bluuebunny/crossref_metadata_2025 huggingface dataset:
Restoration of NADH Oxidation with Menaquinones and Menaquinone Analogues in Membrane Vesicles from the Menaquinone‐Deficient Bacillus subtilis aroD
Authors: BERGSMA Jack , MEIHUIZEN Kor E. , Van OEVEREN Wim , KONINGS Wil N.
Published: March 2005

Membrane vesicles from the menaquinone‐deficient Bacillus subtilis aroD oxidize NADH at a low rate. NADH oxidation can be restored by the addition of slightly water‐soluble menaquinone and ubiquinone analogues up to saturation levels. These saturation levels differ for the different quinone analogues tested from 95 (1,4‐benzo‐ quinone) to 5316 (5‐hydroxy‐1,4‐naphthoquinone, juglon) nmol NADH × min−1× mg membrane protein−1 NADH oxidation in membrane vesicles from B. subtilis aroD restored with water‐soluble quinone analogues supplies the energy for l‐glutamate uptake. Like NADH oxidation the initial rate of l‐glutamate transport increases up to saturation levels. The highest initial rates of l‐glutamate uptake are observed after restoration with quinone analogues with a relatively low standard redox potential.

Functional reconstitution with natural, water‐insoluble, quinones can be achieved effectively by mixing quinone‐containing liposomes with membrane vesicles from B. subtilis aroD and subsequent freezing of the mixture in liquid nitrogen. The rate of NADH oxidation increased with the amount of menaquinone incorporated in the vesicles up to saturation levels. NADH oxidation via these menaquinones also supplies the energy for l‐glutamate uptake. The highest uptake rates can be obtained with menaquinone‐1 and menaquinone‐2. On the basis of efficiencies (mol NADH oxidized/mol l‐glutamate transported) menaquinones and mena‐ quinone analogues can be divided in two classes.

To class 1 belong the menaquinone analogues, menaquinone‐5 and menaquinone‐8. These compounds restore NADH oxidation with low levels of energy transduction. Efficiencies are observed which are comparable with the efficiency observed in membrane vesicles from B. subtilis W23 (120‐140) which contain the natural mena‐ quinone‐7.

To class 2 belong menaquinone‐I and menaquinone‐2, which restore NADH oxidation with high levels of energy transduction. Efficiencies are observed which are in the same range as observed with phenazine methosulphate (9‐13).

A model is proposed in which class 1 compounds feed in electrons from the outside from NADH to the Q‐cycle of the respiratory chain. Class 2 compounds donate electrons to the respiratory chain after cytochrome c and before cytochrome α‐601.
Further Characterization of the Ca2+‐Dependent F‐Actin‐Depolymerizing Protein of Human Serum
Authors: THORSTENSSON Rigmor , UTTER Göran , NORBERG Renée
Published: March 2005

The F‐actin‐depolymerizing factor (ADF) of human serum was purified and identified as a 93000‐daltons protein. The concentration of ADF was calculated to 120–150 μg/ml in four normal sera and about half as much in three sera from leukemia patients treated with cytostatic drugs. In the presence of Ca2+ ADF shortened actin filaments into fragments, the size of which was correlated to the actin: ADF molar ratio, as judged by electron microscopy. The severing of F‐actin was not necessarily followed by an increase in the quantities of monomeric actin, as determined by a DNase I inhibition assay and a sedimentation assay. The findings indicated that ADF shortens filamentous actin by breaking bonds between adjacent actin molecules thereby forming stable ADF‐actin complexes, without a monomeric net release. The effect of ADF on F‐actin was rapid and was reversed upon chelation of Ca2+. ADF cross‐reacted immunologically and exhibited similarity in reaction mechanism with gelsolin, the Ca2+‐dependent F‐actin‐severing protein from macrophages. This implies that the proteins are both structurally and functionally related. The physiological role of ADF may be to handle actin released at cell destruction, probably by forming ADF–G‐actin 1:1 complexes thereby preventing formation of actin filaments.
Biosynthesis of a γ3‐Melanotropin‐like Peptide in the Pars Intermedia of the Amphibian Pituitary Gland
Authors: MARTENS Gerard J. M. , JENKS Bruce G. , VAN OVERBEEKE A. P.
Published: March 2005

This study reveals the biosynthesis of a γ3‐melanotropin‐like peptide in the pars intermedia of the pituitary gland of the aquatic toad Xenopus laevis. Pulse‐chase experiments in vitro showed that this product is synthesized through processing of a prohormone, pro‐opiomelanocortin, and that it is released into the incubation medium. The peptide immunoprecipitated with antiserum to γ3‐melanotropin, appeared to be a glycopeptide and displayed melanotropic activity. This last observation together with the results of tryptic and chymotryptic peptide mapping of the newly synthesized product indicates that Xenopusγ3‐melanotropin is structurally different from the proposed mammalian γ‐melanotropins.
Substrate Specificity of CTP Synthetase from Escherichia coli
Authors: SCHEIT Karl Heinz , LINKE Hans‐Juergen
Published: March 2005

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high‐performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5′‐triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2‐thiouridine 5′‐triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP)=1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5′‐triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product‐like inhibitors supported this view.
Inhibition of Pyruvate Dehydrogenase during the Metabolism of Glutamine and Proline in Hemoglobin‐Free Perfused Rat Liver
Authors: HÄUSSINGER Dieter , GEROK Wolfgang , SIES Helmut
Published: March 2005

A shift of pyruvate dehydrogenase from the active (dephospho) towards the inactive (phospho) form results from the metabolism of added glutamine in isolated perfused rat liver. This is evidenced by a decreased release of 14CO2 from [1‐14C]pyruvate and by direct measurement of enzyme activity in the freeze‐stopped liver tissue. Half‐maximal glutamine concentration for the inhibition of pyruvate oxidation is about 1 mM, i.e. within the physiological range. The effect is reversible and exhibits a time course similar to that of glutamine metabolism as measured by the production of ammonia and urea. The effect is independent of the mode of glutaminase activation, either by ammonium ions or glucagon or by so‐called ‘self‐activation’.

