bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing the heparin‐binding domain of human antithrombin III with V8 protease
Authors: LIU Chen‐Sheng , CHANG Jui‐Yoa
Published: March 2005

From structural analysis on genetically abnormal and chemically modified human antithrombin III [Koide, T., Odani, S., Takahashi, K., Ono, T. and Sakuragawa, N. (1984) Proc. Natl Acad. Sci. USA 81, 289–293; Chang, J.‐Y. and Tran, T. H., (1986) J. Biol. Chem. 261, 1174–1176; Blackburn, M. N., Smith, R. L., Carson, J. and Sibley, C. C. (1984) J. Biol. Chem. 259, 939–941], the heparin‐binding site of antithrombin III has been suggested to be in the region of Pro‐41, Arg‐47 and Trp‐49. In this study the heparin‐binding site was probed by preferential cleavage of V8 protease on heparin‐treated and non‐treated native antithrombin III. The study has been based on the presumption that the heparin‐binding site of antithrombin III is situated at exposed surface domain and may be preferentially attacked during limited proteolytic digestion. Partially digested antithrombin III samples were monitored by quantitative amino‐terminal analysis and amino acid sequencing to identify the preferential cleavage sites. 1‐h‐digested antithrombin III was separated on HPLC and peptide fragments were isolated and characterized both qualitatively and quantitatively. The results reveal that Glu‐Gly (residues 34–35), Glu‐Ala (residues 42–43) and Glu‐Leu (residues 50–51) are three preferential cleavage sites for V8 protease and their cleavage, especially the Glu‐Ala and the Glu‐Leu sites, was drastically inhibited when antithrombin III was preincubated with heparin. Both high‐affinity and low‐affinity antithrombin‐III‐binding heparins were shown to inhibit the V8 protease digestion of native antithrombin III, but the high‐affinity sample exhibited a higher inhibition activity than the low‐affinity heparin. These findings (a) imply that the segment containing residues 34–51 is among the most exposed region of native antithrombin III and (b) support the previous conclusions that this region may play a pivotal role in the heparin binding.
The effect of thrombin on the complex between factor VIII and von Willebrand factor
Authors: HAMER Rob J. , KOEDAM Johannes A. , BEESER‐VISSER Nel H. , SIXMA Jan J.
Published: March 2005

Purified human factor FVIII (FVIII; 6000–8000 U/mg) was radiolabeled and bound to immobilized von Willebrand factor (vWF). The complex was incubated with human thrombin. Thrombin induced a release of 65% of the radioactivity initially bound. Released FVIII fragments and fragments remaining bound during incubation with thrombin were analyzed using gel electrophoresis. This led to the following observations. Released fragments largely consisted of Mr‐70000 and Mr‐50000 fragments; Mr‐90000 and Mr‐80000 fragments were only found in the fractions remaining bound to vWF and decreased with time. In contrast to these digestion products of FVIII, the Mr‐42000 heavy‐chain fragment remained bound to vWF, comprising the larger part of the radioactivity after a 2‐h incubation. No thrombin‐induced cleavages were observed in vWF. Furthermore, vWF‐coated wells preincubated with thrombin were still able to bind 125I‐FVIII.

These results implicate a new concept for the activation of vWF‐bound FVIII. Activation is a multistep process in which several cleavages are necessary to produce and release a coagulant‐active FVIII molecule (FVIIIa), which is probably an Mr‐50000/70000 heterodimer. Inactivation of FVIIIa is likely to be the result of a nonproteolytic dissociation due to loss of the joining divalent cation(s).
Phenylglyoxal modification of arginines in mammalian D‐amino‐acid oxidase
Authors: VANONI Maria A. , PILONE SIMONETTA Mirella , CURTI Bruno , NEGRI Armando , RONCHI Severino
Published: March 2005

