bluuebunny/crossref_metadata_2025_split huggingface dataset:
Involvement of a lysine residue in the N‐terminal Ni2+ and Cu2+ binding site of serum albumins
Authors: SADLER Peter J. , TUCKER Alan , VILES John H. 
Published: March 2005

We report one‐dimensional and two‐dimensional 1H‐NMR studies of the binding of Ni2+, Cu2+, Co2+, Cd2+ and Al3+ to defatted bovine and human serum albumins. The diamagnetic shifts induced by Ni2+, and paramagnetic effects due to Cu2+, were consistent with strong binding to a square‐planar site formed by the three N‐terminal amino acid residues (Asp‐Thr‐His for bovine, and Asp‐Ala‐His for human albumin). In contrast to previous studies on isolated 1–24 N‐terminal peptide, a Lys residue also appeared to be involved in the binding site, and is assigned as Lys4. A second His residue is also close to the Cu2+/Ni2+ binding site in bovine serum albumin and is assigned to His59 (not present in human albumin).Co2+ caused specific perturbation of the resonances for the three N‐terminal residues as well as those for Lys4. This is the first evidence for Co2+ binding to the N‐terminal metal site of serum albumin. Neither Al3+ nor Cd2+ perturbed resonances for the N‐terminal amino acids, but bind elsewhere in the protein.
Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting
Authors: LUMB Michael J. , PURDUE P. Edward , DANPURE Christopher J. 
Published: March 2005

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species‐specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group).Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern‐blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45508 Da. The N‐terminal 22 amino acids comprised the putative mitochondrial‐targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre‐mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase‐protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial‐targeting sequence is excluded from the open reading frame). In vitro studies showed that the 45 kDa polypeptide was imported into rat liver mitochondria and processed to a mature protein of ∼43 kDa, compatible with the cleavage of the N‐terminal 22 amino acids, as is also the case in rat and marmoset. A polypeptide in which the N‐terminal 22 amino acids was absent could not be imported into mitochondria in vitro.
Cloning, sequencing and expression of the gene encoding the coenzyme B12‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli
Authors: BEATRIX Birgitta , ZELDER Oskar , LINDER Dietmar , BUCKEL Wolfgang 
Published: March 2005

The coenzyme B12 (adenosylcobalamin)‐dependent 2‐methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2‐methyleneglutarate to (R)‐3‐methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri.(a) The mgm gene encoding 2‐methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8‐kDa protein of 614 amino acids. It shows significant similarity in its C‐terminal part to that of other cobamide‐dependent enzymes. Probably, this is the coenzyme‐binding region.(b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/PAGE and Western‐blot analysis with rabbit antiserum directed against the native mutase.(c) Cell‐free extracts from E. coli carrying the mgm gene showed 2‐methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B12. Experiments are presented which suggest that the expression product is an apoenzyme.
Functional properties of a hybrid vacuolar H+‐ATPase in Saccharomyces cells expressing the Nephrops 16‐kDa proteolipid
Authors: HARRISON Michael A. , JONES Philip C. , KIM Yong‐In , FINBOW Malcolm E. , FINDLAY John B. C. 
Published: March 2005

