a greater inhibitory effect on invasion of merozoites into human than into rhesus erythrocytes.

The blocking by these sulfated sugars is not related to charge density on the polysaccharides; fucoidan with a relatively low charge density blocks binding of the 135‐kDa polypeptide at 4 μg/ml, while the highly negatively charged chondroitin sulfates do not block binding even at the concentration of 1 mg/ml. Furthermore, fucoidan‐Sepharose bound and removed the 135‐kDa polypeptide from parasite culture supernatants with a selectivity equal to that of the Duffy blood group antigen. The negatively charged sulfate groups on fucoidan and dextran sulfate and the conformation in which they are held possibly mimic similarly charged groups on the Duffy antigen which bind the 135‐kDa P. knowlesi polypeptide.
Characterization of the cell‐cycle‐regulated protein calcyclin from Ehrlich ascites tumor cells
Authors: FILIPEK Anna , GERKE Volker , WEBER Klaus , KUŹNICKI Jacek
Published: March 2005

The nearly complete amino acid sequence obtained for murine calcyclin from Ehrlich ascites tumor cells reveals a very strong similarity with the rat and human sequences previously deduced from corresponding cDNA clones. While mouse and rat calcyclins are identical, the human protein shows at three positions a conservative amino acid replacement. Using a mouse calcyclin affinity matrix, two proteins with molecular masses of about 36 kDa have been purified from Ehrlich ascites tumor cells. The interaction between these two proteins and the immobilized calcyclin is strictly Ca2+‐dependent. Immunological criteria and partial sequence data identify the two calcyclin‐binding proteins as the phospholipid‐binding protein annexin II (p36) and the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase. These observations suggest that calcyclin may exert its physiological function by a Ca2+‐dependent interaction with cellular targets, e.g. annexin II or glyceraldehyde‐3‐phosphate dehydrogenase.
Lys631 residue in the active site of the bacteriophage T7 RNA polymerase
Authors: MAKSIMOVA Tatyana G. , MUSTAYEV Arkady A. , ZAYCHIKOV Evgeny F. , LYAKHOV Dmitry L. , TUNITSKAYA Vera L. , AKBAROV Akhror Kh. , LUCHIN Sergei V. , RECHINSKY Vladimir O. , CHERNOV Boris K. , KOCHETKOV Sergei N.
Published: March 2005

A highly selective affinity labeling of T7 RNA polymerase with the o‐formylphenyl ester of GMP and [α‐32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N‐chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue.

Site‐directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue. Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity. For the Lys → Gly mutant enzyme, anomalous template binding was observed.
Isolation and inactivation of the nuclear gene encoding the rotenone‐insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae
Authors: MARRES C. A. M. , de VRIES S. , GRIVELL L. A.
Published: March 2005

We have recently described the isolation of a mitochondrial rotenone‐insensitive NADH: ubiquinone oxidoreductase from Saccharomyces cerevisiae [de Vries, S. & Grivell, L. A. (1988) Eur. J. Biochem. 176, 377–384]. We now report the isolation of the nuclear gene encoding this single‐subunit enzyme. Null mutants have been constructed by means of one‐step gene disruption. Oxygen‐uptake experiments, performed with mitochondria isolated from the mutant cells, showed that this NADH dehydrogenase catalyzes the oxidation of NADH generated inside the mitochondrion. Inactivation of this NADH dehydrogenase does not affect growth on glucose and ethanol, but growth on lactate, pyruvate and acetate is impaired or absent. This phenotype is discussed in terms of the interplay between different metabolic pathways in yeast.
H+‐translocating inorganic pyrophosphatase of plant vacuoles Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases
Authors: MAESHIMA Masayoshi
Published: March 2005

The effects of divalent cations, especially Ca2+ and Mg2+, on the proton‐translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases. The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+. The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin. Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5= 42 μM). Binding of Mg2+ stabilizes and activates the enzyme. The formation of MgPPi is also an important role of magnesium ion. Apparent Km of the enzyme for MgPPi was about 130 μM.

CaCl2 decreased the enzyme activity to less than 60% at 40 μM, and the inhibition was reversed by EGTA. Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7.2. The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 μM. A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi. It appears that for Ca2+, at least, the inhibitory form is the Ca2+‐PPi complex. Cd2+, Co2+ and Cu2+ also inhibited the enzyme.