The presence of arginine in the active center of D‐amino‐acid oxidase is well documented although its role has been differently interpreted as being part of the substrate‐binding site or the positively charged residue near the N1‐C2= O locus of the flavin coenzyme. To have a better insight into the role of the guanidinium group in D‐amino‐acid oxidase we have carried out inactivation studies using phenylglyoxal as an arginine‐directed reagent. Loss of catalytic activity followed pseudo‐first‐order kinetics for the apoprotein whereas the holoenzyme showed a biphasic inactivation pattern. Benzoate had no effect on holoenzyme inactivation by phenylglyoxal and the coenzyme analog 8‐mercapto‐FAD did not provide any additional protection in comparison to the native coenzyme. Spectroscopic experiments indicated that the modified protein is unable to undergo catalysis owing to the loss of coenzyme‐binding ability. Analyses of time‐dependent activity loss versus arginine modification or [14C]phenylglyoxal incorporation showed the presence of one arginine essential for catalysis. The protection exerted by the coenzyme is consistent with the involvement of an active‐site arginine in the correct binding of FAD to the protein moiety. Comparative analyses of CNBr fragments obtained from apoenzyme, holoenzyme and the 8‐mercapto derivative of D‐amino‐acid oxidase after reaction with phenylglyoxal did not provide unequivocal identification of the essential arginine residue within the primary structure of the enzyme. However, they suggest that it might be localized in the N‐terminal portion of the polypeptide chain and point to a role of phenylglyoxal‐modifiable arginine in binding to the adenylate/pyrophosphate moiety of the flavin coenzyme.
Structural and immunochemical characterization of a ribosomal protein from gram‐positive Micrococcus luteus which is functionally homologous to Escherichia coli ribosomal protein S1
Authors: MURALIKRISHNA Parimi , SURYANARAYANA Tangirala
Published: March 2005

Ribosomes from gram‐positive Micrococcus luteus contain an acidic protein (ML‐S1). ML‐S1 has been purified by chromatography of ribosomes on a poly(U)‐Sepharose column and the purified protein has a mobility in sodium dodecyl sulphate/polyacrylamide gels similar to that of ribosomal protein S1 of Escherichia coli (apparent Mr 72000). Protein ML‐S1 reacted with E. coli anti‐S1 serum with an immunological partial‐identity reaction. ML‐S1 also reacted with antibodies raised against two structural domains of E. coli S1 (the N‐terminal ribosome‐binding domain and central and C‐terminal nucleic‐acid‐binding domain). Weak reaction with antiserum to the nucleic‐acid‐binding domain of E. coli S1 was observed. ML‐S1 was digested with trypsin under mild and exhaustive conditions. Mild digestion resulted in the production of a trypsin‐resistant core (ML‐S1F1) like E. coli S1. The fragment pattern obtained after exhaustive digestion differed appreciably from that obtained with E. coli S1. ML‐S1 bound to poly(U) as strongly as E. coli S1 and also showed appreciable binding to denatured DNA. Addition of ML‐S1 to S1‐depleted ribosomes from E. coli and M. luteus markedly stimulated the poly(U)‐directed polyphenylalanine synthesis. Phage MS2‐RNA‐dependent translation was also found to be stimulated by ML‐S1 although to a much lesser extent than the stimulation by E. coli S1. At a molar excess of ML‐S1 to ribosomes the protein showed a similar inhibitory effect to E. coli S1 on polypeptide synthesis. Our data indicate that ML‐S1 retained the structural domains important for its function despite certain structural differences from E. coli S1.
6‐Hydroxy‐D‐nicotine oxidase of Arthrobacter oxidans
Authors: BRANDSCH Roderich , HINKKANEN Ari E. , MAUCH Ludwig , NAGURSKY Heiner , DECKER Karl
Published: March 2005

The nucleotide sequence of the 6‐hydroxy‐D‐nicotine oxidase (6‐HDNO) gene of Arthrobacter oxidans is presented. This covalently flavinylated enzyme specifically oxidizes 6‐hydroxy‐D‐nicotine to 6‐hydroxy‐N‐methylmyosmine. Coinduced in the presence of nicotine is a 6‐hydroxy‐L‐nicotine‐specific enzyme, 6‐hydroxy‐L‐nicotine oxidase (6‐HLNO), with FAD noncovalently bound to the apoprotein. A comparison of the nucleotidederived amino acid sequence of the 6‐HDNO with the amino acid sequence data obtained from the purified 6‐HLNO polypeptide suggests that the two enantiozymes expressed within the same cell are genetically unrelated. This conclusion is supported by the finding that the FAD‐binding sites of the two enzymes are different. 6‐HLNO exhibits at the amino‐terminus of the polypeptide chain a dinucleotide‐binding site characteristic for many other FAD‐and NAD(P)‐depedent enzymes. No such sequence was found in the nucleotide‐derived amino acid sequence of 6‐HDNO.
Cycloheximide induces accumulation of vasoactive intestinal peptide (VIP) binding sites at the cell surface of a human colonic adenocarcinoma cell line (HT29‐D4)
Authors: LUIS José , MARTIN Jean‐Michel , EL BATTARI Assou , FANTINI Jacques , GIANNELLINI Fernand , MARVALDI Jacques , PICHON Jacques
Published: March 2005

Incubation of monolayers of HT29‐D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 μM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand.