The hydrophobic 16‐kDa polypeptide which forms gap‐junction‐like structures in the crustacean Nephrops norvegicus is a member of a highly conserved family of proteolipids involved in a variety of membrane transport functions in eukaryotic cells. This family also includes the product of the Saccharomyces cerevisiae VMA3 gene which encodes an integral membrane component of the vacuolar membrane H+‐ATPase. The cDNA for the Nephrops proteolipid complements a mutation in the yeast VMA3 gene, resulting in assembly of a hybrid H+‐ATPase comprising yeast catalytic subunits and Nephrops integral membrane components. The hybrid vacuolar ATPase was capable of ATP hydrolysis which was coupled to proton translocation and showed inhibitor binding and enzymological properties similar to those of wild‐type V‐ATPases (Km for ATP, 0.4 mM), suggesting that both yeast and crustacean proteolipids share conserved structure at regions of protein interaction.To facilitate isolation of the Nephrops proteolipid by affinity chromatography on a Ni2+‐binding support, six C‐terminal histidine residues were added to the proteolipid. This modification did not prohibit assembly into the hybrid H+‐ATPase, although the resultant enzyme did have a markedly elevated Km (1.8 mM). The membrane‐bound Vo sector of the ATPase was isolated by the affinity‐chromatography procedure and reconstituted into synthetic vesicles. This complex was found to be impermeable to small cations in the absence of catalytic ATPase subunits either in situ in the vacuolar membrane or in the reconstituted system. The functional significance of this impermeability and the structure/function relationships between proteolipids from different sources are discussed.
Mechanism of inhibition of trypanothione reductase and glutathione reductase by trivalent organic arsenicals
Authors: CUNNINGHAM Mark L. , ZVELEBIL Marketa J. J. M. , FAIRLAMB Alan H. 
Published: March 2005

The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione in mammalian systems, has been shown to interact with anti‐trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p‐(4,6‐diamino‐s‐triazin‐2‐yl)aminophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo‐first‐order kinetics with kinact of 14.3×10−4 s−1 and 1.06×10−4 s−1 and Ki of 17.2 μM and 9.6 μM for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time‐dependent loss of the characteristic charge‐transfer absorbance band at 530 nm is observed upon addition of arsenical to pre‐reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH4 form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme‐inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur‐arsenic bond leads to the binding of the arsenical to the charge‐transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide‐binding sites.Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.
Substrate and inhibitor specificity of mevalonate kinase determined with substrate analogues
Authors: WILDE Jürgen , EGGERER Hermann 
Published: March 2005

Substrate analogues of mevalonate kinase were prepared and their kinetic properties determined with the enzyme from pig liver. The analogues are derived from mevalonate by substitutions of hydroxyl and/or methyl at C3(group 1), by systematic prolongation of C6 to yield 3‐alkyl‐3,5‐dihydroxyvalerates (group 2) and by methylation at C2, C6 and C4 (group 3).Compounds of group 1 were inhibitors of mevalonate kinase, their Ki values ranging over 140 mM to 2.5 mM and decreasing in the series CH2 > C(CH3)2 > CH(OH) > CH(CH3) > C(OH)‐CO2H > C(OH)CH2OH. Three of these analogues, the competitive inhibitors 3,5‐dihydroxyvalerate, 3‐methyl‐5‐hydroxy‐valerate and 3,3‐dimethyl‐5‐hydroxyvalerate are also (poor) substrates of the kinase.Substances of group 2 contain an additional hydrophobic group and are noncompetitive inhibitors with Ki values decreasing systematically from 7.7 mM to 0.7 mM in the sequence 3‐n‐propyl, 3‐n‐butyl, 3‐n‐pentyl and 3‐n‐hexyl derivative of 3,5‐dihydroxyvalerate.The methylated mevalonates of group 3 are competitive inhibitors as well as good substrates of the kinase, their Ki=Km values ranging over 4.3–0.2 mM and their maximal velocities related to mevalonate varying between 7–65%.All of these analogues have in common the carbon chain and 5‐hydroxy group of the substrate and could, therefore, fit to the active site of mevalonate kinase and be phosphorylated by ATP. The results, however, indicate a strong dependence of such interaction on the ligands at C3 of the substrate. Enzymic activity as well as affinity are lost if both of these ligands are replaced by hydrogen; partial restoration occurs at a low level if only one of the ligands is concerned. In the presence of the ligands even monomethylation at C2 and C4 yields enzymically active products. Oxidation of C6 of the substrate yields inhibitors but methylation yields a reasonably good substrate. Thus 3,5‐dihydroxy‐3‐ethylvalerate fits into the active site whereas higher C6 homologues are only inhibitory and probably bind to a hydrophobic region of the enzyme. Some of these results are shown to have parallels in the enzymic behaviour of fluorinated mevalonates.
The kinetics and thermodynamics of the binding of cytochalasin B to sugar transporters
Authors: WALMSLEY Adrian R. , LOWE Allan G. , HENDERSON Peter J. F. 
Published: March 2005

