bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and properties of phosphoribosyl‐diphosphate synthetase from Bacillus subtilis
Authors: ARNVIG Kirsten , HOVE‐JENSEN Bjarne , SWITZER Robert L.
Published: March 2005

Phosphoribosyl‐diphosphate (PPRibP) synthetase from Bacillus subtilis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthetase, on a plasmid. The Mr of the subunit (34000) and its amino‐terminal amino acid sequence (14 residues) were in complete agreement with expectations from the nucleotide sequence of the prs gene. The Mr of the native enzyme (280000 ± 10000) was consistent with an octameric quaternary structure. No tendency toward multiple states of aggregation of the enzyme was seen. The purified enzyme required Mg2+ and inorganic phosphate for activity; Mn2+ supported only 30% the activity seen with Mg2+. Michaelis constants for ATP and ribose 5‐phosphate (Rib5P) were 0.66 mM and 0.48 mM, respectively. Of several end products tested, only ADP was strongly inhibitory; GDP was a weak inhibitor. ADP inhibition displayed homotropic cooperativity and was enhanced by increasing saturation of the enzyme with ATP. These observations strongly suggest a specific allosteric site for ADP binding. A comparison of physical and kinetic properties of bacterial and mammalian PPRibP synthetases is presented.
Phosphorylation of connexin 32, a hepatocyte gap‐junction protein, by cAMP‐dependent protein kinase, protein kinase C and Ca2+/calmodulin‐dependent protein kinase II
Authors: SAEZ Juan C. , NAIRN Angus C. , CZERNIK Andrew J. , SPRAY David C. , HERTZBERG Elliot L. , GREENGARD Paul , BENNETT Michael V. L.
Published: March 2005

Phosphorylation of connexin 32, the major liver gap‐junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP‐dependent protein kinase (cAMP‐PK), by protein kinase C (PKC) and by Ca2+/calmodulin‐dependent protein kinase II (Ca2+/CaM‐PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP‐PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. Ca2+/CaM‐PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL‐amine (residues 228–239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP‐PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM‐PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP‐PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20‐deoxy‐20‐oxophorbol 12,13‐dibutyrate (PDBt) resulted in increased 32P‐incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP‐PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap‐junction proteins.
Heat capacity and entropy changes of troponin C from bullfrog skeletal muscle induced by calcium binding
Authors: IMAIZUMI Masamoto , TANOKURA Masaru
Published: March 2005

In order to elucidate the structural changes of bullfrog skeletal muscle troponin C (TnC) caused by Ca2+ binding, microcalorimetric titrations were performed in both the absence and presence of Mg2+, at pH 7.0, and at 5°, 15°, and 25°C. The results indicate that, in the absence of Mg2+, Ca2+ binding to sites 1 and 2 gives rise to a large hydrophobic effect, a sequestering of nonpolar groups on the surface of molecule to the interior, and a tightening of the molecular structure as a whole. In contrast, hydrophobic groups were exposed from the interior to the surface of molecule and the molecular mobility was increased, upon Ca2+ binding to site 3. Ca2+ binding to site 4 induced much less change in the conformation of TnC molecule than that to the other sites. The presence of 5 mM Mg2+ dramatically reduced the magnitude of the conformational changes in TnC on Ca2+ binding to sites 1 and 2. On the other hand, the conformational changes by Ca2+ binding to sites 3 and 4 were not affected by Mg2+.

Ca2+ binding to site 3 of TnC, thus, causes the conformational changes distinctly different from those upon Ca2+ binding to the other sites. The conformational changes upon Ca2+ binding to site 3 of TnC are also clearly distinct from the Ca2+ ‐induced conformational changes of parvalbumins, but are similar to those of calmodulin. Site 3 of TnC may, therefore, be the regulatory site in muscle contraction, and the exposure or generation of hydrophobic groups on the surface of molecule as well as the increase in molecular mobility may be the common characteristics of the regulation by Ca2+ ‐binding proteins.
Fructose 1‐phosphate and the regulation of glucokinase activity in isolated hepatocytes
Authors: DAVIES Dewi R. , DETHEUX Michel , VAN SCHAFTINGEN Emile
Published: March 2005

Fructose 1‐phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1‐phosphate and fructose.

The concentration of fructose 1‐phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05–1 mM) caused a concentration‐dependent and transient increase in the fructose 1‐phosphate content. Glucagon (1 μM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1‐phosphate in cells incubated with fructose, whereas the addition of 0.1 μM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1‐phosphate concentration.

Sorbitol had a similar effect to fructose in causing the formation of fructose 1‐phosphate. D‐Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier. The effect of D‐glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon.