The increase in 125I‐VIP‐binding capacity was dose‐dependent between 0.35 μM and 17.5 μM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5–100 μM) a plateau corresponding to a twofold increase in VIP‐binding capacity was reached independently of the extent of protein synthesis inhibition.

We found that VIP receptors of HT29‐D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide).

After inactivation of 90% of cell‐surface VIP receptors by α‐chymotrypsin, we observed a biphasic kinetic of reappearance of VIP‐binding sites. 40% of VIP‐binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide‐treated cells. However, the slow recovery was inhibited in cycloheximide‐treated cells probably because a pool of immature receptors was depleted by the drug before the α‐chymotrypsin treatment.

Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.
Vasodilator‐stimulated protein phosphorylation in platelets is mediated by cAMP‐ and cGMP‐dependent protein kinases
Authors: WALDMANN Rainer , NIEBERDING Maria , WALTER Ulrich
Published: March 2005

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin‐regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 μM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masscs, Mr, of 240000, 68 000, 50 000, and 22 000 in intact platelets.

These proteins were also phosphorylated in response to low concentrations (1–2 μM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 μM) and the 8‐bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50000 which was also phosphorylated in response to low concentrations (1–2 μM) of cGMP in platelet membranes. An additional protein (Mr 24000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50000 was stimulated both in intact platelets by cyclic‐nucleotide‐elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50‐kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50‐kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50‐kDa protein phosphorylated in platelet membranes by cGMP‐ and cAMP‐dependent protein kinases, respectively.

Regulation of protein phosphorylation mediated by cAMP‐ and cGMP‐dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.
The obtention of simian virus 40 recombinants carrying d(CG · GC)n, d(CA · GT)n and d(CT · GA)n sequences
Authors: CASASNOVAS Jose M. , ELLISON Michael J. , RODRIGUEZ‐CAMPOS Antonio , AZORIN Fernando
Published: March 2005

A general strategy for the introduction of simple repeating DNA sequences into the simian virus 40 (SV40) has been developed. SV40 recombinants carrying d(CG · GC)5, d(CA · GT)30 or d(CT · GA)22 insertions at either the TaqI site (position 4739) or the HpaII site (position 346) were obtained and the stability of the inserted DNA sequences studied. The palindromic potentially Z‐DNA‐forming d(CG · GC)n sequence was found to be highly unstable when compared to either d(CA · GT)n or d(CT · GA)n.
Peroxisomal oxidation of L‐2‐hydroxyphytanic acid in rat kidney cortex
Authors: DRAYE Jean Pierre , VAN HOOF François , de HOFFMANN Edmond , VAMECQ Joseph
Published: March 2005

A previously unreported metabolite of mammalian phytanic acid catabolism, 2‐oxophytanic acid, was identified by gas chromatography/mass spectrometry analysis. The formation of 2‐oxophytanic acid was demonstrated to result from the oxidation of L‐2‐hydroxyphytanic acid, a reaction catalysed by a rat‐kidnev‐corted H2O2‐generating oxidase. The pH optimum for the L‐2‐hydroxyphytanate oxidase activity was 8.5 and its apparent Km and Vm were about 0.15 mM and 0.35 μmol min1 (g tissue)−1, respectively. L‐2‐Hydroxyisocaproate, a substrate of rat kidney L‐α‐hydroxyacid oxidase type B, inhibited the formation of 2‐oxophytanate from L‐2‐hydroxyphytanic acid. Fractionation studies have indicated that 40% of L‐2‐hydroxyphytanate oxidase was associated with a particulate fraction and that the activity distribution of the oxidase closely paralleled that of catalase, a well known peroxisomal marker enzyme.
The photochemical reaction center of Chloroflexus aurantiacus is composed of two structurally similar polypeptides
Authors: SHIOZAWA Judith A. , LOTTSPEICH Friedrich , FEICK Reiner
Published: March 2005

A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chloroflexus aurantiacus. The procedure consisted of three chromatography steps. The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl‐N‐methylglucamide (Mega‐9). The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less. Under non‐denaturing conditions, a pigmented protein band having a Mr of 52000–55000 was observed in sodium dodecyl sulfate gels. When the isolated complex was heat‐dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar Mr (24000 and 24500) were observed. Two protein bands were also observed in two‐dimensional isoelectric focusing/sodium‐dodecyl‐sulfate polyacrylamide gel electropheresis; the PI values of the two polypeptides were 6.5 and 6.7. Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides.

The N‐terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria.

The Chloroflexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins. Hence, it appears to be the smallest photochemically active reaction center isolated to date.
Protein‐structural heterogeneity in a non‐allosteric monomeric insect hemoglobin monitored by proton magnetic resonance spectroscopy
Authors: PEYTON David H. , KRISHNAMOORTHI R. , LA MAR Gerd N. , GERSONDE Klaus , SMITH Kevin M. , PARISH Daniel W.
Published: March 2005

Proton NMR has revealed two modes of structural heterogeneity in the monomeric hemoglobin I of Chironomus thummi thummi, CTT I; rotational disorder caused by a 180° rotation of the heme about the α, γ‐meso axis (primary heterogeneity), which varies for each preparation or reconstitution of this hemogobin, and a ‘silent’ amino acid replacement [Thr/Ala exchange in position 98(FG4)] in the vicinity of the heme group, which is invariant under all experimental conditions. The heme rotational disorder (primary heterogeneity) can be removed by reconstitution of CTT I with the symmetrical protoheme III. The secondary splitting is not affected; the ratio of intensities of the two types of resonance remains constant. The 8‐methyl and 3‐methyl and one of the α‐vinyl proton resonances for the major heme rotational component and the 5‐methyl and 1‐methyl and one of the α‐vinyl proton resonances for the minor heme rotational component have been identified and assigned by reconstitution with deuterium‐labeled heme. Decoupling experiments have been employed to assign vinyl β protons in cis and trans position to the respective vinyl α protons. Hyperfine shifts for the heme protons exhibited no pH influence above pH 6, in accord with the lack of the alkaline Bohr effect. Below pH 6, pH effects are most strongly reflected by the 8‐methyl and 5‐methyl proton resonances possibly reflecting titration of the propionate groups.
Proton‐motive‐force‐driven formation of CO from CO2 and H2 in methanogenic bacteria
Authors: BOTT Michael , THAUER Rudolf K.
Published: March 2005

Cell suspensions of methanogenic bacteria (Methanosarcina barkeri, Methanospirillum hungatei, Methanobrevibacter arboriphilus, and Methanobacterium thermoautotrophicum) were found to form CO from CO2 and H2 according to the reaction: CO2+ H2→ CO + H2O; ΔG0=+20 kJ/mol. Up to 15000 ppm CO in the gas phase were reached which is significantly higher than the equilibrium concentration calculated from ΔG0 (95 ppm under the experimental conditions). This indicated that CO2 reduction with H2 to CO is energy‐driven and indeed the cells only generated CO when forming CH4. The coupling of the two reactions was studied in more detail with acetate‐grown cells of M. barkeri using methanol and H2 as methanogenic substrates. The effects of the protonophore tetrachlorosalicylanilide (TCS) and of the proton‐translocating ATPase inhibitor N,N′‐dicyclohexylcarbodiimide (cHxN)2C were determined. TCS completely inhibited CO formation from CO2 and H2 without affecting methanogenesis from CH3OH and H2. In the presence of the protonophore the proton motive force Δp and the intracellular ATP concentration were very low. (cHxN)2C, which partially inhibited methanogenesis from CH3OH and H2, had no effect on CO2 reduction to CO. In the presence of (cHxN)2C Δp was high and the intracellular ATP content was low. These findings suggest that the endergonic formation of CO from CO2 and H2 is coupled to the exergonic formation of CH4 from CH3OH and H2 via the proton motive force and not via ATP. CO formation was not stimulated by the addition of sodium ions.
A structural and dynamic model for the nicotinic acetylcholine receptor
Authors: KOSOWER Edward M.
Published: March 2005

Folding of the five polypeptide subunits (α2βγδ), of the nicotinic acetylcholine receptor (AChR) into a functional structural model is described. The principles used to arrange the sequences into a structure include: (1) hydrophobicity → membrane‐crossing segments; (2) amphipathic character → ion‐carrying segments (ion channel with single group rotations); (3) molecular shape (elongated, pentagonal cylinder) → folding dimensions of exobilayer portion; (4) choice of acetylcholine binding sites → specific folding of exobilayer segments; (5) location of reducible disulfides (near agonist binding site) → additional specification of exobilayer arrangement; (6) genetic homology → consistency of functional group choices; (7) noncompetitive antagonist labeling → arrangement of bilayer helices. The AChR model is divided into three parts: (a) exobilayer consisting of 11 antiparallel β‐strands from each subunit; (b) bilayer consisting of four hydrophobic and one amphiphilic α‐helix from each subunit; (c) cytoplasmic consisting of one (folded) loop from each subunit.