When oxoglutarate, glutamate and ammonia are generated intracellularly from proline, there is also an inhibition of 14CO2 release from [1‐14C]pyruvate. Proline metabolism is stimulated about twofold by addition of glucagon. This is accompanied by a further decrease of pyruvate dehydrogenase activity, whereas addition of glucagon alone has no effect.

There is good agreement between 14CO2 production from [1‐14C]glutamine and oxoglutarate production as calculated from nitrogen balance measurements. This indicates that oxoglutarate formed from glutamine is predominantly oxidized by oxoglutarate dehydrogenase. Furthermore, there is a close correlation between oxidation of oxoglutarate derived from glutamine or proline and the degree of inhibition of pyruvate dehydrogenase. This correlation holds also when oxoglutarate production from glutamine or proline is increased by ammonium ions or glucagon. Thus, it is concluded that increased flux through oxoglutarate dehydrogenase is accompanied by a decreased flux through pyruvate dehydrogenase in rat liver.
Interference of Ligands on the Phenylalanyl‐tRNA Synthetase from Yeast
Authors: THIEBE Rainer
Published: March 2005

The present paper reports a study of the mutual interactions between the substrates, the intermediate, and the products of the aminoacylation reaction, when bound to the phenylalanyl‐tRNA synthetase from yeast. The following conclusions can be drawn.

tRNAPhe displaces Phe‐tRNAPhe from the synthetase by lowering the affinity of the enzyme for the amino‐acylated tRNA.

Phe‐tRNAPhe and Phe‐AMP compete for the catalytically active site of the enzyme.

Chemically synthesized Phe‐AMP, when added to the synthetase, primarily forms a low‐affinity complex with the enzyme. The transformation of this complex into the high‐affinity catalytic complex is a very slow process.

These findings confirm a previous study, based on steady‐state kinetics. A schematic representation of the aminoacylation process is given. It summarizes the present and previous results and illustrates a rather complex ‘flip‐flop’ mechanism.
Mechanism of Synthesis of Adenosine(5′)tetraphospho(5′)adenosine (AppppA) by Aminoacyl‐tRNA Synthetases
Authors: GOERLICH Ottmar , FOECKLER Renate , HOLLER Eggehard
Published: March 2005

Synthesis of radioactive adenosine(5′)tetraphospho(5′)adenosine in the presence of aminoacyl‐tRNA synthetases of procaryotes or eucaryotes was measured by thin‐layer chromatography on poly(ethyleneimine)‐cellulose sheets. Catalysis by the majority of aminoacyl‐tRNA synthetases was observed. They were classified into a fast group (specificity for Lys, Phe, His), a slow group (Ser, Ile, Leu, Asp, Tyr, Val) and a non‐catalytic group (Trp, Arg). The presence of the cognate amino acid is required.

The mechanism was verified by direct synthesis of the tetraphosphate from preformed aminoacyladenylate and by its reverse reaction from chemically synthesized adenosine(5′)tetraphospho(5′)adenosine. Essentials are as follows. (1) Aminoacyladenylate is synthesized from a first molecule of MgATP. (2) A second ATP adds as substrate, Michaelis‐Menten constants being 11 mM and 50 mM in the case of Escherichia coli lysine‐specific and phenylalanine‐specific enzymes, respectively; these values are two orders of magnitude higher than for the first ATP. (3) This is followed by the synthesis of adenosine(5′)tetraphospho(5′)adenosine from adenylate and the second ATP; the reaction is rate‐determining in catalysis, rate constants being 0.5 s−1 and 0.26 s−1 respectively. (4) The equilibrium constant, K∼ 0.02, does not favour the synthesis of the tetraphosphate from ATP; removal of accumulating pyrophosphate by the action of added inorganic pyrophosphatase increases yields substantially. (5) The presence of Mg2+ ions is essential; in a second role, they can strongly activate the catalytic reaction. (6) Zn2+ is a more potent activator, although it is not essential; its effect was to decrease the value of the Michaelis‐Menten constant and to increase that of the catalytic rate constant; activation was observed only for a minority of aminoacyl‐tRNA synthetases (Phe, Lys).

The synthesis of adenosine(5′)tetraphospho(5′)adenosine and of adenosine(5′)triphospho(5′)adenosine and its reverse are discussed with regard to possible biological functions.
Presence of an Amino Acid Oxidase in Photosystem II of Anacystis nidulans
Authors: PISTORIUS Elfriede K. , VOSS Helga
Published: March 2005

Membrane fractions from the blue‐green algae Anacystis nidulans can catalyze O2 evolution with ferricyanide as acceptor in the light and the dark oxidation of l‐arginine. The two reactions are both inhibited by o‐phenanthroline, but they respond to certain trivalent, divalent and monovalent cations (like e.g. La3+ or Ca2+ or K+) in different ways. These metals stimulate the Hill reaction with ferricyanide as acceptor, but totally inhibit l‐arginine oxidation. The order of effectiveness is M3+ > M2+ > M+. The effect of e.g. Ca2+ on the dark oxidation of l‐arginine mirrors the effect of Ca2+ on the light‐driven O2 evolution with ferricyanide, as though these two activities are mutually exclusive, i.e. one process is inhibited to the extent that the other is activated. The enzyme responsible for the dark oxidation of l‐arginine has been purified from A. nidulans and shown to be a flavo‐enzyme [Pistorius, E. K. and Voss, H. (1980) Biochim. Biophys. Acta, 611, 227–240].