The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases. Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme. Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immuno‐reactivity.
Single administration of hepatotoxic chemicals transiently decreases the gap‐junction‐protein levels of connexin 32 in rat liver
Authors: MIYASHITA Tetsukazu , TAKEDA Akira , IWAI Masaru , SHIMAZU Takashi
Published: March 2005

Effects of a single administration of hepatotoxic chemicals on the major gap‐junction protein of liver (connexin 32) were studied in rats. The connexin‐32 content was analyzed by immunoblotting and immunohistochemistry using an affinity‐purified monoclonal antibody against connexin 32. The connexin‐32 content decreased dramatically to less than 10% of the control value 24 h after injection of 25 mg/kg dimethylnitrosamine and returned to the normal level 240 h later. Injection of CCl4 at a dose of 1 ml/kg also caused a transient reduction of connexin‐32 content in the liver, as seen after the dimethylnitrosamine injection. The decrease in hepatic connexin‐32 content was inversely correlated to the increase in plasmic alanine‐aminotransferase activity which has been used as an index of acute liver injury. The changes in connexin 32 were essentially similar to those observed in regenerating liver after partial hepatectomy. However, incorporation of [3H]thymidine into liver DNA after dimethyl‐nitrosamine injection was significantly less than that obtained after partial hepatectomy. The 5′‐nucleotidase activity of the plasma‐membrane fraction of the liver was not significantly altered by the injection of dimethyl‐nitrosamine. These results suggest that liver gap‐junction protein is specifically reduced by acute liver injury and that this kind of decrease in connexin 32 is not simply related to cell proliferation, unlike the decrease after partial hepatectomy.
Primary structure of O‐linked carbohydrate chains in the cellulosome of different Clostridium thermocellum strains
Authors: GERWIG Gerrit J. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , (MORGENSTERN) Ely MORAG , LAMED Raphael , BAYER Edward A.
Published: March 2005

The cell‐free forms of the multiple cellulase‐containing protein complex (cellulosome), isolated from the celluloytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5–7% (by mass), consisting of O‐linked oligosaccharide chains. The carbohydrate chains were liberated by alkaline‐borohydride treatment and fractionated as oligosaccharide alditols via gel‐permeation chromatography and HPLC. The fractions were investigated by 500‐MHz 1H‐NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast‐atom‐bombardment mass spectrometry (FAB‐MS). In addition to the previously described major oligosaccharide,

image

[Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027 – 10351, the following partial structures of this compound could be established:

image

Cell‐free and cell‐associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested.
Investigation of the membrane‐active peptides melittin and glucagon by photochemically induced dynamic‐nuclear‐polarization (photo‐CIDNP) NMR
Authors: MAURER Till , LÜCKE Christian , RÜTERJANS Heinz
Published: March 2005

The photochemically induced dynamic‐nuclear‐polarization (photo‐CIDNP) NMR technique was used to investigate the membrane‐active peptides melittin and glucagon. The experiments were performed both in the absence and presence of phospholipid vesicles in order to study the topography of the membrane‐bound state. From the results it can be concluded that the melittin peptide chain is oriented in such a way that the single tryptophan residue (Trp19) reaches into the membrane. In the case of glucagon, a binding interaction with vesicle membranes is indicated within the pH range 2–10, whereby the single tryptophan residue (Trp25) is buried in the lipid bilayer and the tyrosine and histidine residues are exposed to the aqueous solvent.
The mechanism of Ca2+‐related control of gluconeogenesis in perfused liver
Authors: STERNICZUK Anna , HRENIUK Stan , SCADUTO Russell C. , LaNOUE Kathryn F.
Published: March 2005