The exobilayer strands can form a closed ‘flower’ (the ‘resting state’) which is opened (‘activated’) by agonists bound perpendicular to the strands. Rearrangement of the agonists to a strand‐parallel position and partial closing of the ‘flower’ leads to a desensitized receptor. The actions of acetylcholine and succinoyl and suberoyl bis‐cholines are clarified by the model. The opening and closing of the exobilayer ‘flower’ controls access to the ion channel which is composed of the five amphiphilic bilayer helices. A molecular mechanism for ion flow in the channel is given. Openings interrupted by short duration closings (50 μs) depend upon channel group motions. The unusual photolabeling of intrabilayer serines in α, β and δ subunits but not in γ subunits near the binding site for non‐competitive antagonists is explained along with a mechanism for the action of these antagonists such as phencyclidine. The unusual α 192Cys–193Cys disulfide may have a special peptide arrangement, such as a cis‐peptide bond to a following proline (G.A. Petsko and E.M. Kosower, unpublished results). The position of phosphorylatable sites and proline‐rich segments are noted for the cytoplasmic loops.

The dynamic behavior of the AChR channel and many different experimental results can be interpreted in terms of the model. An example is the lowering of ionic conductivity on substitution of bovine for Torpedoδ M2 segment. The model represents a useful construct for the design of experiments onAChR.
Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole
Authors: GOULAS Philippe
Published: March 2005

Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6‐[N‐(8‐amino‐3,6‐dioxaoctyl)carbamoylmethyl]‐NAD+ in order to alkylate the carboxylic acid group of Asp‐273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6‐[N‐(8‐amino‐3,6‐dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prothetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated.
Characterization of a soluble Mr‐30000 catalytic fragment of the neuronal calmodulin‐dependent protein kinase II
Authors: LEVINE Harry , SAHYOUN Naji E.
Published: March 2005

Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the α (Mr 50000) subunit of the neuronal cytoskeletal calmodulin‐dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I‐Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated α subunit. Catalytic fragments of both forms of the enzyme comigrate on two‐dimensional SDS‐PAGE/isoelectric focusing with pI 6.7–7.2.

The fragment phosphorylates microtubule‐associated protein (MAP‐2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP‐2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I‐calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment.

Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25600 with a frictional ratio (f/fo) of 1.12; the Mr value determined by SDS‐PAGE was 30000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin‐dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.
Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine: pyruvate aminotransferase of rat liver
Authors: ODA Toshiaki , MIYAJIMA Hiroaki , SUZUKI Yoshie , ICHIYAMA Arata
Published: March 2005

The nucleotide sequence of the mRNA coding for the precursor of mitochondrial serine:pyruvate aminotransferase of rat liver was determined from those of cDNA clones. The mRNA comprises at least 1533 nucleotides, except the poly(A) tail, and encodes a polypeptide consisting of 414 amino acid residues with a molecular mass of 45834 Da. Comparison of the N‐terminal amino acid sequence of mitochondrial serine: pyruvate aminotransferase with the nucleotide sequence of the mRNA showed that the mature form of the mitochondrial enzyme consisted of 390 amino acid residues of 43210 Da. The amino acid composition of mitochondrial serine:pyruvate aminotransferase deduced from the nucleotide sequence of the cDNA showed good agreement with the composition determined on acid hydrolysis of the purified protein. The extra 24 amino acid residues correspond to the N‐terminal extension peptide (pre‐sequence) that is indispensable for the specific import of the precursor protein into mitochondria. In the extension peptide there are four basic amino acids distributed among hydrophobic amino acids and, as revealed on helical wheel analysis, the putative α‐helical structure of the peptide was amphiphilic in nature. The secondary structures of the mature serine: pyruvate aminotransferase and three other aminotransferases of rat liver were predicted from their amino acid sequences. Their secondary structures exhibited a common feature and so we propose the specific lysine residue which binds pyridoxal phosphate as the active site of serine: pyruvate aminotransferase.
Studies on the linkage between spin equilibria and protein structure in carp ferric hemoglobin
Authors: NOBLE Robert W. , DE YOUNG Alice , VITALE Stefano , MORANTE Silvia , CERDONIO Massimo
Published: March 2005