Active photosystem II particles have also been isolated from Anacystis membrane fractions. They can catalyze 2,6‐dichloroindophenol reduction with diphenylcarbazide as donor in the light. They retain l‐arginine oxidation activity in the presence of EDTA. Both reactions are inhibited by o‐phenanthroline. Moreover, it could be shown that one of the protein bands which has been shown to be characteristic for photosystem II particles [Koenig, F. and Vernon, L. P. (1981) Z. Naturforsch. C36, 295–304], co‐chromatographs with the purified l‐amino‐acid oxidase on SDS gels. An antibody prepared to the purified l‐amino‐acid oxidase also gave a precipitin line with detergent‐treated photosystem II particles.

The experiments show that the l‐amino‐acid oxidase is part of photosystem II particles in A. nidulans. This protein might have a dual role: acting as an electron transferring protein in photosynthesis in the presence of certain metal ions (M3+ > M2+ > M+) and as an l‐amino‐acid oxidase in the absence of these.
Preparation of Apoprotein from Spinach Ferredoxin‐NADP+ Reductase
Authors: ZANETTI Giuliana , CIDARIA Dante , CURTI Bruno
Published: March 2005

Ferredoxin‐NADP+ reductase resolved into apoprotein and flavin by incubation with 2.5 M CaCl2 at pH 7.5 and 2°C. Essential factors to recover a reconstitutable apoprotein are dithiothreitol, glycerol and guanidine/HCl. The apoprotein is stable for at least a week at −20°C.

The release of the prosthetic group from the protein by the Ca2+ ions is a multi‐step process. Three different effects of these ions are identifiable: (a) a rapid 20–25% inhibition of catalytic activity, probably caused by an increase in the ionic strength of the medium; other cations can produce it as well; (b) a slower induction of a conformational change in the protein which causes complete loss of activity and exposure to solvent of the flavin moiety; the FAD is finally released from the protein; (c) complete conversion of FAD to FMN, which blocks reconstitution to holoenzyme, caused by the well‐known hydrolytic action of Ca2+ ions on the pyrophosphate bridge of FAD

Binding of FAD by the apoferredoxin‐NADP+ reductase is very rapid and it is complete in a few minutes even at 0°C. A Kd of 3.4 × 10−9 M has been determined by fluorescence titration. The reconstituted holoenzyme has catalytic activity, spectral and fluorescence properties nearly identical to the native enzyme. The gel electrophoresis and isoelectrofocusing patterns of the two enzymes are very similar. Removal of factors from the apoprotein solution such as dithiothreitol and glycerol promotes the appearance of protein aggregates.
Chemical Modification Studies on Ricinus communis (Castor Bean) Agglutinin
Authors: KHAN M. Islam , SUROLIA Avadhesha
Published: March 2005

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide‐binding properties was studied.

Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide‐binding activity of Ricinus agglutinin.

Modification of tryptophyl, arginyl and carboxyl‐group‐containing residues did not lead to any loss in the activity of the agglutinin.

Acetylation of tyrosyl groups with N‐acetylimidazole strongly reduced the hemagglutinating and saccharide‐binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin.

The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.
4‐Aminobutyrate Aminotransferase
Authors: CHURCHICH Jorge E.
Published: March 2005

The analogues 5‐phospho‐pyridoxal‐aminooxyacetate and 4‐vinyl‐pyridoxal 5‐phosphate inhibit reduced 4‐aminobutyrate aminotransferase reconstituted with pyridoxal 5‐phosphate, but they do not affect the catalytic activity of the holoenzyme. The binding of the 5‐phosphopyridoxal adducts to the holoenzyme was monitored by measuring the fluorescence properties of the protein and the ligands. The presence of nonequivalent binding sites in reduced samples of enzyme containing 1 mol and 1.8 mol P‐pyridoxyl residues/mol dimer was detected by steady and nanosecond fluorescence spectroscopy. The spectroscopic properties (fluorescence lifetime τ= 1 ns, polarization = 0.36 and quantum yield = 0.01) of the first molecule of 5‐phospho‐pyridoxyl are different from the properties of the second molecule of 5‐phospho‐pyridoxyl covalently bound to the enzyme (τ= 2.1 ns, P= 0.16, QT= 0.1). The spectroscopic properties of pyridoxal 5‐phosphate and 5‐phospho‐pyridoxal‐aminooxyacetate bound to different sites on the holoenzyme can be used to deduce proximity relationships between the catalytic sites of 4‐aminobutyrate aminotransferase. On the basis of resonance energy transfer measurements, it is proposed that a distance of 27 Å (2.7 nm) separates the two catalytic binding sites.
The Structural Isomerisation of Human‐Muscle Adenylate Kinase as Studied by 1H‐Nuclear Magnetic Resonance
Authors: KALBITZER Hans Robert , MARQUETANT Rainer , RÖSCH Paul , SCHIRMER R. Heiner
Published: March 2005

Human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3.) was studied by 1H‐nuclear magnetic resonance spectroscopy. The C‐2 and C‐4 proton resonances of the active‐center histidine His‐36 could be identified; the pK of His‐36 was determined as 6.1. The pK of His‐189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase [McDonald et al. (1975) J. Biol. Chem. 250, 6947–6954].

