bluuebunny/crossref_metadata_2025_split huggingface dataset:
Description of enzyme kinetics in reversed micelles
Authors: VERHAERT Raymond M. D. , HILHORST Riet , VERMUË Marian , SCHAAFSMA Tjeerd J. , VEEGER Cees 
Published: March 2005

In the literature measurements of kinetic data of enzymes in reversed micelles have been interpreted in two ways. In the first, all enzyme parameters are expressed with respect to the total volume of the reversed micellar solution. In the second, the enzymatic conversion is related only to the fraction of the volume consisting of aqueous solution (pseudophase model). In this paper equations are derived describing the rate of an enzymatic reaction for three different kinds of enzymes: enzymes obeying Michaelis‐Menten kinetics, enzymes following a ping‐pong bi‐bi mechanism and enzymes which convert substrates according to an ordered mechanism. In deriving these equations, a distinction is made between intermicellar exchange reactions of substrate(s) and product(s) and the enzymatic reaction which takes place in the waterpool of a reversed micelle. In the description, all intrinsic rate constants of the enzyme are assumed to be independent of its environment. The rate equations show that the presence and efficiency of the intermicellar exchange reaction, which supplies the enzyme with substrate and removes product, can affect the rate of an enzymatic reaction under common experimental conditions.Whereas kinetic parameters derived from double‐reciprocal plots often seem to be affected by enclosure in reversed micelles, these apparent deviations from kinetics in aqueous media can be explained by the model presented here as arising from exchange phenomena. Neither the experimentally determined maximum enzyme velocity, vmax, nor the Michaelis constants are affected by the incorporation of the enzyme in reversed micelles. The deviations of kinetic parameters from the aqueous values are shown to depend strongly on the concentration of reversed micelles, the intermicellar exchange rate and the volume fraction of water, a dependence in agreement with findings reported in the literature.
Ribofuranosyl‐benzimidazole derivatives as inhibitors of casein kinase‐2 and casein kinase‐1
Authors: MEGGIO Flavio , SHUGAR David , PINNA Lorenzo A. 
Published: March 2005

5,6‐Dichloro‐1‐(β‐d‐ribofuranosyl)benzimidazole (DiCl‐RB) is a powerful inhibitor of casein kinase‐2 (CK‐2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414–3420]. Here a series of 17 analogues of DiCl‐RB has been employed for studying the specificity and the mode of action of this family of CK‐2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6‐dibromo derivative (DiBr‐RB) being fivefold more effective than DiCl‐RB (Ki= 2 μ M cf. 10 μ M, with GTP as substrate), whereas the difluoro derivative (DiF‐RB) is nearly as ineffective as unsubstituted 1‐(β‐d‐ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl‐Bz) is an inhibitor almost as good as DiCl‐RB. Inhibition of CK‐2 by DiCl‐RB and by its analogues, DiCl‐Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr‐RB, with ATP and GTP, are 6 μ M and 2 μ M, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP.DiBr‐RB has been assayed for its inhibitory capacity toward several protein kinase other than CK‐2. Protein kinase‐C, cAMP‐dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr‐RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK‐2. Casein kinase‐1 however is nearly as sensitive as CK‐2 to DiBr‐RB. Inhibition of CK‐1 is also of the competitive type with respect to ATP (Ki= 14 μ M). Although the inhibitory spectrum of CK‐1 by the various analogues is reminiscent of that observed with CK‐2, a remarkable difference is revealed by 5′‐phosphorylation of ribose which increases the Ki with CK‐2 while decreasing that with CK‐1.
Localization and characterization of three different β‐adrenergic receptors expressed in Escherichia coli
Authors: CHAPOT Marie‐Pierre , ESHDAT Yuval , MARULLO Stefano , GUILLET Jean‐Gérard , CHARBIT Alain , STROSBERG A. Donny , DELAVIER‐KLUTCHKO Colette 
Published: March 2005