The effects of protein conformation on the spin‐state equilibria of several derivatives of carp hemoglobin have been examined. This has been done by measuring the pH dependence of the paramagnetic susceptibilities of these derivatives in the presence and absence of inositol hexakisphosphate, P6‐inositol. In all cases the addition of P6‐inositol at low pH and the lowering of the pH in the presence of P6‐inositol shift the spin‐state equilibrium in favor of the high‐spin electronic configuration. The P6‐inositol and pH dependence of these magnetic properties parallels the pH and P6‐inositol dependence of the conformational state of the hemoglobin as determined in earlier studies and further supports a thermodynamic linkage between the electronic state of the iron atoms and the quaternary structure of the hemoglobin molecule.
Activation and transfer of novel synthetic 9‐substituted sialic acids
Authors: GROSS Hans Jürgen , BÜNSCH Almuth , PAULSON James C. , BROSSMER Reinhard
Published: March 2005

In this report several NeuAc analogues differently modified at position C‐9 were tested as substrates for CMPsialic acid synthase from bovine brain: the hydroxy group at C‐9 was replaced by an amino, acetamido, benzamido, hexanoylamido and azido group.

The synthase was partially purified by chromatography on CDP‐hexanolamine –Sepharose. CMP‐glycosides synthesized were measured by analytical HPLC at 275 nm.

Each NeuAc analogue was activated to the respective CMP‐glycoside: Km‐values varied from 0.8 mM to 4.6 mM, the Km for NeuAc was 1.4 mM. Thus affinity of the enzyme was influenced only moderately by chemical modification at C‐9.

CMP‐glycosides were synthesized on a preparative scale with good yield and characterized by analytical HPLC. In addition, 500‐MHz 1H‐NMR data of CMP‐9‐amino‐NeuAc and CMP‐9‐acetamido‐NeuAc were obtained.

Each CMP‐activated NeuAc analogue was a suitable donor substrate for Galβ1‐4GlcNAc α2,6‐sialyl‐transferase from rat liver. Transfer was determined by the thiobarbituric acid method and by analytical HPLC at 200 nm. The results demonstrate that synthetic, not naturally occurring, non‐labelled NeuAc analogues can be incorporated into glycoprotein with high yield.
Sequence‐specific 1H‐NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica
Authors: OTTING Gottfried , STEINMETZ Wayne E. , BOUGIS Pierre E. , ROCHAT Hervé , WÜTHRICH Kurt
Published: March 2005

Sequence‐specific assignments of the 1H‐nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two‐dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25°C and 45°C for all but one back bone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side‐chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497–508]. The 1H/2H‐exchange kinetics were measured in 2H2O at 20°C for the amide protons and the N‐terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double‐stranded antiparallel β‐sheet comprising the residues 2–4 and 11–13, and a triple‐stranded antiparallel β‐sheet consisting of the residues 20–26, 35–39, and 49–55. The two peripheral strands of the triple‐stranded β‐structure were found to be connected by a right‐handed cross‐over, and the locations of several tight turns were also identified.
Structural variations in the alanine‐rich antifreeze proteins of the pleuronectinae
Authors: SCOTT Gary K. , DAVIES Peter L. , SHEARS Margaret A. , FLETCHER Garth L.
Published: March 2005

The sequence and activity of antifreeze proteins from two righteye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97‐residue preproportion. Edman degradation identified the N‐terminus of the 48‐amino‐acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5′ and 3′ untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11‐amino‐acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11‐amino‐acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice‐binding residues per molecule.
The dynamical structure of the RNA in alfalfa mosaic virus studied by 31P‐nuclear magnetic resonance
Authors: KAN Jaap H. , CREMERS Alfons F. M. , HAASNOOT Cornelis A. G. , HILBERS Cornelius W.
Published: March 2005

The structure of the viral RNA in alfalfa mosaic virus (AIMV) was investigated by means of 31P‐nuclear magnetic resonance (NMR). It was found that the 31P‐NMR line width of AIMV Top a particles is significantly smaller than that of the larger Bottom particles. At low temperatures, the totational correlation time of the 31P nuclei essentially equals the tumbling rate of the virus particle, indicating that the RNA is contained rigidly inside the virion. At more elevated temperatures, the NMR line width sharpens more than expected on the basis of viscosity changes and the RNA exhibits internal mobility. The occurrence of internal mobility is paralleled by an increased internal mobility of the N‐terminal part of the coat protein, as could be observed by 1H‐NMR spectroscopy. The influence of EDTA on the 31P‐NMR line width appeared to be negligible, which is in agreement with the idea that AIMV does not ‘swell’ like several other RNA‐containing plant viruses.
Cultured Ito cells of rat liver express the α2‐macroglobulin gene
Authors: ANDUS Tilo , RAMADORI Giuliano , HEINRICH Peter C. , KNITTEL Thomas , MEYER ZUM BÜSCHENFELDE Karl‐Hermann
Published: March 2005

Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize α2‐macroglobulin was analyzed at different times after isolation and by pulse‐chase experiments. Newly synthesized α2‐macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. α2‐Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5–11 days after the isolation of the cells, increasing amounts of α2‐macroglobulin were synthesized. The results of pulse‐chase experiments performed on day 7 showed that radioactively labeled α2‐macroglobulin decreased in the intracellular compartment and increased in the culture medium. α2‐Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non‐reducing conditions. Furthermore, when unlabeled α2‐macroglobulin was added during the immunoprecipitation a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with α2‐macroglobulin. α2‐Macroglobulin‐specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells.