Both fructose and D‐glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3H2O from [2‐3H]glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier. Glucagon and ethanol antagonised the effect of low concentrations of fructose or D‐glyceraldehyde on the detritiation of glucose. These results support the proposal that fructose 1‐phosphate mediates the effects of fructose, D‐glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179–184].
Primary structure of sorghum malate dehydrogenase (NADP) deduced from cDNA sequence
Authors: CRÉTIN Claude , LUCHETTA Philippe , JOLY Cécile , DECOTTIGNIES Paulette , LEPINIEC Loïc , GADAL Pierre , SALLANTIN Marc , HUET Jean‐Claude , PERNOLLET Jean‐Claude
Published: March 2005

Malate dehydrogenase (NADP) (NADP‐MDH) is an important enzyme of the photosynthetic CO2 fixation pathway of C4 plants. We have isolated two clones from a sorghum λgt11 cDNA library (CM3, 932 bp, and CM7, 1441 bp). Nucleotide sequence analysis of the cDNAs CM3 and CM7 showed the existence of two NADP‐MDH mRNA species encoding different enzyme subunits. Microsequencing of the N‐terminus of the mature protein indicated that a specific cleavage of 13 amino acids occurred during the purification steps of the enzyme. The full‐length cDNA CM7 contains a large open reading frame encoding an NH2‐terminal transit peptide of 40 amino acids and a mature protein of 389 amino acids (42.207 kDa). Alignment of the NADP‐MDH sequence with those of several malate dehydrogenases revealed some similarities with NAD‐MDHs.
Fluoroaluminates do not affect the guanine‐nucleotide binding centre of the peptide chain elongation factor EF‐Tu
Authors: KRAAL Barend , de GRAAF J. Martien , MESTERS Jeroen R. , van HOOF Peter J. M. , JACQUET Eric , PARMEGGIANI Andrea
Published: March 2005

EF‐Tu is often referred to as a model for guanine‐nucleotide‐binding regulatory proteins (G‐proteins), since X‐ray diffraction analysis of its GTP‐binding domain shows a detailed location of the ‘consensus’ amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the γ‐phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF‐Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed AIF−4 behaves as a γ‐phosphate analogue in G‐proteins, then EF‐Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences.
A novel tubulin from Mimosa pudica
Authors: PAL Mita , ROYCHAUDHURY Asis , PAL Amita , BISWAS Susweta
Published: March 2005

From Mimosa pudica fresh leaves and pulvinar callus cells, we have purified tubulin protein using an anion‐exchange resin, DEAE‐Sephadex A‐50, followed by ammonium sulfate fractionation and Sephadex G‐200 gel filtration. The purified protein consisted of α and β subunits and trace quantities of other proteins. When analysed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis at pH 8.8, both α and β subunits of tubulin almost comigrated with their counterparts from goat brain. Two‐dimensional electrophoresis revealed that this tubulin contains one major α‐tubulin having a pI value of 7.1 and three β species having pI values of 6.70, 6.46 and 6.40. Morphologically normal microtubules were observed by electron microscopy; self‐polymerization in vitro, even in the cold, can also be achieved. Radioimmunoassays, and also immunoblotting with the antibodies raised against α‐ and β‐tubulins of this plant, showed that the nature of α‐tubulin is different from that obtained from other sources. This is an example of plant tubulin where strong colchicine binding at 1 μM was observed. This protein constitutes 5–6% of the total extractable protein in the leaves. We propose that movement of the leaves of this plant may be regulated by the presence of a high amount of this protein.
Control analysis of systems having two steps catalyzed by the same protein molecule in unbranched chains
Authors: CASCANTE Marta , CANELA Enric I. , FRANCO Rafael
Published: March 2005

The analysis of the control of a metabolic pathway having an enzyme catalyzing two different reactions (or a protein displaying two different activities) has been performed. For such systems although the summation theorems are valid, the flux and concentration connectivity theorems of the metabolic control analysis are not valid. Another general relationship of control analysis is shown to be more widely obeyed and holds in these systems. An exemplary case, where the enzyme catalyzes two irreversible reactions, demonstrates that the level of one internal intermediate is constant, i.e. it does not depend upon the independent variables of the system.
Conformational preferences of [Leu5]enkephalin in biomimetic media
Authors: PICONE Delia , D'URSI Annamaria , MOTTA Andrea , TANCREDI Teodorico , TEMUSSI Piero A.
Published: March 2005

The conformation of [Leu5]enkephalin has been studied by 1H‐NMR spectroscopy in media more like the actual environment in which the agonist‐receptor interaction takes place than water, i.e. in three cryoprotective mixtures (dimethylformamide/water, methanol/water and ethylene glycol/water), in aqueous SDS and in two neat solvents, dimethylformamide and acetonitrile, whose dielectric constants (36.7 and 37.5) are intermediate between that of water and that of the lipid phase. In all cases examined, contrary to the studies in water or dimethylsulfoxide, we were able to detect numerous nuclear Overhauser effects, indicating that the media employed favour well‐defined structures and/or reduce the internal motions of the peptide.