A kinetic expression for rat‐liver mitochondrial aspartate formation in situ was developed in order to determine whether hormonally induced decreases in 2‐oxoglutarate levels can regulate hepatic gluconeogenesis from lactate via control of aspartate formation. Previous studies from this laboratory showed that 2‐oxoglutarate can inhibit aspartate production by isolated mitochondria. These present studies were designed to probe the physiological significance of the decrease in 2‐oxoglutarate levels observed when Ca2+‐mobilizing gluconeogenic hormones are administered to isolated perfused rat livers. First, estimates were made of the kinetic constants which determine the rate of aspartate formation in isolated mitochondria. The concentrations of the substrates and products of this process were then measured in perfused livers. From these values, it was possible to estimate aspartate efflux from mitochondria in situ. The calculated rates of aspartate production were increased by decreases in 2‐oxoglutarate levels which occurred when glucagon or phenylephrine was added to the perfused livers. Glucagon also effected an inhibition of pyruvate kinase, evidenced by the fact that the calculated rate of aspartate efflux equalled the rate of gluconeogenesis (the difference between the two is equivalent to the pyruvate‐kinase flux). By contrast, in control livers and with phenylephrine stimulation, aspartate formation was higher than gluconeogenesis suggesting significant pyruvate‐kinase flux in this condition. The calculations also show a correlating increase in flux through pyruvate carboxylase (30% with phenylephrine, 15% with glucagon, compared with ∼ 50% increases in gluconeogenic flux). The mechanism of this increase is discussed.
Processing of the precursor of protamine P2 in mouse Identification of intermediates by their insolubility in the presence of sodium dodecyl sulfate
Authors: ELSEVIER Susan M. , NOIRAN Joseph , CARRE‐EUSEBE Danièle
Published: March 2005

Two basic proteins, protamines P1 and P2, are present in chromatin of mouse spermatozoa. Protamine P1, the less abundant protein in mouse, has a homolog in most mammals, and its synthesis follows a conventional route. In contrast, protamine P2 has been found only in certain other mammals, including humans, and it is synthesized as a precursor nearly twice as long as the mature protein. Processing of this precursor is not yet understood, although it necessarily takes place in elongating spermatids and is likely to play a role in the chromatin condensation occurring in these haploid cells.

We have fractionated basic proteins from mouse testis chromatin and have identified six proteins on electrophoretic gels which, like protamines, are insoluble in SDS. All six were also soluble at the same trichloroacetic acid concentration as protamine P2 and were present in chromatin of elongating spermatids. Radioactive labelling patterns acquired by these SDS‐insoluble proteins during translation in vitro of testis RNA indicate that the largest represents the precursor of protamine P2, and suggest that the others represent intermediates generated by proteolytic cleavage of the precursor. Results from pulse 3H labelling in vivo were also consistent with the conclusion that a precursor/product relationship exists between these proteins and protamine P2. Conclusions concerning the kinetics of processing have, in addition, been drawn from this data. Hypotheses concerning possible functional roles played by the precursor are presented.
Structure and expression of the attacin genes in Hyalophora cecropia
Authors: SUN Shao‐Cong , LINDSTRÖM Ingrid , LEE Jong‐Youn , FAYE Ingrid
Published: March 2005

To study the regulation of the immune genes in insects, we have cloned and sequenced the attacin gene locus of the giant silk moth Hyalophora cecropia. The locus contains one acidic and one basic attacin gene as well as two pseudogenes, which are remnants of basic attacin genes. A small insertion element was found within the locus. The two functional attacin genes are transcribed in opposite directions and have two introns inserted at homologous positions. A common sequence, GGGGATTCCT, is found at nucleotide position –48 in the acidic gene and at nucleotide position –58 in the basic gene. Interestingly, this decanucleotide is similar to the consensus of the NF‐kB‐binding site. Expression studies revealed that both attacins are strongly induced by phorbol 12‐myristate 13‐acetate, lipolysaccharide and bacteria. However, only the acidic attacin gene showed a clear response to injury.
A proton‐NMR study of a site‐directed mutation (His388 → Glu) in the interdomain region of yeast phosphoglycerate kinase
Authors: GRAHAM Heidi C. , WILLIAMS Robert J. P. , LITTLECHILD Jennifer A. , WATSON Herman C.
Published: March 2005

Proton NMR has been used to study a site‐directed mutant of yeast phosphoglycerate kinase in which the interdomain residue His388 has been replaced by a glutamine residue. Using 1H‐NMR spectroscopy, it was found that 3‐phosphoglycerate binding to the mutant protein induces different conformational effects to those observed for the wild‐type enzyme. These differences are not only located at the 3‐phosphoglycerate binding site but are also seen as long‐range effects at the surface of the protein. Measurements of the Kd for 3‐phosphoglycerate from the NMR experiments show that the mutant enzyme has a 30‐times reduced affinity for this substrate as compared with the wild‐type enzyme. These data are consistent with the suggestion that an aromatic residue at position 388 plays an important role in the proposed hinge‐bending mechanism.
Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes
Authors: de LAMOTTE‐GUERY Frédéric , MIGINIAC‐MASLOW Myroslawa , DECOTTIGNIES Paulette , STEIN Mariana , MINARD Philippe , JACQUOT Jean‐Pierre
Published: March 2005