A pH‐dependent structural isomerization as shown by X‐ray crystallography in the pig enzyme [Pai et al. (1977) J. Mol. Biol. 114, 37–45] was not observed for human adenylate kinase in solution. However, the binding of adenosine(5′)pentaphospho(5′)adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s−1 and decreases by addition of Mg2+. On the basis of these values and the known dissociation constant it is likely that the binding of Ap5A is a diffusion‐controlled process, Kon being 108 M−1 s−1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which has been studied by NMR spectroscopy and X‐ray diffraction in parallel, is suitable for analysing the induced fit postulated by Jencks for all kinase‐catalyzed reactions.
Uptake, Metabolism and Excretion of Orally and Intravenously Administered, Double‐Labeled N‐GIycoloylneuraminic Acid and Single‐Labeled 2‐Deoxy‐2,3–dehydro‐N‐acetylneuraminic Acid in Mouse and Rat
Authors: NÖHLE Ulrich , BEAU Jean‐Marie , SCHAUER Roland
Published: March 2005

N‐Glycoloyl‐d‐[2‐14C,9‐3H]neuraminic acid enzymically prepared from sodium [2‐14C]pyruvate and N‐glycoloyl‐d‐[6‐3H]mannosamine by N‐acetylneuraminate lyase in 52% yield was orally administered to 20‐day‐old fasted mice. In the course of 6 h 98% of the total radioactivity belonging to the originally administered N‐glycoloyl‐neuraminic acid was resorbed from the intestine and appeared in the urine. 6 h after application total 0.7% of the 3H and 0.5% of the 14C radioactivity were recovered in blood, liver, spleen, kidney and brain. The difference between the 14C and the 3H quantities was detected as exhaled 14CO2. After intravenous injection of N‐glycoloylneuraminic acid into rats, 90% of the radioactivity was excreted in the urine within 10 min corresponding to the originally applied substance. After 6 h, 1.3% of the 3H and 1% of the 14C radioactivity were left in blood and in liver, spleen, kidney and brain.

An efficient preparation of 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid and 2‐deoxy‐2,3‐dehydroneuraminic acid from N‐acetylneuraminic acid is described, as well as the synthesis of a tritium‐labelled 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid. The unsaturated neuraminic acid derivative behaved similarly to N‐glycoloylneuraminic acid after oral and intravenous application. 99% of the radioactivity orally administered was resorbed from the intestine and excreted in the urine without chemical alteration in the course of 6 h. About 1% of the radioactivity administered orally to mice and 1.7% of the same compound administered intravenously to rats were still found in blood, liver, spleen, kidney and brain after this time.

The results suggest that the small amount of N‐glycoloylneuraminic acid retained in the organism was at least partially cleaved by N‐acetylneuraminate lyase. The high excretion rate of unchanged 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid and the fact that it does not act as a substrate for the lyase supposes that there is no further metabolism of this sialic acid under the conditions described. It may be concluded from these observations that N‐glycoloylneuraminic acid occurring in food cannot be used directly for the biosynthesis of glycoconjugates, which is important from an immunological point of view. The results presented here are compared with those obtained after application of double‐labeled N‐acetylneuraminic acid in former studies [U. Nöhle and R. Schauer (1981) Hoppe‐Seyler's Z. Physiol. Chem. 362, 1495–1506].
Structure‐Function Relationship in the Allosteric l‐Lactate Dehydrogenases from Lactobacillus casei and Lactobacillus curvatus
Authors: MAYR Ulrich , HENSEL Reinhard , DEPARADE Matthias , PAULY Hans E. , PFLEIDERER Gerhard , TROMMER Wolfgang E.
Published: March 2005

The activation of the allosteric l‐lactate dehydrogenases from Lactobacillus casei and Lactobacillus curvatus induced by binding of the effectors fructose 1,6‐bisphosphate [Fru(1,6)P2] and Mn2+ is correlated with conformational changes as indicated by alterations of the tryptophan and tyrosine absorption and by an alteration of the tryptophan fluorescence of the proteins. Both enzymes contain four NADH and Fru(1,6)P2 binding sites per tetramer as determined by fluorescence measurements. In the case of the L. casei enzyme at least four Mn2+ binding sites were determined for the tetrameric state by electron spin resonance (ESR) spectroscopy. The modification of tryptophan of the L. casei enzyme with dimethyl‐(2‐hydroxy‐5‐nitrobenzyl)‐sulfonium bromide suggests that the alteration of the tryptophan absorption is due to a tryptophan residue being located in the interior of the protein, whereas the alteration of the tryptophan fluorescence is due to a second tryptophan residue which is located on the surface of the enzyme. Thus the effector‐induced conformational changes may cause structural alterations in an inner as well as in an outer region.

To obtain information about the distance between the coenzyme and Mn2+ binding sites, ESR spectra were recorded of the spin‐labeled NADH analogs bound to the L. casei enzyme in the presence and absence of Mn2+. The analogs were substituted at C‐6 or C‐8 with a 4‐(2,2,6,6‐tetramethyl‐piperidinyl‐1‐oxyl)‐amino group. The oxidized forms of both derivatives, labeled by a nitroxide radical, were shown to be active coenzymes. However, no spin‐spin interaction between the spin label and Mn2+ could be observed, indicating that the Mn2+ binding site is at least 1.5–2.0 nm apart from the adenine moiety of the coenzyme. Although a direct interaction between the metal and coenzyme is unlikely because of this large distance, binding of the effectors to the L. casei enzyme causes changes of the fluorescence of enzyme‐bound NADH. Thus, the bound coenzyme appears to be affected by the conformational changes in the L. caseil‐lactate dehydrogenase induced by Fru(1,6)P2 and Mn2+.
The Reactions between Active and Inactive Forms of Wheat Ribulosebisphosphate Carboxylase and Effectors
Authors: GUTTERIDGE Steven , PARRY Martin A. J. , SCHMIDT C. N. Godfrey
Published: March 2005

The processes of activation and deactivation of ribulose‐1,5‐bisphosphate carboxylase purified from wheat have been investigated. Two forms of the enzyme are indistinguishable in terms of ribulose‐1,5‐bisphosphate carboxylation and oxidation but exhibit different rates of activation. One form is slowly activated in saturating CO2 and Mg2+ at moderate temperatures (t0.5∼ 120 min at 25°C), the other form rapidly activated (t0.5∼ 8 s).