Data from both organic solvents and cryoprotective mixtures suggest a 4 → 1 β turn as the most probable structure of [Leu5]enkephalin in solution, whereas in SDS/H2O micelles the structural picture appears completely different, suggesting the presence of a 5 → 2 β turn. The existence of two different preferred conformations of enkephalins may possibly be related to their ability to be effective towards both μ and δ opioid receptors.
Sequence of proton abstraction and stereochemistry of the reaction catalyzed by naphthoate synthase, an enzyme involved in menaquinone (vitamin K2) biosynthesis
Authors: IGBAVBOA Urule , LEISTNER Eckhard
Published: March 2005

The enzymic conversion of the coenzyme A ester of 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid (i. e.o‐succinylbenzoic acid) to 1,4‐dihydroxy‐2‐naphthoic acid is a cyclization reaction which is part of menaquinone (vitamin K2) biosynthesis. This conversion, which is probably a two‐step process, was investigated using chirally labelled samples of the coenzyme A ester of 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid. To synthesize these, the following enzymes were employed: isocitrate: NADP+ oxidoreductase (EC 1.1.1.42), isocitrate glyoxylate‐lyase (EC 4.1.3.1), 2‐oxoglutarate dehydrogenase complex (which includes EC 1.2.4.2), 4‐(2′‐carboxyphenyl)‐4‐oxobutyrate synthase system and 4‐(2′‐carboxyphenyl)‐4‐oxobutyrate: CoA ligase.

Isocitrate: NADP+ oxidoreductase was employed to generate the two enantiometric samples of 2‐oxoglutarate enantiotopically labelled at C‐3. These samples were converted enzymically to succinate with retention of configuration at C‐2 and C‐3, and to 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid with retention of configuration at C‐3. Isocitrate glyoxylate‐lyase and isocitrate NADP+ oxidoreductase were employed to generate samples of 2‐oxoglutarate enantiotopically tritiated at C‐4 or at C‐3 and C‐4. The four variously labelled samples of 2‐oxoglutarate were enzymically converted to the coenzyme A ester of 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid.

The resulting variously labelled coenyzme A esters were incubated with naphthoate synthase to investigate the ring closure reaction. In the first step the 2HRc atom of the oxobutvric moiety of the coenzyme A ester is equilibrated with solvent protons in a fast and reversible reaction. Subsequently the 2HSi and 3HSi atoms are removed whereas the 3HRe atom becomes the proton at C‐3 of 1,4‐dihydroxy‐2‐naphthoic acid. The second step in this ring closure reaction is the rate‐limiting step.
Expression and characterization of the chick nicotinic acetylcholine receptor α‐subunit in insect cells using a baculovirus vector
Authors: ATKINSON Allan E. , EARLEY Fergus G. P. , BEADLE David J. , KING Linda A.
Published: March 2005

A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) α‐subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co‐transfection of Spodoptera frugiperda cells with infectious, wild‐type AcNPV DNA and the transfer vector. Polyhedrin‐negative, recombinant viruses were identified which expressed the nAChR α‐subunit. The insect cell‐expressed α‐subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin‐binding assays. The levels of expression were low, approximately 1 – 2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor α‐subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR α‐subunit and carried the binding site for the snake venom toxin, α‐bungarotoxin. Bound α‐bungarotoxin was displaced in competition binding assays by α‐cobra toxin, carbamylcholine and d‐tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR α‐subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound α‐bungarotoxin.
Internalization of the cytotoxic molecules of T101 F(ab′)2— (ricin‐A‐chain) immunotoxin into human T‐leukemic cells
Authors: RAVEL Sophie , CASELLAS Pierre
Published: March 2005

We have investigated the internalization step of an immunotoxin and its relationship with cytotoxicity, with the F(ab′)2—T101(ricin‐A‐chain) immunotoxin, directed against the CD5 antigen expressed on leukemic CEM cells.

We first demonstrated that the biological action of the conjugate was related to its entry into the cell by an energy‐dependent endocytotic process. We also found that during the first hours of cell intoxication, internalization is not the rate‐limiting step of immunotoxin cytotoxicity. Internalization becomes limiting in cell intoxication only when the entry rate is low.

Lastly we show that ammonium chloride, which strongly enhances immunotoxin potency, acts on internalized molecules for a very short time, suggesting that this enhancer affects an early intracellular step.
The multifunctional 6‐methylsalicylic acid synthase gene of Penicillium patulum
Authors: BECK Joachim , RIPKA Sabine , SIEGNER Axel , SCHILTZ Emil , SCHWEIZER Eckhart
Published: March 2005

6‐Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150‐fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P. patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322‐bp‐long open reading frame coding for a protein of 1774 amino acids and 190731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5′ and 3′ portions of the gene. The same cDNA sequences revealed the presence of a 69‐bp intron within the N‐terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5′‐ and 3′‐splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Saccharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2‐oxoacyl reductase, 2‐oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS‐ and FAS‐specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. patulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.
Primary structure of the bovine analogues to human apolipoproteins CII and CIII
Authors: BENGTSSON‐OLIVECRONA Gunilla , SLETTEN Knut
Published: March 2005