After fusion with the N‐proximal portion of the outer membrane protein LamB, three β‐adrenergic receptors, the human β1‐ and β2‐ and turkey β1‐adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti‐peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti‐peptide antibodies.The functional expression in E. coli of three β‐adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G‐protein‐coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes.
Isolation and characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellifera)
Authors: CASTEELS Peter , AMPE Christophe , RIVIERE Lise , van DAMME José , ELICONE Christopher , FLEMING Mark , JACOBS Frans , TEMPST Paul 
Published: March 2005

Honeybees (Apis mellifera) are frequently exposed to and likely to be infected by plant‐associated bacteria. We mimicked this process by injecting bees with live bacteria and isolated five induced antibacterial substances by comparative liquid chromatographic mapping of the hemolymph. Three of these antibiotics belong to a unique family of small (18 amino acids) peptides: the apidaecins [Casteels et al. (1989) EMBO J. 8, 2387–2391].We have now characterized a fourth bee immune response peptide. The complete sequence was established by Edman degradation of the peptide and fragments thereof. It is 34 amino acids long and contains 10 proline residues. The amino‐terminal half is related to the apidaecins; similar proline motifs are also present in the aminoterminal quarter of the much longer fly diptericins.The newly identified peptide's broad spectrum, lower specific activities against Gram‐negative plant pathogens and its inability to inhibit bacterial growth at medium ionic strength are different from the apidaecins. Moreover, the highest observed specific activity was against an apidaecin‐resistant Xanthomonas strain. In contrast to the immediate action of apidaecins, bactericidal activity is delayed. We propose the name ‘abaecin’ for this new antibacterial response peptide.
Intraorganelle localization and substrate specificities of the mitochondrial acyl‐CoA:sn‐glycerol‐3‐phosphate O‐acyltransferase and acyl‐CoA:1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferase from potato tubers and pea leaves
Authors: FRENTZEN Margrit , NEUBURGER Michel , JOYARD Jacques , DOUCE Roland 
Published: March 2005

The mitochondrial sn‐glycerol‐3‐phosphate and 1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn‐glycerol‐3‐phosphate O‐acyltransferase behaved like a soluble protein of the intermembrane space.The sn‐glycerol‐3‐phosphate O‐acyltransferase of both potato and pea mitochondria used sn‐glycerol‐3‐phosphate but not dihydroxyaetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1‐acyl‐sn‐glycerol‐3‐phosphate which subsequently served as substrate for the second acylation reaction at its C‐2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl‐CoA than with the corresponding acyl‐(acyl carrier protein) thioesters.When different acyl‐CoA thioesters were offered separately, the sn‐glycerol‐3‐phosphate O‐acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1‐acyl‐sn‐glycerol‐3‐phosphate O‐acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl‐CoA thioesters. Furthermore, these enzymes preferentially used oleoyl‐ and linoleoyl‐CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn‐glycerol‐3‐phosphate O‐acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.
A two‐dimensional 1H NMR study on Megasphaera elsdenii flavodoxin in the reduced state
Authors: van MIERLO Carlo P. M. , VERVOORT Jacques , MÜLLER Franz , BACHER Adelbert 
Published: March 2005

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41°C at pH 8.3. Spin systems were sequentially assigned using phase‐sensitive two‐dimensional‐correlated spectroscopy and phase‐sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against 2H2O were obtained.Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the 1H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring.The majority of the nuclear Overhauser effect contacts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin.
Purification and characterization of Escherichia coli RNase I
Authors: MEADOR James , CANNON Barbara , CANNISTRARO Vincent J. , KENNELL David 
Published: March 2005