In the presence of the effectors 6‐phosphogluconate or NADPH, significantly lower concentrations of the activating co‐factors can achieve full activation of both enzyme species. However, with another effector, fructose 1,6‐bisphosphate, for the slowly activating species the mode of action is the same as with 6‐phosphogluconate or NADPH, whereas the activation of the rapidly activating species is significantly inhibited. The substrate, ribulose 1,5‐bisphosphate, also inhibits this rapid activation process.

A mechanism is proposed for the reactions involving activation that accounts for the differential rates of activation and the response to effectors.
Crosslinking by Thiol Disulfide Interchange of 5,5′‐Dithiobis(2‐nitrobenzoic acid)‐Treated Light Chain and Heavy Chain of Rabbit Skeletal Myosin
Authors: MOCZ Gabor , BIRO Endre N. A. , BALINT Miklos
Published: March 2005

Interchain disulfide crosslinks between the heavy‐chain fragment in heavy meromyosin and myosin light chain 2, generated by 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2), are formed under appropriate ionic conditions at neutral pH as revealed by liberation of the chromogenic 2‐nitro‐5‐thiobenzoic acid. The presence of the original or of a slightly digested light chain 2 reduces the rate of the reaction of heavy meromyosin with Nbs2‐modified light chain 2 by 32–39%, if Ca2+ is present. Dodecyl sulfate/polyacrylamide gel electrophoresis in absence of reducing agents shows that Nbs2‐modified light chain 2 attaches to the heavy chain in the region of the 21‐kDa fragment of heavy meromyosin, which contains the essential thiol groups and which has been located at the subfragment 1/sub‐fragment 2 junction of myosin [Balint, M., Wolf, I., Tarcsafalvi, A., Gergely, J. and Sreter, F. A. (1978) Arch. Biochem. Biophys. 190, 793–799]. Modification of thiol‐1 groups with iodoacetamide as well as crosslinking the thiol‐1 and thiol‐2 groups by the bifunctional reagent p‐N,N′‐phenylenedimaleimide prior to incubation with Nbs2‐modified light chain 2 has no substantial effect on the crosslinking reaction. This indicates that other thiol groups are involved in the binding of Nbs2‐modified light chain 2 to the heavy chain. An examination of K+, Ca2+, Mg2+ and actin‐activated Mg2+ ATPase activities of heavy meromyosin that had been crosslinked with Nbs2‐modified light chain 2 shows only a slight change in comparison with intact heavy meromyosin, indicating that crosslinking had not altered significantly the hydrolytic site. Crosslinking of Nbs2‐modified light chain 2 to light‐chain‐2‐deficient heavy meromyosin restored the original light‐chain‐2‐dependent Ca2+ sensitivity of the tryptic fragmentation of heavy meromyosin, suggesting that crosslinking takes place at the proper binding site for light chain 2.
Effect of Caffeine on Glucose‐Induced Inactivation of Gluconeogenetic Enzymes in Saccharomyces cerevisiae
Authors: TORTORA Paolo , BURLINI Nedda , HANOZET Giorgio M. , GUERRITORE Andrea
Published: March 2005

The mechanism of catabolite inactivation of three gluconeogenetic enzymes, fructose‐1,6‐bisphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase, has been studied in the yeast Saccharomyces cerevisiae. The glucose‐induced inactivation of the three enzymes is remarkably retarded by pre‐incubation of the cells with different caffeine concentrations; however, a full conservation of activity has never been obtained, even at the highest drug concentration. Caffeine modifies the metabolic effects produced in the yeast cell by exposure to glucose. It reduces the consumption rate of glucose; changes the glycolytic intermediate pattern, giving rise to a crossover point at the level of the phosphofructokinase/fructose‐bisphosphatase cycle; and increases the ATP level and the energy charge. Moreover, it substantially reduces the peak of intracellular cAMP content that immediately follows glucose entry; the magnitude of this effect is dependent on the drug concentration. The effect on the change of intracellular cAMP level appears, among all metabolic effects determined by caffeine, the only plausible one to explain the interference with catabolite inactivation of enzymes. Actually a strong negative correlation between residual activity of each of the three investigated enzymes and intracellular cAMP level has been demonstrated. The existence of a common mechanism of action of cAMP, as the mediating factor for catabolite inactivation of all three enzymes, is proposed.
Adrenocorticotropic Hormone and α‐Melanocyte‐Stimulating Hormone Induce Secretion and Protein Phosphorylation in the Rat Lacrimal Gland by Activation of a cAMP‐Dependent Pathway
Authors: JAHN Reinhard , PADEL Ulrike , PORSCH Paul‐Hermann , SÖLING Hans‐Dieter
Published: March 2005

In the present study a cAMP‐dependent pathway leading to exocytosis in the rat lacrimal gland was investigated in detail. Using a lobule system in vitro, adrenocorticotropic hormone (corticotropin) and α‐melano‐cyte‐stimulating hormone (α‐melanotropin) stimulated protein discharge almost equally effective as dibutyryl‐adenosine 3′,5′‐monophosphate (Bt2cAMP) and 3‐isobutyl‐1‐methylxanthine but less potent than carbamoyl‐choline. Maximal stimulation was obtained at a hormone concentration of 20 nM for both peptides indicating that the active sequence is part of the first 13 amino acids of the corticotropin molecule. 3‐Isobutyl‐1‐methyl‐xanthine (40 μM) potentiated the effect of corticotropin on secretion. In contrast to the action of carbamoyl‐choline, corticotropin‐induced protein discharge was not inhibited by omission of Ca2+ ions from the incubation medium.