Two major isoforms of the bovine analogue to human apolipoprotein (apo) CII were purified from plasma. They were both as effective as human apo CII in activating lipoprotein lipase. Amino acid sequencing revealed that one form contained 79 amino acid residues, and corresponded to human pro apo CII. The other form lacked the first six residues at its N‐terminus. This was apparently due to cleavage of the ‐Gln‐Asp‐ linkage in the sequence H2N‐Ala‐His‐Val‐Pro‐Gln‐Gln‐Asp‐Glu‐, analogous to cleavages described for human apo AI and apo CII. Previous studies with human apo CII have shown that the ability to activate lipoprotein lipase resides in the C‐terminal third of the molecule. This was highly conserved in the bovine analogue: of the 30 last residues, 21 are identical. Five residues in this part of human apo CII have been reported to be essential for activation of lipoprotein lipase. Only one of these, Tyr63, is present in the bovine sequence. The bovine structure contains a threonine at position 61, instead of serine in the human, and the four last residues are ‐Ser‐Gly‐Lys‐Asp instead of the allegedly necessary ‐Lys‐Gly‐Glu‐Glu. Three differently sialylated isoforms of the bovine analogue to human apolipoprotein CIII were also isolated and partially sequenced. All three lacked the first three N‐terminal residues as compared to sequences from other species (man, dog and rat). Sequence differences were more pronounced at the ends than in the central parts of the apo CIII molecules.
The spectrum of N‐linked oligosaccharide structures detected by enzymic microsequencing on a recombinant soluble CD4 glycoprotein from Chinese hamster ovary cells
Authors: YUEN Chun‐Ting , CARR Steven A. , FEIZI Ten
Published: March 2005

Structures of the N‐linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N‐glycosylation sites, Asn271 and Asn300, at both of which evidence for the presence of complex type biantennary sialo‐oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S.A., Hemling, M.E., Folena‐Wasserman, G., Sweet, R. W., Anumula, K., Barr, J. R., Huddleston, M. J. & Taylor, P. (1989) J. Biol. Chem. 264, 21286–21295]. Among oligosaccharides released from sCD4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase‐treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase‐treated oligosaccharides of sCD4 as follows:

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where ± indicates residues present on only a proportion of chains. The spectrum of oligosaccharide structures released from each glycosylation site was assessed as being similar to that of total oligosaccharides on the basis of their chromatographic profiles on the lectin columns and on Bio‐Gel P‐4.
cDNA cloning of human‐milk bile‐salt‐stimulated lipase and evidence for its identity to pancreatic carboxylic ester hydrolase
Authors: NILSSON Jeanette , BLÄCKBERG Lars , CARLSSON Peter , ENERBÄCK Seven , HERNELL Olle , BJURSELL Gunnar
Published: March 2005

We have isolated and sequenced cDNA clones covering the entire coding sequence of human‐milk bile‐salt‐stimulated lipase, as well as 996 nucleotides of the 3′ end of the pancreatic enzyme carboxylic ester hydrolase. The deduced amino acid sequence of the lipase starts with a 23‐residue leader peptide. The open reading frame continues with 722 amino acid residues. The sequence contains in the C‐terminal part a proline‐rich repeat, 16 repeats of 11 amino acid residues each. The mRNA was estimated to be approximately 2500 nucleotides from Northern blot and of similar size in mammary and pancreatic tissues. Data obtained indicate that the lipase and the carboxylesterase are identical and coded for by the same gene. The cDNA is 2428 bases long, which indicates that a near full‐length copy of the transcript has been isolated. Comparisons with other enzymes show that the lipase is a new member of the supergene family of serine hydrolases. It is not only closely related (and in its N‐terminal half virtually identical) to lysophospholipase from rat pancreas and cholesterol esterase from bovine pancreas, but also shows a high degree of similarity to several esterases, e.g. acetylcholine esterase. In contrast, no such similarity could be found to typical lipases.
Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method
Authors: RĘDOWICZ Maria J. , SOBIESZEK Apolinary , STRZELECKA‐GOŁASZEWSKA Hanna
Published: March 2005

It was previously shown that tryptic digestion of subfragment 1 (S1) of skeletal muscle myosins at 0°C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH2‐terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25°C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH2‐terminus is involved in the communication between the sites of nucleotide and actin binding.