Under the conditions where α2‐macroglobulin was synthesized in Ito cells, no synthesis of α1‐macroglobulin, α1‐inhibitor 3, α1‐proteinase inhibitor, α1‐acid glycoprotein, α1‐acute‐phase globulin (T‐kininogen) and albumin could be demonstrated. It is concluded that α2‐macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases.
Amino acid sequence similarity between malate dehydrogenases (NAD) and pea chloroplast malate dehydrogenase (NADP)
Authors: FICKENSCHER Karl , SCHEIBE Renate , MARCUS Frank
Published: March 2005

Purified pea chloroplast malate dehydrogenase (NADP) was reduced, S‐pyridylethylated with 4‐vinyl‐pyridine and cleaved with trypsin. The resulting peptides were separated by reversed‐phase high‐performance liquid chromatography. Several of these peptides were subjected to automated Edman degradation. The sequences obtained were compared to the published primary structures of malate dehydrogenase from the thermophilic bacterium Thermus flavus and with the sequence of heart mitochondrial and cytoplasmic malate dehydrogenase (NAD). Most peptides from choroplast malate dehydrogenase (NADP) showed high homology with sequences of the other malate dehydrogenases, especially with those of the bacterial enzyme. One of the sequenced peptides contains the active‐site histidine residue which is conserved in all malate dehydrogenases. Our results suggest a common evolutionary origin for all malate dehydrogenases despite their different coenzyme specificities and regulatory properties. The sequenced peptides which revealed no homology were either located at the amino‐terminal or at the carboxy‐terminal region of chloroplast malate dehydrogenase (NADP). These novel sequences are most likely plant‐specific extensions of an ancestral malate dehydrogenase and may be responsible for the unique light‐dependent activation of the chloroplast enzyme.
Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b‐558
Authors: FRIDÉN Henrik , RUTBERG Lars , MAGNUSSON Kerstin , HEDERSTEDT Lars
Published: March 2005

Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b‐558, a 23‐kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b‐558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C‐terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His‐113 excludes this residue as an axial heme ligand. A base‐pair exchange of G to A in the ribosome‐binding sequence of sdhC was found to reduce cytochrome b‐558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild‐type amount of membrane‐bound succinate dehydrogenase in B. subtilis still allows growth on non‐fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.
Chemical modification of phosphorylase b by tetranitromethane
Authors: CARUSO Carla , DI PRISCO Guido , CACACE Marcello G.
Published: March 2005

Tetranitromethane, C(NO2)4, a reagent for tyrosyl residues, was found to inactivate irreversibly rabbit skeletal muscle glycogen phosphorylase b. Under the chosen conditions seven tyrosyl residues, namely Tyr‐75, 203, 262, 280, 403, 552 and 647, were found to be nitrated. Inactivation was prevented by the presence of the allosteric activator 5′‐AMP during nitration. Under these latter conditions one of the reactive tyrosyl residues was not modified by C(NO2)4; thus, this residue appeared to be essential for either catalytic activity or allosteric activation.

Tryptic digests of phosphorylase b, reacted with C(NO2)4 in the absence and presence of 5′AMP, were fractionated by gel filtration. The peptide mixtures were further purified by reverse‐phase HPLC. One of the peptides contained the tyrosyl residue which was modified by C(NO2)4 only in the absence of 5′AMP. The sequence of this peptide was determined. The amino acid residue which is responsible for the loss of activity upon reaction with C(NO2)4 was identified in the amino acid sequence of phosphorylase b as tyrosine‐75.