The kinetics of the binding of cytochalasin B to the proton‐linked l‐arabinose (AraE) and d‐galactose (GalP) symporters from Escherichia coli and to the human erythrocyte glucose transporter (GLUT1) have been investigated by exploiting the changes in protein fluoresence that occur upon binding the ligand. Steady‐state mesurements yielded Kd values of 1.1, 1.9 and 0.14 μM for the AraE, GalP and GLUT1 proteins, respectively. The association and dissociation rate constants for the binding of cytochalasin B have been determined by stopped‐flow spectroscopy. In each case, the apparent Kd was calculated from the corresponding rate constants, yielding values of 1.5, 0.4 and 1.6 μM for AraE, GalP and GLUT1, respectively. The differences between these apparent Kd values and those measured by fluorescence titration is interpreted in terms of the following three step mechanism where CB represents cytochalasin B:
The transporter is proposed to alternate between two different conformational forms (T1 and T2), with cytochalasin B binding only to the T2 conformation, to induce a further conformational transition of the transporter to the T3 form. The values for the overall dissociation constants show that the T1 conformation is favoured by AraE and GalP in the absence of ligands, but the T2 conformation is favoured by GLUT1. Thus, the binding of cytochalasin B to GLUT1 alters the equilibrium towards the T3(CB) conformational state, producing the observed tight binding, in contrast to the changes in the equilibrium observed with the binding of cytochalasin B to AraE and GalP. A thermodynamic analysis of these conformational transitions has been performed. The T1 and T2 conformations may represent transporter states in which the binding site is facing outwards and inwards, respectively.
Stimulation of cGMP‐dependent protein kinase Iα by a peptide from its own sequence
Authors: HUGGINS John P. , GANZHORN Axel J. , SAUDEK Vladimir , PELTON John T. , ATKINSON R. Andrew 
Published: March 2005

The structure of cGMP‐dependent protein kinase Iα‐(546–576)‐peptide amide (peptide‐546) and its effects on cGMP‐dependent protein kinase Iα (G‐kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide‐546 was predicted to form part of the protein/peptide binding site of G‐kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP‐dependent protein kinase analogous to peptide‐546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp‐2 and Phep‐11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide‐546. Peptide‐546 is a reasonably potent G‐kinase activator, increasing the turnover number with the peptide substrate Arg‐Lys‐Arg‐Ser‐Arg‐Lys‐Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate.Peptide‐546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G‐kinase. The activation does not seem to result from an interaction between peptide‐546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide‐546 on G‐kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2‐trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2‐trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80°C with no clear melting transition.Assignment of the resonances in the 1H‐NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N‐terminal residues are less structured but show some helical character.Calculation of the structure of the peptide using nuclear Overhauser effects (NOEs) as distance constraints in distance geometry and molecular dynamics simulations revealed a well defined helical structure in the C‐terminal region preceded by a less well defined proline‐rich segment and N‐terminal region. On the basis of these results, peptide‐546 may adopt a conformation similar to that expected by analogy with cAMP‐dependent protein kinase, and we propose that residues 546–576 of G‐kinase are involved in enzyme autoinhibition.
Stability and proteolytic domains of Nef protein from human immunodeficiency virus (HIV) type 1
Authors: FREUND Jens , KELLNER Roland , HOUTHAEVE Tony , KALBITZER Hans Robert 
Published: March 2005