We also show here for the first time that the S1–S2 junction in gizzard myosin can be cleaved by chymotrypsin and that this cleavage (observed in papain‐produced S1 devoid of the regulatory light chain) is also temperature‐dependent but insensitive to nucleotides and actin. It is suggested that the temperature‐dependent alteration in the flexibility of the head‐rod junction, which is apparent from these and similar observations on skeletal muscle myosin [Miller, L. & Reisler, E. (1985) J. Mol. Biol. 182, 271–279; Rȩdowicz, M. J. & Strzelecka‐Golaszewska, H. (1988). Eur. J. Biochem. 177, 615–624], may contribute to the temperature dependence of some steps in the cross‐bridge cycle.
Primary structure of the oligo‐1,6‐glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene
Authors: WATANABE Kunihiko , KITAMURA Kazuhisa , IHA Hidekatsu , SUZUKI Yuzuru
Published: March 2005

The gene coding for Bacillus cereus ATCC7064 (mesophile) oligo‐1,6‐glucosidase was cloned within a 2.8‐kb SalI—EcoRI fragment of DNA, using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. E. coli C600 bearing the hybrid plasmid pBCE4 accumulated oligo‐1,6‐glucosidase in the cytoplasm. The cloned enzyme coincided absolutely with B. cereus oligo‐1,6‐glucosidase in its Mr (65000), in its electrophoretic behavior on a polyacrylamide gel with or without sodium dodecyl sulfate, in its isoelectric point (4.5), in the temperature dependence of its stability and activity, and in its antigenic determinants.

The nucleotide sequence of B. cereus oligo‐1,6‐glucosidase gene and its flanking regions was determined with both complementary strands of DNA (each 2838 nucleotides). The gene consisted of an open reading frame of 1674 bp commencing with a ATG start codon and followed by a TAA stop codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 558 amino acid residues with a Mr of 66010. The amino acid composition and Mr were comparable with those of B. cereus oligo‐1,6‐glucosidase. The predicted N‐terminal sequence of 10 amino acid residues agreed completely with that of the cloned ligo‐1,6‐glucosidase.

The deduced amino acid sequence of B. cereus oligo‐1,6‐glucosidase was 72% and 42% similar to those from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) oligo‐1,6‐glucosidase and from Saccharomyces carlsbergensis CB11 α‐glucosidase, respectively. Predictions of protein secondary structures along with amino acid sequence alignments demonstrated that B. cereus oligo‐1,6‐glucosidase may take the similar (α/β)8‐barrel super‐secondary structure, a barrel of eight parallel β‐strands surrounded by eight α‐helices, in its N‐terminal active site domain as S. carlsbergensisα‐glucosidase and Aspergillus oryzaeα‐amylase.
Inhibition of human immunodeficiency virus 1 reverse transcriptase by 3′‐azidothymidine triphosphate
Authors: HEIDENREICH Olaf , KRUHØFFER Mogens , GROSSE Frank , ECKSTEIN Fritz
Published: March 2005

In the presence of oligo(dT) · poly(rA) as primer‐template, 3′‐azidothymidine triphosphate (N33′‐ddTTP) is a substrate for human immunodeficiency virus 1 reverse transcriptase with an apparent Km value of 3.0 μM. This compares with an apparent Km for thymidine monophosphate (dTMP) incorporation of 2.5 μM. The apparent Vmax value for 3′‐azidothymidine monophosphate (N33′‐ddTMP) incorporation is 50 times lower than that of dTMP incorporation. Kinetic analysis of the inhibition of reverse transcriptase by N33′‐ddTTP shows competitive inhibition with thymidine triphosphate (dTTP) with a Ki of 41 nM and an uncompetitive pattern of inhibition with template‐primer having a Ki of 140 nM. This indicates incorporation of the analogue into the primer and inhibition of the enzyme by formation of a dead‐end complex. The 3′‐azidothymidine‐terminated primer‐template [N33′‐ddT‐(dT)15· poly(rA)] is a potent competitive inhibitor versus primer‐template with a Ki of 2.4 nM and shows mixed‐type inhibition against dTTP with a Ki of 8 nM. The low inhibition constant for this chain‐terminated primer suggests that such oligonucleotides can act as potent inhibitors of enzyme catalysis.
Direction of the DNA‐unwinding reaction catalysed by Escherichia coli DNA helicase II
Authors: GEORGI‐GEISBERGER Petra , HOFFMANN‐BERLING Hartmut
Published: March 2005

The direction of the DNA‐unwinding reaction catalysed by Escherichia coli DNA helicase II was studied using gapped linear DNA molecules with short duplex ends as substrate. The results suggest that DNA helicase II unwinds with 3′– 5′ polarity relative to the single strand of the DNA partial duplex. At high enzyme DNA ratio the enzyme also unwinds the duplex connected to the 3′ end of the single strand and, as further studies show, fully duplex linear DNA. The fraction of DNA unwound decreases as the length of the duplex substrate increases. The preference of DNA helicase II for a short duplex can obscure the fact that the typical substrate is duplex connected to the 5′ end of a single strand.
Site‐specific N‐glycosylation of ovine lutropin
Authors: WEISSHAAR Gerhard , HIYAMA Jun , RENWICK Alistair G. C.
Published: March 2005

The Asn‐linked carbohydrate structures of the heterodimeric glycoprotein hormone lutropin from ovine pituitary glands have been investigated at each of its three glycosylation sites using one‐ and two‐dimensional 400‐MHz 1H‐NMR spectroscopy.