The endoribonuclease, RNase I, was purified from the periplasm of Escherichia coli. Based on PAGE, it has molecular mass of approximately 27 kDa with a migration rate indistinguishable from that of the recently reported RNase M from E. coli. The amino acid sequence of the two enzymes must be very similar based on two‐dimensional mapping of their tryptic peptides and suggests either a post‐transcriptional modification to yield different proteins from the same gene or evolution of two genes by gene duplication. However, while RNase I could degrade each of the four ribonucleotide homopolymers, only poly(U) or poly(C) were good substrates for RNase M with possibly some hydrolysis of poly(A). The reaction rate for poly(C) hydrolysis with RNase M was about ten times faster than for poly(U), while for RNase I the rates were about equal.Besides differences in specificity, RNase M was only located in the spheroplasts while RNase I found in the periplasm of growing cells. In terms of function, RNase I is known to cause degradation of rRNA during periods of stress or non‐growth, whereas it has been proposed that RNase M is the endonuclease for mRNA degradation in growing cells.
Induction of the 4‐aminobutyrate and urea‐catabolic pathways in Saccharomyces cerevisiae
Authors: VISSERS Stephan , ANDRE Bruno , MUYLDERMANS Francine , GRENSON Marcelle 
Published: March 2005

In the yeast Saccharomyces cerevisiae, induction of the 4‐aminobutyrate‐catabolic pathway by 4‐aminobutyrate requires two positive regulatory factors, encoded by the UGA3 and the UGA35 genes respectively. In addition to this, expression of one gene of this pathway, namely the UGA4 gene encoding the 4‐aminobutyrate‐specific permease, is controlled negatively by the product of the UGA43 gene [Vissers, S., André, B., Muyldermans, F. & Grenson, M. (1989) Eur. J. Biochem. 181, 357–361].We show here that the products of two of these regulatory genes, UGA35 and UGA43, also control the expression of the genes encoding the urea‐catabolic pathway, although the 4‐aminobutyrate and urea‐catabolic pathways are synthesised under specific conditions and do not share any enzymatic step or metabolite: the UGA35 gene is shown to be identical to the DURL gene which was previously identified as a positive regulatory factor of the urea‐catabolic pathway; the UGA43 gene product is shown to behave like a negative regulatory factor of this pathway.In contrast to UGA35/DURL and UGA43, the positive regulatory factors encoded by the UGA3 gene and the previously identified DURM gene specifically control 4‐aminobutyrate and urea catabolisms respectively.Northern hybridization experiments suggest that the UGA35/DURL and UGA43 common regulatory factors act at the transcriptional level.Our results show that the expression of two biochemically distinct nitrogenous catabolisms, as triggered by their respective inducers, seems to involve multiple regulatory factors, some of which are common to the two catabolic pathways.
Heat capacity and entropy changes of the major isotype of the toad (Bufo) parvalbumin induced by calcium binding
Authors: TANOKURA Masaru 
Published: March 2005

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0, and at 5°, 15°, and 25° C. The two sites in a molecular were equivalent on Mg2+‐Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25°C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin of Ca2+ and Mg2+ binding and Mg2+ ‐Ca2+ exchange have been estimated by the empirical method of Strurtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236–2240].Although no major conformational changes were noted between Ca2+ ‐ and Mg2+‐ bound forms of toad pervalbumin, the conformational difference was larger in Ca2 (or Mg2+) binding to site 1 than site 2. This may indicate that the metal‐free form is much less stable than any with Ca2+ (or Mg2+) bound at one site at least. On Mg2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former inidcates the sequestering of nonpoloar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2+ ‐Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2+ ‐C2+ exchange.
Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding
Authors: SVENSSON Birte , CLARKE Anthony J. , SVENDESEN Ib , MØLLER Hanne 
Published: March 2005

Functionally important carboxyl groups in glucoamylase G2 form Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose‐inhibited enzyme with 1‐ethyl‐3(4‐azonia‐4,4‐dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantions. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC‐labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme‐acarbose complex were also identified.Asp176, Glu179 and Glu180 are invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and α‐amylase. The partially radiolabelled Asp112 corresponds in Taka‐amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.
Nuclear magnetic relaxation studies of the role of the metal ion in Mn2+‐substituted aminoacylase I
Authors: HEESE Dirk , BERGER Stefan , RÖHM Klaus‐Heinrich 
Published: March 2005