The tissue content of cAMP was not significantly affected, by corticotropin or 40 μM 3‐isobutyl‐1‐methyl‐xanthine. However, a combination of both agonists led to a rapid 2.5–5‐fold increase of the tissue cAMP level in vitro. Adenylate cyclase activity of a lacrimal membrane fraction was effectively stimulated by corticotropin as well as by α‐melanotropin. The following hormones and neurotransmitters were ineffective with respect to induction of secretion or activation of adenylate cyclase: isoproterenol (20 μM), dopamine (10 μM), secretin (0.1 μM) glucagon (10 nM), vasoactive intestinal peptide (0.1 μM), substance P (0.1 μM), pancreozymin (0.1 μM) and methioninenkephalin (0.1 μM).

Corticotropin, α‐melanotropin, Bt2cAMP and 3‐isobutyl‐1‐methylxan thine induced the phosphorylation of three membrane‐bound proteins (Mr 35000, 26000 and 20000) which corresponded almost exactly to the EC‐protein and the proteins II and III respectively described previously for the parotid gland [Jahn, R., Unger, C. and Söling, H.D. (1980) Eur. J. Biochem. 112, 345–352]. In accordance with an earlier report [Jahn, R. and Soling, H.D. (1981) FEBS Lett. 131, 28–30] proteins II and III were not phosphorylated during cholinergic stimulation. The combination of submaximal concentrations of agonists acting via the same or via different mechanisms resulted in an additive enhancement of secretion as well as of phosphorylation of the EC‐protein.
Activity of Cyclic‐AMP Phosphodiesterase in Permeabilised Cells of Bakers' Yeast
Authors: LONDESBOROUGH John
Published: March 2005

Yeast cyclic‐AMP phosphodiesterases were assayed in situ, using cells permeabilised with cytochrome c, to get information about the kinetics of these enzymes at the high concentrations of macromolecules occurring in vivo. Protamine treatment was not suitable, because it perturbed the intracellular localisations of both the (Mg‐dependent) low‐Km enzyme and the (EDTA‐insensitive) high‐Km enzyme. The pH‐dependence of Km and V for EDTA‐insensitive activity in situ agreed well with the behaviour of pure high‐Km enzyme, except that near pH 8 Hofstee plots were bent slightly upwards both for activity in situ and with crude broken‐cell preparations. Hofstee plots of Mg‐dependent activity in situ were distinctly concave, and could be resolved mathematically into two activities, one (accounting for about 30% of the Mg‐dependent V) with a Km close to the value in vitro of 0.2 μM, and the other with an apparent Km of 3 μM. The 3 μM Km activity probably represents a fraction of the low‐Km enzyme that is particle‐bound at the high protein concentrations occurring in situ.
Purification of Penicillin‐Binding Protein 3 from Streptococcus pneumoniae
Authors: HAKENBECK Regine , KOHIYAMA Masamichi
Published: March 2005

Penicillin‐binding protein 3 from wild‐type Streptococcus pneumoniae has been purified to homogeneity by solubilization with Triton X‐100 and successive column chromatography. The penicillin‐binding activity during the fractionation procedure was monitored with a rapid filter binding assay using [3H]propionylampicillin and penicillin‐binding protein 3 identified after fluorography of dodecyl sulfate gels. The purified protein showed penicillin‐sensitive d, d‐carboxypeptidase activity.
Purification, Properties and Subunit Structure of Arginase from Iris Bulbs
Authors: BOUTIN Jean‐Pierre
Published: March 2005

Arginase (l‐arginine amidinohydrolase, EC 3.5.3.1) from Iris hollandica bulbs has been purified approximately 20000‐fold. The purification procedure involved extraction from a particulate fraction (probably mitochondria), DEAE‐Sephacel chromatography, aminohexyl‐Sepharose 4B chromatography and gel filtration on Ultrogel AcA 34. Optimum assay conditions were determined, i.e. pH 9.0, 0.5 mM Mn2+, 0.5 mM dithio‐threitol. The arginase is a slightly acidic protein (PI ∼ 5.6) highly specific for l‐arginine.

The arginase was almost completely inactivated by dialysis against a Mn2+‐free buffer, 70% of the activity was then recovered after a treatment with 0.1 mM Mn2+ and 1 mM dithiothreitol at 40°C for 45 min. The Mn2+ was found essential for the preservation of the arginase activity during the enzyme assay and for activation. However, the optimum Mn2+ concentration for activation was reduced to one‐tenth and a better activity recovery was obtained in the presence of 1 mM dithiothreitol. The same dithiothreitol and Mn2+ requirement was observed to preserve arginase activity on storage.

The native arginase showed an apparent Mr, of about 191000 as estimated by gel filtration and polyacrylamide gradient electrophoresis in the presence of 1 mM Mn2+. The subunit apparent Mr, was 36500 as estimated by dodecylsulfate electrophoresis. The arginase dissociated into reactivatable oligomers (apparent Mr, = 59000 ± 5000 and 120000) during electrophoresis on polyacrylamide gradient gels, carried out in a Mn2+‐free electrophoresis buffer. The intramolecular cross‐linkage of subunits by glutaraldehyde treatment showed that the purified arginase dissociated into dimers when losing its Mn2+. These results suggest that the iris bulb arginase is a hexamer, which can be dissociated into activatable dimers when losing its Mn2+. Such a structure has never been shown with other arginases.
Reduction of Respiratory‐Chain Cytochrome b by Lactate in Saccharomyces cerevisiae
Authors: BRIQUET Michel , PURNELLE Bénédicte , BEATTIE Diana S. , GOFFEAU André
Published: March 2005

Cytochrome b of yeast mitochondria can be reduced by a part of the electrons resulting from the oxidation of lactate enantiomers.