Highly purified, biologically active ovine lutropin (oLH) was dissociated and separated into its α and β subunits (oLHα, glycosylated at Asn56 and Asn82; oLHβ glycosylated at Asn13). Oligosaccharides from intact oLHβ and from glycopeptides obtained after tryptic digestion of oLHα were released by hydrazinolysis and subsequently fractionated according to charge and size by anion‐exchange and ion‐suppression amine‐adsorption HPLC, respectively. 1H‐NMR analysis revealed, that monosulphated, mostly hybrid‐type, oligosaccharides predominate at both glycosylation sites of oLHα, whereas a disulphated, diantennary N‐acetyllactosamine‐type structure accounts for more than 60% of total oligosaccharides in the β subunit. Furthermore, the saccharides attached to the β subunit are almost completely fucosylated (Fucα1–6) at the reducing terminal GlcNAc, whereas the sugar chains in oLHα are either approximately 50% fucosylated (Asn82) or contain fucose only to a minor extent (Asn56).

The results clearly indicate a distinct subunit‐ and site‐specific synthesis of oligosaccharides in ovine lutropin and suggest that biosynthesis is effectively influenced by the surrounding polypeptide chain(s) at a given site.
Primary structure of profilins from two species of Echinoidea and Physarum polycephalum
Authors: TAKAGI Takashi , MABUCHI Issei , HOSOYA Hiroshi , FURUHASHI Kiyoshi , HATANO Sadashi
Published: March 2005

Profilin is a small G‐actin‐binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast. Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined. Two echinoid profilins were composed of 139 amino acid residues, N‐termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE [Mabuchi, I. & Hosoya, H. (1982) Biomed. Res. 3, 465–476]. On the other hand, Physarum profilin was composed of 124 amino acid residues, the N‐terminus was acylated, and the calculated molecular mass was 13132 Da.

The sequences of C. japonicus and A. crassispina profilins were homologous (84% identical). However, the similarity of these profilins with those from other organisms was low. The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins.

The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N‐terminal region, which was suggested to be a common actin‐binding region. The C‐terminal region was also conserved, although to a lesser extent than the N‐terminal region.
Specific interaction of vitronectin with the cell‐secreted protease inhibitor glia‐derived nexin and its thrombin complex
Authors: ROVELLI Giorgio , STONE Stuart R. , PREISSNER Klaus T. , MONARD Denis
Published: March 2005

Interaction of vitronectin with glia‐derived nexin (GDN), thrombin, and the complex GDN‐thrombin was demonstrated in direct binding assays that indicated the formation of binary and ternary complexes. The concentration of vitronectin necessary to obtain 50% saturation of the immobilized GDN‐thrombin complex binding sites (EC50) was about 1 nM. Under similar experimental conditions, the EC50 of vitronectin for the immobilized antithrombin‐III–thrombin complex was about fivefold higher. A tight complex was also formed between vitronectin and immobilized GDN (EC50∼ 1.5 nM) but when vitronectin was immobilized, GDN displayed a reduced affinity for vitronectin (EC50∼ 10 nM). These results suggest differences between the immobilized and free conformations of GDN and/or vitronectin. In contrast, vitronectin displayed negligible affinity for antithrombin III. Biotinylated GDN was used to characterize further the binding of GDN or the GDN‐thrombin complex to vitronectin. The interaction of the biotinylated GDN‐thrombin complex with immobilized vitronectin (EC50∼ 2 nM) was completely blocked by nonbiotinylated complexes of thrombin with either GDN or antithrombin III, whereas free GDN, free thrombin and the GDN‐trypsin complex were only weak competitors. Activesite‐blocked urokinase and the complex GDN‐urokinase also strongly competed for binding of the biotinylated GDN‐thrombin complex to vitronectin. Binding of biotinylated GDN to immobilized vitronectin was specific, saturable and was competed with decreasing efficiency by the GDN‐thrombin complex, free GDN and free antithrombin III. These interactions between the adhesive component vitronectin and the serine protease inhibitor GDN may relate to localized control of thrombin and/or urokinase action at certain extravascular sites. These results are discussed in terms of binding sites for vitronectin on GDN, thrombin, and the GDN‐thrombin complex.
Characterization of the transcription‐initiation site and of the promoter region within the 5′ flanking region of the human aldolase C gene
Authors: BUONO Pasqualina , MANCINI Francesco P. , IZZO Paola , SALVATORE Francesco
Published: March 2005

Several aldolase C clones from a human genomic library have been identified using a mouse aldolase C cDNA as a hybridization probe. The most complete fragment of the clones identified is 14 kb long and contains the complete aldolase C gene. The nucleotide sequence analysis of more than 5 kb includes the intron/exon organization structure of the gene and the 3′ and 5′ flanking regions. Although no human cDNA is yet available, a canonical polyadenylation signal at the 3′ end of the gene indicates the proximity of the poly(A) addition site.