Substitution of the essential Zn2+ ions of porcine kidney aminoacylase I (EC 3.5.1.14) by Mn2+ did not markedly affect the kinetic properties of the enzyme. Using Mn2+as a paramagnetic probe, we were able to study the conformations of bound ligand by measuring the enhancement of ligand proton relaxation in 1H NMR. In addition, the effects of inhibitors on the paramagnetic enhancement of water proton relaxation rates were examined.The results of both approaches, in agreement with kinetic evidence, suggest that the metal center of aminoacylase I is too distant from the ligand binding site to allow direct participation of the metal in substrate binding or catalysis. We, therefore, propose that the metal ion of aminoacylase I plays a purely structural role.
Lactate dehydrogenase from the extreme thermophile Thermotoga maritima
Authors: WRBA Alexander , JAENICKE Rainer , HUBER Robert , STETTER Karl Otto 
Published: March 2005

Lactate dehydrogenase was isolated from the extreme thermophilic eubacterium Thermotoga maritima. The enzyme is stereospecific for L(+)‐lactate. It represents a homotetramer of 144 kDa molecular mass, with a sedimentation coefficient of s20,w∼ 7 S.Under physiological temperature conditions, the enzyme shows high catalytic efficiency with a broad pH optimum at pH 7.0 ± 1.0, and long‐term stability up to 80°C. The coenzyme, NAD+, and the effector fructose 1,6‐bisphosphate [Fru(1,6]P2] increase the thermal stability: at 90°C (pH 6.0), the liganded enzyme exhibits a half‐life of thermal inactivation of 150 min. The enhanced rigidity of the enzyme at ambient temperature is reflected by an anomalously high stability toward guanidine denaturation: the midpoint of the equilibrium transition being 1.6 M guanidine hydrochloride. Under optimum conditions of the enzyme assay, the Michaelis constants (Km) for NADH, NAD+, pyruvate and L(+)‐lactate at 55°C, and in the absence of Fru (1,6)P2, are 0.03 mM, 0.09 mM and 410 mM, respectively; Fru(1,6)P2, as a positive shifts the Km values for pyruvate and L(+)‐lactate to 0.06 mM and 25 mM, respectively. The Km values for the coenzyme are not affected. Neither Mn2+ nor other divalent cations have any activating effect.In contrast to lactate dehydrogenases from eukaryotes, the N‐terminus of the enzyme from Th. maritima is not acetylated. Comparison of the 30 N‐terminal amino acid residues with lactate dehydrogenase from Thermus aquaticus shows a high degree of similarity. This also holds if the two lactate dehydrogenases are compared with the glyceraldehyde‐3‐phosphate dehydrogenases from the same organisms.
Characterization of a soluble form of human CD4
Authors: HARRIS Reed J. , CHAMOW Steven M. , GREGORY Timothy J. , SPELLMAN Michael W. 
Published: March 2005

CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen‐presenting cells bearing the class II major histocompatability (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells.This 368‐amino‐acid glycoprotein contains two potential sites of N‐linked glycosylation (Asn‐271 and Asn‐300) and six cysteine residues. Amino‐terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P. J. et al. (1985) Cell 42, 93–104]. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed‐phase HPLC after digestion of S‐carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin‐digested rCD4 confirmed that the carboxy‐terminus of the protein was Pro‐368. Enzymatic digestion of non‐reduced rCD4 generated disulfide‐bonded fragments that demonstrated the presence of disulfide bonds between Cys‐16 and Cys‐84, Cys‐130 and Cys‐159, and between Cys‐303 and Cys‐345.The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N‐acetylglucosamine and N‐acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N‐glycosidase F demonstrated that both potential N‐glycosylation sites are utilized. The tryptic map of rCD4 treated with endo‐β‐N‐acetylglucosamine H demonstrated that only complex‐type oligosaccharides are attached to Asn‐271, while Asn‐300 has high‐mannose or hybrid structures attached in addition to complex‐type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn‐300 or Asn‐271 while no galactosamine was observed. This suggests that rCD4 contains no O‐linked oligosaccharides.