A new over‐expression system has been set up for Escherichia coli thioredoxin, yielding 55 mg purified protein/10 g fresh cells. This system has been used to produce thioredoxin modified by site‐directed mutagenesis. Taking advantage of the structural and enzymatic similarity between E. coli and spinach m‐type thioredoxin, Asp61 of E. coli thioredoxin has been changed into Asn in order to investigate the impact of the suppression of a charged residue on the interaction of thioredoxin with target enzymes. The modification did not significantly alter the structure of the protein. Neither the rate of reduction of insulin and 5,5′‐dithio‐bis(2‐nitrobenzoic acid) by the reduced thioredoxin, nor the reduction by NADPH‐dependent thioredoxin reductase, have been modified. The major effect of the mutation was observed for chloroplast enzyme activation with thioredoxin reduced by dithiothreitol and with thioredoxin reduced by ferredoxin‐dependent thioredoxin reductase in a light‐activation reconstituted chloroplast system. The substitution of the negatively charged Asp61 by the neutral Asn led to an increase in the efficiency of spinach fructose‐1,6‐bisphosphatase activation by the dithiothreitol‐reduced thioredoxin, and to an increase in both spinach fructose‐1,6‐bisphosphatase and corn NADP‐dependent malate dehydrogenase activities in the light‐activation system. This suggests that the suppression of the negative charge improves the reactivity of thioredoxin with chloroplast enzymes such as fructose‐1,6‐bisphosphatase and ferredoxin‐dependent thioredoxin reductase.
The open/closed conformational equilibrium of aspartate aminotransferase
Authors: PICOT Daniel , SANDMEIER Erika , THALLER Christina , VINCENT Michael G. , CHRISTEN Philipp , JANSONIUS Johan N.
Published: March 2005

Aspartate aminotransferase undergoes major shifts in the conformational equilibrium of the protein matrix during transamination. The present study defines the two conformational states of the enzyme by crystallographic analysis, examines the conditions under which the enzyme crystallizes in each of these conformations, and correlates these conditions with the conformational behaviour of the enzyme in solution, as monitored by a fluorescent reporter group.

Cocrystallization of chicken mitochondrial aspartate aminotransferase with inhibitors and covalent coenzyme‐substrate adducts yields three different crystal forms. Unliganded enzyme forms triclinic crystals of the open conformation, the structure of which has been solved (space group P1) [Ford, G. C., Eichele, G. & Jansonius, J. N. (1980) Proc. Natl Acad. Sci. USA 77, 2559–2563; Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H. & Christen, P. (1984) J. Mol. Biol. 174, 487–525]. Complexes of the enzyme with dicarboxylate ligands form monoclinic or orthorhombic crystals of the closed conformation. The results of structure determinations of the latter two crystal forms at 0.44 nm resolution are described here. In the closed conformation, the small domain has undergone a rigid‐body rotation of 12–14° which closes the active‐site pocket.

Shifts in the conformational equilibrium of aspartate aminotransferase in solution, as induced by substrates, substrate analogues and specific dicarboxylic inhibitors, can be monitored by changes in the relative fluorescence yield of the enzyme labelled at Cys166 with monobromotrimethylammoniobimane. The pyridoxal and pyridoxamine forms of the labelled enzyme show the same fluorescence properties, whereas in the apoenzyme the fluorescence intensity is reduced by 30%. All active‐site ligands, if added to the labelled pyridoxal enzyme at saturating concentrations, cause a decrease in the fluorescence intensity by 40–70% and a blue shift of maximally 5 nm. Comparison of the fluorescence properties of the enzyme in various functional states with the crystallographic data shows that both techniques probe the same conformational equilibrium.