The respiration of d‐lactate and l‐lactate is 30–40% inhibited by antimycin A.

Reduction of cytochrome b is observed in submitochondrial particles in the presence of low concentration of d‐lactate and l‐lactate (half‐optimal concentration of 4.7 mM and 2.4 mM respectively) in the presence of different bc1 inhibitors.

Reduction of cytochrome b and c1 occurs in purified complex III of yeast in the presence of l‐lactate and added l‐lactate:NAD+ oxidoreductase.

In the particles obtained from yeast grown in lactate the oxidation of l‐lactate involves the reduction of a pigment absorbing at 558 nm.
Primary Structure of Parvalbumin from Rat Skeletal Muscle
Authors: BERCHTOLD Martin W. , HEIZMANN Claus W. , WILSON Kenneth J.
Published: March 2005

The primary structure of parvalbumin from rat skeletal muscle has been determined principally by automated sequencing of tryptic peptides using 4‐N,N‐dimethylaminoazobenzene 4′‐isothiocyanate as the Edman reagent on a solid‐phase sequencer. Remaining positions and most peptide overlaps were identified by analysis of peptides arising from CNBr, chymotryptic and Staphylococcus aureus protease cleavages and through digestions with carboxypeptidases A, B and Y. Reverse‐phase high‐performance liquid chromatography on C‐18 supports was employed for all peptide separations. Structural homology between rat and rabbit parvalbumins helped to confirm the alignments of the tryptic peptides T4‐T3, T2‐T6 and to define the position of the Lys triplet (36–38). A comparison of the two mammalian proteins revealed 14 amino acid differences, which are all located on the surface of the molecule. A prediction of the secondary structure has been made and found to be very similar for the rat and rabbit proteins with the exception of the sequence region 72–78, located between the Ca2+, Mg2+‐binding CD and EF domains.
The Chemistry of Favism‐Inducing Compounds
Authors: CHEVION Mordechai , NAVOK Tikva , GLASER Gad , MAGER Jacob
Published: March 2005

Isouramil and divicine are pyrimidine aglycones of two glucosides found in broad beans. They have been shown to be strong reducing agents. Their reaction with oxygen in a (gas) saturated solution, 26°C, is characterized by τ1/2= 1 min and 3 min respectively. Hydrogen peroxide is formed in this reaction stoichiometrically (1:1). The pyrimidines lose two hydrogen and form an intermediate that is structurally analogues to alloxan. This intermediate is not stable, and in the absence of reducing agents it decomposes, possibly by ring‐cleavage.

In the presence of reduced glutathione the intermediate is reduced and can now react with oxygen once again. Thus, the pyrimidines cycle between the two states and the net reaction is the catalytic oxidation of glutathione by molecular oxygen; in each cycle 4 molecules of glutathione are dissipated.

The possible involvement of these pyrimidines in the pathogenesis of favism may be in a similar mechanism. Red blood cells deficient in glucose‐6‐phosphate dehydrogenase cannot cope with such an oxidative challenge exerted by the pyrimidines. Consequently an irreversible cellular damage can take place leading to the enhanced sequestration of these red blood cells by the reticuloendothelial system.
Mitochondrial Transcripts in Glucose‐Repressed Cells of Saccharomyces cerevisiae
Authors: BALDACCI Giuseppe , ZENNARO Elisabetta
Published: March 2005

We have compared mitochondrial transcripts from yeast Saccharomyces cerevisiae strain D273‐10B grown in the presence of 2% galactose (non‐repressed cells) or 15% glucose (glucose‐repressed cells).

The ethidium‐bromide‐stained electrophoretic pattern of mitochondrial RNAs from glucose‐repressed cells shows a clear decrease of tRNAs. In addition, some RNA bands appear to be specific for a single growth condition. To identify these RNA species we have performed hybridization experiments with 32P‐labelled mitochondrial DNA from petite mutant cells. The mitochondrial repeat units of the mutants retained only one of the following genes: oxi1, oxi2, oxi3, oli2, cob and oli1. Unchanged amounts of oxi2 and oli2 transcripts and reduced concentrations of oli1 and oxi1 putative mRNAs are present in glucose‐repressed cells. In the same growth condition we observe a decreased processing of a precursor RNA species from the split cob gene and reduced amounts of transcripts corresponding to the first, second and fifth intron of the split oxi3 gene. The oxi3 first and second introns, whose transcripts are the most variable, include long open reading frames in their nucleotide sequence, but at present it is not known whether the corresponding RNA species have a functional role. Our results show that their concentrations are related to the growth condition.
Messenger Ribonucleoproteins of Cells Infected by Simian Virus 40 Contain Large T‐Antigen
Authors: MICHEL Marcel R. , SCHWYZER Martin
Published: March 2005