Of the other residues modified in the presence and in the absence of C(NO2)4, tyrosine‐403 contributes to the glycogen‐storage site whereas Tyr‐280 is close to the α‐d‐glucose‐binding site. These residues, exposed to the solvent both in the presence and in the absence of 5′ AMP, are not essential for catalytic activity.
In vitro synthesis and processing of a bean pathogenesis‐related (PR4) protein
Authors: DE TAPIA Marc , DIETRICH André , BURKARD Gérard
Published: March 2005

When bean plants (Phaseolus vulgaris var. Saxa) are treated with mercuric chloride or infected with alfalfa mosaic virus, they produce pathogenesis‐related (PR) proteins. We report here that functional mRNA encoding bean PR4 protein is only present when synthesis of this protein has been induced. Treatment with mercuric chloride results in a rapid induction of functional bean PR4 mRNA (within 2–3 h), whereas in virus‐infected plants this mRNA can only be detected the second day following the infection. Bean PR4 protein is synthesized in vitro, using this mRNA in a rabbit reticulocyte lysate system, as a precursor of 35 kDa. This precursor can be processed into a polypeptide having the same molecular mass (33.5 kDa) as the in vivo PR4 protein by the addition to the cell‐free translation system of canine pancreatic microsomal membranes.
Amino‐acid sequence of human α2‐antiplasmin
Authors: LIJNEN H. Roger , HOLMES William E. , VAN HOEF Berthe , WIMAN Björn , RODRIGUEZ Henri , COLLEN Désiré
Published: March 2005

The amino‐acid sequence of human α2‐antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature α2‐antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659–1664], 444 residues were identified by amino‐acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369).

α2‐Antiplasmin contains two disulfide bridges (Cys64‐Cys104 and Cys31‐Cys113) and four glucosamine‐based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. α2‐Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.
Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6‐bisphosphate and of glycolytic intermediates in Trypanosoma brucei
Authors: VAN SCHAFTINGEN Emile , OPPERDOES Fred R. , HERS Henry‐Géry
Published: March 2005

Upon differential centrifugation of cell‐free extracts of Trypanosoma brucei, 6‐phosphofructo‐2‐kinase and fructose‐2,6‐bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6‐phosphofructo‐2‐kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycèrol 3‐phosphate. The Km of fructose‐2,6‐bisphosphatase was 7 μM; this enzyme was inhibited by fructose 1,6‐bisphosphate (Ki= 10 μM) and, less potently, by fructose 6‐phosphate, phosphoenolpyruvate and glycerol 3‐phosphate. Melarsen oxide inhibited 6‐phosphofructo‐2‐kinase (Ki < 1 μM) and fructose‐2,6‐bisphosphatase (Ki= 2 μM) much more potently than pyruvate kinase (Ki > 100 μM).

The intracellular concentrations of fructose 2,6‐bisphosphate and hexose 6‐phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6‐bisphosphate, ATP, hexose 6‐phosphate and fructose 1,6‐bisphosphate. These studies indicate that the concentration of fructose 2,6‐bisphosphate is mainly controlled by the concentration of the substrates of 6‐phosphofructo‐2‐kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6‐bisphosphate on pyruvate kinase.

At micromolar concentrations, melarsen oxide blocked almost completely the the formation of fructose 2,6‐bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6‐phosphates. At higher concentrations (3–10 μM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolpyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.
Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae
Authors: TSUKAGOSHI Yuko , NIKAWA Jun‐ichi , YAMASHITA Satoshi
Published: March 2005

The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Saccharomyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme. The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant. Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type.

Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase. The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis.

The cloned DNA was subcloned into a 2.5‐kb DNA fragment. The nucleotide sequence which contained CCT was determined by the dideoxy chain‐termination method. The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49379.31. Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product.

Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N‐ and C‐terminal regions.

The primary translation product contains a region showing local sequence homology with nucleotidyl‐transfer enzymes such as DNA polymerase (Escherichia coli), CDPdiacylglycerol pyrophosphatase (E. coli), 3‐deoxy‐manno‐octulosonate cytidylyltransferase (E. coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related. Statistically significant sequence homology was also noted between the human c‐fos gene product and the enzyme.
Characterization of binding equilibrium data by a variety of fitted isotherms
Authors: BRODERSEN Rolf , NIELSEN Frede , CHRISTIANSEN Jørn C. , ANDERSEN Kim
Published: March 2005

Experimental binding equilibrium data, resulting from measurement of ligand binding to macromolecular carriers, are usually described by fitting of binding constants. These constants are often highly variable, as illustrated in the present paper by two examples, binding of salicylate to human serum albumin and of oxygen to hemoglobin. In order to avoid over‐interpretation of binding constants it is pointed out that the best‐fit solution, obtained by least squares fitting, may be supplemented by several, e. g. thirty, acceptable solutions. It is further shown how the 30 sets of acceptable binding constants, plotted as Klotz' affinity profiles, can serve for evaluation of cooperative effects.