The conformational change that closes the active site seems to be ligand‐induced in the reaction of the pyridoxal form of the enzyme and syncatalytic in the reverse reaction with the pyridoxamine enzyme.
Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae
Authors: STEVEN John , COTTINGHAM Ian R. , BERRY Sarah J. , CHINERY Simon A. , GOODEY Andrew R. , COURTNEY Michael , BALLANCE D. James
Published: March 2005

Expression of plasminogen activator inhibitor 2 (PAI‐2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N‐terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI‐2 was purified by copper‐chelate and anion‐exchange chromatography to > 95% pure and was fully active. The recombinant PAI‐2 formed SDS‐stable complexes with urokinase and tissue‐type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI‐2 (second‐order rate constant for uPA, 2.4 × 106 M−1 s−1, and for two‐chain tPA, 0.7 × 105 M−1 s−1). As is the case for placental PAI‐2, the N‐terminus of the yeast‐derived recombinant PAI‐2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI‐2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.
Separation of the poly(glycerophosphate) lipoteichoic acids of Enterococcus faecalis Kiel 27738, Enterococcus hirae ATCC 9790 and Leuconostoc mesenteroides DSM 20343 into molecular species by affinity chromatography on concanavalin A
Authors: LEOPOLD Klaus , FISCHER Werner
Published: March 2005

This study shows for the first time microheterogeneity of 1,3‐linked poly(glycerophosphate) lipoteichoic acids. The lipoteichoic acids investigated were those of Enterococcus faecalis Kiel 27738 (I), Enterococcus hirae (Streptococcus faecium) ATCC 9790 (II), and Leuconostoc mesenteroides DMS 20343 (III). Lipoteichoic acids II and III are partially substituted by mono‐, di‐, tri‐, and tetra‐α‐D‐glucopyranosyl residues with (1 → 2) interglycosidic linkages. Lipoteichoic acid I is substituted with α‐kojibiosyl residues only. Lipoteichoic acids I and III additionally carry D‐alanine ester.

Lipoteichoic acids were separated on columns of concanavalin‐A—Sepharose according to their increasing number of glycosyl substituents per chain. It was evident that all molecular species are usually glycosylated and that alanine ester and glycosyl residues occur on the same chains. The chain lengths of lipoteichoic acid I and II vary between 9–40 glycerophosphate residues, whereas those of lipoteichoic acid III appear to be uniform (33 → 2 residues). Molecular species differ in the extent of glycosylation but their content of alanyl residues is fairly constant. All lipoteichoic acids contain a small fraction (5–15%) different in composition from the bulk and most likely reflecting an early stage of biosynthesis.

Two procedures for chain length determination of poly(glycerophosphate) lipoteichoic acids are described.
High‐mobility‐group proteins P1, I and Y as substrates of the M‐phase‐specific p34cdc2/cyclincdc13 kinase
Authors: MEIJER Laurent , OSTVOLD Anne‐Carine , WALAAS S. Ivar , LUND Terje , LALAND Søren G.
Published: March 2005

All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M‐phase‐specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA‐binding high‐mobility‐group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG‐like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M‐phase‐specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2‐binding p13suc1‐Sepharose and affinity purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr‐Pro‐Xaa‐Lys/Arg). HMG I is phosphorylated invivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA‐binding proteins HMG I, Y and P1 are natural substrates for the M‐phase‐specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle.
Characterization of sequences involved in mediating degradation of ornithine decarboxylase in cells and in reticulocyte lysate
Authors: ROSENBERG‐HASSON Yael , BERCOVICH Zippi , KAHANA Chaim
Published: March 2005

Mouse ornithine decarboxylase is a 461‐amino‐acid protein that is extremely labile. A set of contiguous inframe deletions were introduced into its C‐terminal hydrophilic region. The resulting mutant proteins were expressed in cos monkey cells using an expression vector based on simian virus 40 (SV40) or by in vitro translation in reticulocyte lysate. The degradation of wild‐type and mutant proteins was determined in transfected cos cells and in a degradation system based on reticulocyte lysate. Deletion mutants lacking segments of the C‐terminus (amino acids 423–461, 423–435, 436–449 and 449–461) were converted into stable proteins in both experimental systems. The mutant lacking amino acids 295–309 was significantly stabilized in transfected cos cells, but was rapidly degraded in reticulocyte‐lysate‐based degradation mix. Our results suggest that the carboxyl‐terminal region encompassing amino acids 423–461 and perhaps also amino acids 295–309 may constitute a signal recognized by the proteolytic machinery that degrades ornithine decarboxylase.
Primary structure of a new actin‐binding protein from human seminal plasma
Authors: SCHALLER Johann , AKIYAMA Kazuko , KIMURA Hiroshi , HESS Daniel , AFFOLTER Michael , RICKLI Egon E.
Published: March 2005