In monkey or mouse cells undergoing lytic or transforming infection with simian virus 40, about 10% of large tumor antigen (T‐antigen) molecules were consistently present in the cytoplasm and 90% in the nucleus. The bulk of cytoplasmic large T‐antigen cosedimented in linear sucrose gradients with polyribosomes (150–500 S) and with messenger ribonucleoproteins sedimenting within 20–80 S. As determined by centrifugation in discontinuous sucrose gradients, its apparent density (1.2–1.3 g/ml) corresponded to that of ribonucleoproteins. In contrast, the bulk of nuclear T‐antigen sedimented between 5–20 S and its apparent density (1.1 g/ml) corresponded to that of free protein. Nuclear T‐antigen added before cell fractionation did not bind to cytoplasmic constituents. After dissociation of purified polyribosomes with puromycin/KCl or EDTA, cytoplasmic large T‐antigen cosedimented with the released messenger ribonucleoproteins containing poly(A)‐rich messenger RNA. Upon hydrolysis of the RNA with RNase A, large T‐antigen exhibited the sedimentation properties and density of free protein. The results suggest that cytoplasmic large T‐antigen is associated with messenger ribonucleoproteins.
Modulation of the Nuclear‐Envelope Nucleoside Triphosphatase by Poly(A)‐rich mRNA and by Microtubule Protein
Authors: BERND August , SCHRÖDER Heinz C. , ZAHN Rudolf K. , MÜLLER Werner E. G.
Published: March 2005

Nuclear envelopes contain a nucleoside triphosphatase which is thought to be involved in the supply of energy for nucleo‐cytoplasmic RNA transport. This enzyme is stimulated most efficiently by poly(A) and to a lesser extent by poly(G) and poly(dT). Half‐maximal stimulation of the enzyme from rat liver nuclei, which was associated with the poly(A)‐specific endoribonuclease IV and was free from poly(A) polymerase and endoribonuclease V activity, was determined to occur at a concentration of 1.1 × 106 poly(A) molecules/nuclear ghost. Double‐reciprocal plot analyses revealed a 2.8‐fold stimulation of the enzyme by poly(A). Poly(A) in the hybrid form had no influence on the activity of the nucleoside triphosphatase. Stimulation by oligo(A) required a minimum chain length of 18 nucleotide units. Naturally occurring RNA species enhanced the nucleoside triphosphatase activity, provided that they contained a poly(A) segment. Using poly(A)‐rich mRNA, half‐maximal stimulation was determined to proceed at 0.5 × 106 molecules/nuclear ghost. Removal of the poly(A) segment from mRNA abolished the stimulatory effect on the enzyme.

Microtubule protein was found to inhibit the nucleoside triphosphatase efficiently. At a concentration of 2.0 mg/ml, polymerized microtubule protein reduced the enzyme activity by 96%. Dimeric tubulin was less inhibitory, while actin was without any significant effect.

From these findings it is suggested that a possible nucleoside‐triphosphatase‐mediated transport of poly(A)‐rich mRNA through nuclear envelopes is controlled, first, by the poly(A) segment of this RNA species and, secondly, by cytoplasmic microtubules.
Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β2 Subunits
Authors: WILHELM Peter , PILZ Ingrid , LANE Andrew N. , KIRSCHNER Kasper
Published: March 2005

The α and β2 subunits of tryptophan synthase were investigated by small‐angle X‐ray scattering. The molecular parameters are: radius of gyration, α: 1.95 nm, β2: 3.01 nm; maximum particle diameter, α: 5.8 nm, β2: 10.5 nm; and hydrated volume, α: 60 nm3, β2 160 nm3. The shape of the α subunit can best be described by a circular cylinder, slightly tapered at one end. An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the β2 subunit.

The α2β2 enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3. No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the α and β2 subunits. Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the α2β2 complex.
Regulation of the Aminoacyl‐tRNA Synthetase Complex of Rat Liver by Phosphorylation/Dephosphorylation in vitro and in vivo
Authors: DAMUNI Zahi , CAUDWELL F. Barry , COHEN Philip
Published: March 2005

Incubation of liver extracts with MgATP led to complete inactivation of the aminoacyl‐tRNA synthetase complex provided that sodium fluoride was included. If sodium fluoride was omitted no inactivation was observed. The arginyl‐tRNA and isoleucyl‐tRNA synthetases were inactivated more slowly than the leucyl‐tRNA, lysyl‐tRNA and methionyl‐tRNA synthetases. Gel filtration on Sepharose 4B separated the aminoacyl‐tRNA synthetase complex (Ve/Vo= 1.2) from an inactivating factor that required MgATP (Ve/Vo= 2.8).

The inactivated aminoacyl‐tRNA synthetase complex was eluted from Sepharose‐4B at the same position as the activated complex, and was resolved from a reactivating factor that required Mg2+ and was inhibited by sodium fluoride (Ve/Vo= 2.5). The arginyi‐tRNA and isoleucyl‐tRNA synthetases were reactivated more rapidly than the leucyl‐tRNA, lysyl‐tRNA and methionyl‐tRNA synthetases.

The aminoacyl‐tRNA synthetase complex could also be reactivated using highly purified preparations of either protein phosphatase 1 or protein phosphatase 2A. Inhibitor 2, a specific inhibitor of protein phosphatase 1, prevented reactivation of the complex by protein phosphatase 1, but not by protein phosphatase 2A. The relative rates of reactivation of the different activities of the complex by either protein phosphatase 1 or protein phosphatase 2A were very similar to those observed with the reactivating factor.

The aminoacyl‐tRNA synthetase complex was largely in the activated state in livers of normally fed rats, but overnight starvation produced substantial conversion to the inactivated form of the complex. Incubation of hepatocytes from normally fed rats with glucagon and isobutylmethylxanthine for 20 min also caused a substantial decrease in the activation state from approximately 80% to approximately 30%. Conversely, when hepatocytes prepared from starved rats were incubated with insulin for 30 min, almost complete reactivation of the complex was achieved.

The results indicate that the aminoacyl‐tRNA synthetase complex is regulated by a phosphorylation dephosphorylation mechanism in vitro and in vivo. The effects of hormones on the activation state of the complex are discussed in the light of the observation that the inactivating factor is not cyclic‐AMP‐dependent protein kinase.