We have analyzed the 5′ noncoding region by S1 mapping and primer‐extension experiments. The transcription‐initiation site for the human aldolase C gene in brain tissue was located about 1300 bp upstream from the methionine initiation codon.

Preliminary functional assays of the promoter by transfection into rat glioma cells have indicated that promoter elements lie between positions −161 and −416 from the start point of transcription.
Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus
Authors: NAKAYA Kazuyasu , OMATA Kumiko , OKAHASHI Ikuko , NAKAMURA Yasuharu , KOLKENBROCK Hansjörg , ULBRICH Norbert
Published: March 2005

A very basic secreted protein whch displays antifungal activity was isolated from the medium of the mold Aspergillus giganteus. The protein consists of 51 amino acid residues whose sequence was determined as Ala ‐Thr‐Tyr‐Asn‐Gly‐Lys‐Cys‐Tyr‐Lys‐Lys‐Asp‐Asn‐Ile‐Cys‐Lys‐Tyr‐Lys‐Ala‐Gln‐Ser‐Gly‐Lys‐Thr‐Ala‐Ile‐Cys‐Lys‐Cys‐Tyr‐Val‐Lys‐Lys‐Cys‐Pro‐Arg‐Asp‐Gly‐Ala‐Lys‐Cys‐Glu‐Phe‐Asp‐Ser‐Tyr‐Lys‐Gly‐Lys‐Cys‐Tyr‐Cys. Disulfide bonds were formed between Cys7–Cys33, Cys14–Cys40, Cys26–Cys28 and Cys49–Cys51. These results suggest that the antifungal protein forms a loop structure and is similar to phospholipase A2.
Kinetic analysis of the human erythrocyte glyoxalase system using 1H NMR and a computer model
Authors: RAE Caroline , BERNERS‐PRICE Susan J. , BULLIMAN Brian T. , KUCHEL Philip W.
Published: March 2005

1H NMR was used with methylglyoxal, purified by an HPLC technique, to study the kinetics of the human erythrocyte glyoxalase system. 1H NMR enabled the direct measurement of the time‐dependent changes in concentrations of the two hydrates of methylglyoxal, which have not previously been directly measurable, as well as measurement of substrates and products of the glyoxalase enzyme system in the human red blood cell. A computer model of the reaction scheme was developed and NMR data numerically analyzed, thus allowing a complete kinetic description of the reactions. The rate constants describing the chemical equilibria between the hydrated species of methylglyoxal were determined by this numerical analysis or by a saturation‐transfer technique, and found to be much slower (by several orders of magnitude) than previously determined by other methods. The kinetic parameters describing the enzyme‐catalyzed reactions were also determined from experiments using a dilute haemolysate that was added to solutions of methylglyoxal and reduced glutathione (GSH). The maximal velocity of glyoxalase 1 is threefold greater (Vmax= 70.4 ± 4.7 mmol · min−1· 1 packed cells−1) than glyoxalase 2 (Vmax= 24 ± 5 mmol · min−1· 1 packed cells−1) and it exhibits threefold‐greater affinity for its substrate (Km= 0.46 ± 0.04 mM) than the second enzyme (Km= 1.5 ± 0.4 mM). Both enzymes are subject to competitive inhibition; glyoxalase 1 by reduced glutathione (KGSHi= 7.88 ± 0.16 mM) and glyoxalase 2 by the hemithioacetal (HTA) of methylglyoxal and GSH (KHTAi= 0.29 ± 0.04 mM).
The three cysteine residues of cytoplasmic aspartyl‐tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity
Authors: KERN Daniel , MEJDOUB Hafedh , VINCENDON Pascale , BOULANGER Yves , REINBOLT Joseph
Published: March 2005

Cytoplasmic aspartyl‐tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63 000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence). Thiolspecific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions. Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N‐ethylmaleimide and 5,5′‐dithiobis(2‐nitrobenzoate) reacted with Cys512 and Cys255, respectively. Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected. Site‐directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously. For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII λ gene. The resulting substitution in the amino‐terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S. cerevisiae. Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme, ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.
Secretin receptors in the neuroglioma hybrid cell line NG108‐15
Authors: GOSSEN Denis , TASTENOY Michèle , ROBBERECHT Patrick , CHRISTOPHE Jean
Published: March 2005

Secretin receptors in membranes from the neuroblastoma‐glioma hybrid cell line NG108‐15 were investigated by 125I‐secretin binding and adenylate cyclase activation. On both parameters the corresponding relative potencies of parent peptides were, respectively: secretin > helodermin > peptide histidine isoleucinamide = vasoactive intestinal peptide. With secretin analogs and secretin fragments, the order of potency for binding was: secretin = [Val5]secretin > [Ala2]secretin = [Ala11]secretin > [Ala4,Val5]secretin > [Ala4]secretin > [d‐Phe4]secretin > [d‐Phe2]secretin = secretin (2–27) > secretin (3–27) > secretin (7–27). Also, on adenylate cyclase, [d‐Phe4]secretin, [d‐Phe2]secretin, secretin (2–27) and secretin (3–27) were partial agonists while secretin (7–27) was ineffective.