Secretory actin‐binding protein (SABP), a glycoprotein from human seminal plasma, was isolated according to Akiyama and Kimura [Akiyama, K. & Kimura, H. (1990) Biochim. Biophys. Acta 1040, 206–210]. The complete amino acid sequence of SABP was determined with the aid of fragments generated by trypsin, Staphylococcus aureus V8 protease and pepsin. The single polypeptide chain of SABP contains 118 amino acids with a calculated Mr of 13506 and pyroglutamic acid as the N‐terminal residue. A single N‐glycosidic carbohydrate moiety is located at Asn77. The carbohydrate composition shows an unusually high amount of fucose. The arrangement of the two disulfide bonds is Cys37–Cys63 and Cys61–Cys95.

Sequence comparison revealed a high degree of similarity with a 14‐kDa submandibular gland protein from mouse (45% identity and 64% similarity). SABP is identical with a prolactin‐inducible protein and a protein termed gross cystic disease fluid protein 15 (sequences translated from cDNA clones), both from human breast tissues.

Although SABP was also detected in saliva, in extracts of the submandibular gland and seminal vesicles, little is known of its function.
An alternatively processed mRNA generated from human hepatocyte growth factor gene
Authors: MIYAZAWA Keiji , KITAMURA Akiko , NAKA Daiji , KITAMURA Naomi
Published: March 2005

We recently reported the isolation and sequencing of cDNA for human hepatocyte growth factor (hHGF) [Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. & Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967–973]. In the present study, we report the sequence of another cDNA clone for a shorter form of hHGF mRNA. Comparison of the sequence with that of the hHGF cDNA revealed that the two sequences are identical in their 5′ ends up to 865 nucleotides downstream from the translation‐initiation site, then completely diverge from each other. By Northern blot analysis, the hHGF‐related 1.5‐kb mRNA, which corresponded to the newly isolated cDNA variant, was identified in human placenta. Sequence analysis of a human genomic HGF clone showed that the diverged 3′‐terminal portion of the mRNA is generated by alternative RNA‐processing events utilizing a specific exon. The mRNA could encode a short hHGF molecule of 290 amino acids corresponding to the N‐terminal portion of hHGF which consists of 728 amino acids. In order to examine the effect of the predicted translation product on hepatocyte growth, an expression plasmid for the cDNA variant was constructed and transfected into Cos cells. Immunoblotting analysis showed that the transfected Cos cells produced a protein of about 33 kDa. The protein product did not stimulate DNA synthesis by rat hepatocytes in primary culture.
Isolation and analysis of the human 46‐kDa mannose 6‐phosphate receptor gene
Authors: KLIER Hans‐Jürgen , von FIGURA Kurt , POHLMANN Regina
Published: March 2005

From a genomic library in EMBL 3, two overlapping clones for the human 46‐kDa mannose 6‐phosphate receptor (MPR46) were isolated, which span the entire coding sequence. The human MPR46 gene is distributed over 12 kb and is divided into seven exons (110–1573 bp). All the intron/exon borders agree with the consensus sequences of splice junctions. Exon 1 codes for a 5′ untranslated sequence. The ATG initiation codon begins with the second nucleotide in exon 2. A signal sequence of 26 amino acid residues is followed by the extracytoplasmic (luminal) domain, which extends to exon 5. The transmembrane domain of the receptor spans exons 5 and 6 and the cytoplasmic domain is encoded by exons 6 and 7. The latter domain also codes for an extended 3′ untranslated sequence.

The transcription‐initiation site was defined by primer extension. The sequence upstream of the cap site has strong promoter activity and contains structural elements characteristic of promoters found in housekeeping genes.

No correlation between the genomic organization and known protein domains of the MPR46 was apparent. Moreover, the sequence of about 150 amino acids within the luminal domain of MPR46, which is homologous to the 15 repeats that constitute the luminal domain of the 300‐kDa mannose 6‐phosphate receptor (MPR300), does not correlate with intron/exon borders. MPR46 and MPR300 have therefore diverged from a common ancestral gene before introduction of the present intron sequences.