Proteolytic experiments in conjunction with 1H‐NMR spectroscopy show that the Nef (negative factor) protein from human immunodeficiency virus type 1 probably consists of two main domains, the N‐terminal anchor domain at amino acid positions 2–65 and the C‐terminal core domain at positions 66—206. The N‐terminal domain is likely to be located at the surface of the protein, while the C‐terminal domain has a compactly folded core and is stable in the absence of the anchor domain. It is conceivable that the core domain represents a functional domain of the Nef protein, activated after the removal of the membrane anchor by the human‐immunodeficiency‐virus protease or cellular proteases.Nef is stable at pH 5–12 and denatures at 317–322 K. The Nef protein remains in its native conformation in dimethyl‐sulfoxide/water mixtures up to 35% (by vol.), and in acetonitrile/water up to 14% (by vol.). Nef refolds spontaneously after denaturation with urea or guanidinium hydrochloride. The 1H‐NMR parameters and pKa values of five of the nine histidine residues and one of the seven tyrosine residues were determined and were found in four cases to be typical for residues which are not located in the interior of the protein.
(Rp)‐ and (Sp)‐8‐piperidino‐adenosine 3′,5′‐(cyclic)thiophosphates discriminate completely between site A and B of the regulatory subunits of cAMP‐dependent protein kinase type I and II
Authors: ØGREID Dagfinn , DOSTMANN Wolfgang , GENIESER Hans‐Gottfried , NIEMANN Percy , DØSKELAND Stein Ove , JASTORFF Bernd 
Published: March 2005

8‐Piperidino‐cAMP has been shown to bind with high affinity to site A of the regulatory subunit of cAMP‐dependent protein kinase type I (AI) whereas it is partially excluded from the homologous site (AII) of isozyme II [Øgreid, D., Ekanger, R., Suva, R. H., Miller, J. P., and Døskeland, S. O. (1989), Eur. J. Biochem. 181, 19–31]. To further increase this selectivity, the (RP)‐ and (SP)‐diastereoisomers of 8‐piperidino‐cAMP[S] were synthesized and analyzed for their potency to inhibit binding of [3H]cAMP to site A and site B from type I (rabbit skeletal muscle) and type II (bovine myocardium) cAMP‐dependent protein kinases.(SP)‐8‐Piperidino‐cAMP[S] showed an enhanced relative affinity for site AI, thus being by far the best A‐selective compound (more than 100‐fold) tested for this isozyme. In contrast, the (RP)‐ isomer was less selective for AI than 8‐Piperidino‐cAMP itself. The reduction in affinities for BII, compared to 8‐piperidino‐cAMP, were 10‐fold and 50‐fold for the (SP)‐ and (RP)‐isomer, respectively. Both isomers were almost completely excluded from AII, with affinities about 1000‐fold lower than 8‐piperidino‐cAMP itself. The (RP)‐isomer selected BII with an affinity about 10000 times higher than for AII, whereas the (SP)‐isomer showed a preference of about 70000‐fold in favour of BII. 8‐Piperidino‐cAMP as well as its (SP)‐isomer activated both types of holoenzyme protein kinases whereas the (RP)‐isomer acted as an antagonist of cAMP‐induced activation.The study concludes that the combination of piperidino‐ and exocyclic sulfur substitutions generate cAMP analogs that completely discriminate between site A and B of cAMP‐dependent protein kinases.
Hydrolysis of lactosylceramide by human galactosylceramidase and GM1‐β‐galactosidase in a detergent‐free system and its stimulation by sphingolipid activator proteins, sap‐B and sap‐C Activator proteins stimulate lactosylceramide hydrolysis
Authors: ZSCHOCHE Alexandra , FÜRST Werner , SCHWARZMANN Günter , SANDHOFF Konrad 
Published: March 2005

Two exo‐β‐galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1‐β‐galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap‐B and sap‐C, rather than detergents, to stimulate the reaction.While sap‐B was a better activator for the reaction catalyzed by GM1‐β‐galactosidase, sap‐C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome‐integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2>C4>C6>C8>C10>C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap‐B‐stimulated hydrolysis of liposomal ganglioside GM1 by GM1‐β‐galactosidase and sap‐C‐stimulated degradation of liposomal galactosylceramide by galactosylceramidase.Kinetic and dilution experiments indicated that sap‐B forms water‐soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1‐β‐galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561–572]. GM1‐β‐galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap‐C and sap‐B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.