The differentiating agent N6,2′‐O‐dibutyryladenosine 3′,5′‐monophosphate (1 mM) increased the density of secretin receptors and secretin‐stimulated adenylate cyclase activity after a lag period of 4 h. After incubation for 24 h, receptor number and enzyme activity were increased 4‐ and 3‐fold, respectively. These effects were inhibited totally by 1 μg/ml cycloheximide and halved by 5 μg/ml actinomycin D. They were mimicked by 1 mM sodium butyrate but were not reproduced by either 8‐bromoadenosine 3′,5′‐monophosphate or the phosphodiesterase inhibitor rac‐4‐(3‐Butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone.
Transmembrane signaling during erythropoietin‐ and dimethylsulfoxide‐induced erythroid cell differentiation
Authors: KURAMOCHI Satomi , SUGIMOTO Yoshikazu , IKAWA Yoji , TODOKORO Kazuo
Published: March 2005

Erythropoietin is a glycoprotein factor which specifically regulates the proliferation and differentiation of erythroid progenitor cells. We have investigated here the biochemical mechanisms of erythroid differentiation on mouse erythroleukemia SKT6 cells which can be induced to differentiate either with erythropoietin or dimethyl sulfoxide (Me2SO). cAMP‐elevating agents, such as forskolin and 3‐isobutyl‐1‐methyl‐xanthine, caused spontaneous erythroid differentiation, and these agents showed the stimulatory effects on erythropoietin‐ or Me2SO‐induced differentiation. An adenylate cyclase inhibitor, 2′,5′‐dideoxyadenosine, blocked erythropoietin‐induced differentiation. The intracellular cAMP level was rapidly increased by addition of erythropoietin but not by Me2SO. These observations suggest that erythroid differentiation induced by erythropoietin is mediated, at least in part, through the cAMP‐dependent pathway. When the effect of erythropoietin and Me2SO on the intracellular Ca2+ level was examined using fura 2, no acute change was observed. Measurements of the levels of inositol 1,4,5‐trisphosphate and diacylglycerol following stimulation with erythropoietin or Me2SO showed that phosphatidylinositol turnover did not change significantly after erythropoietin stimulation but decreased gradually after Me2SO induction. Taken together, these results indicate that a complex signaling network including the cAMP‐dependent pathway is involved in the erythroid differentiation process.
Geometry of binding of the benzamidine‐ and arginine‐based inhibitors Nα‐(2‐naphthyl‐sulphonyl‐glycyl)‐dl‐p‐amidinophenylalanyl‐piperidine (NAPAP) and (2R,4R)‐4‐methyl‐1‐[Nα‐(3‐methyl‐1,2,3,4‐tetrahydro‐8‐quinolinesulphonyl)‐l‐arginyl]‐2‐piperidine carboxylic acid (MQPA) to human α‐thrombin
Authors: BODE Wolfram , TURK Dušan , STÜRZEBECHER Jörg
Published: March 2005

The X‐ray crystal structure of the trypsin complex formed with Nα‐(2‐naphthyl‐sulphonyl‐glycyl)‐dl‐p‐amidinophenylalanyl‐piperidine (NAPAP) was determined with X‐ray data to 0.18‐nm resolution and crystallographically refined. NAPAP binds into the active site of trypsin in a quite compact form: the p‐amidinophenylalanine moiety of the d‐stereoisomer binds into the specificity pocket; the glycyl group is hydrogen bonded with Gly216; the naphthyl group stands perpendicular to the indole moiety of Trp215; the piperidine ring is tightly packed between this naphthyl moiety and His57; in consequence the carboxy‐terminal amido bond of NAPAP is located in such a way that it is not susceptible to the active‐site Ser195. NAPAP and (2R,4R)‐4‐methyl‐1‐[Nα‐(3‐methyl‐1,2,3,4‐tetrahydro‐8‐quinolinesulphonyl)‐l‐arginyl]‐2‐piperidine carboxylic acid (MQPA) [Matzusaki, T., Sasaki, C., Okumura, C. & Umeyama (1989) J. Biochem. (Tokyo) 105, 949–952] were transferred in their trypsin‐binding conformations to human α‐thrombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R. & Hofsteenge, J. (1989) EMBO J. 8, 3467–3475] and energy minimized. Both synthetic inhibitors fit perfectly into the much more restricted active site of thrombin. The accommodation of the S‐aryl moieties in the ‘aryl‐binding site’ and of the piperidine rings in the S2 subsite of thrombin are particularly favourable. The preference of thrombin for distinctly substituted piperidine derivatives and its generally higher (compared with trypsin) affinity for benzamidine and arginine‐based inhibitors can be accounted for by these thrombin inhibitor models.