bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient and Selective P‐nitrophenyl‐ester‐hydrolyzing Antibodies Elicited by a P‐nitrobenzyl Phosphonate Hapten
Authors: Tawfik Dan S. , Lindner Ariel B. , Chap Rachel , Eshhar Zelig , Green Bernard S. 
Published: July 2004

A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)‐phosphonate transition‐state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl‐ester, were also found to catalyze the hydrolysis of the analogous p‐nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np‐ester is higher in terms of rates (kcat as expected from the higher intrinsic reactivity of Np‐esters); however, the rate acceleration (kcat/kuncat) is close to or lower than that observed with the Nbzl‐ester. Unexpectedly, the affinity to the Np‐ester substrate (1/kM) and therefore kcat/KM are significantly higher. The best example is antibody D2.4 having a kcat/KM value of 64 s−1. M−1 with the Nbzl‐ester and 9400 s−1. M−1 with the Np‐ester. Moreover, due to a lower product inhibition by p‐nitrophenol relative to p‐nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np‐ester. The differential affinity of these antibodies to the Nbzl‐phosphonate TSA versus the Nbzl‐ester substrate (KS/KTSA or KM/Ki) correlates well with the observed rate enhancement (kcat/kuncat). For the Np‐ester, however, stabilisation of the transition state (as reflected by Ks/kTSA and by the catalytic proficiencies, kcat/KM/kuncat) does not fully account for the catalytic power (kcat/kuncat), indicating a more complex catalytic mechanism than simply transition‐state stabilization.A comparison of the kinetic parameters of D2.4 with other Np‐ester‐hydrolyzing antibodies raised against Np‐phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl‐phosphonate hapten. These results appear to be general: anti‐(Nbzl‐phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np‐esterases. They demonstrate the use of an expanded TSA‐hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.
Intermediates in the Assembly of Bacteriorhodopsin Investigated by Time‐Resolved Absorption Spectroscopy
Authors: Booth Paula J. , Farooq Amjad 
Published: July 2004

The in vitro folding and assembly kinetics of bacteriorhodopsin have been studied by absorption spectroscopy. Folding is initiated by rapid stopped‐flow mixing of denatured apoprotein (bacterioopsin) in SDS micelles and mixed dimyristoylglycerophosphocholine/Chaps micelles containing retinal. The apparent mixing rate of the two types of micelles has been determined by time‐resolving the changes in light scattering by the micelles. Micelle mixing appears to occur in two stages: a fast phase with an apparent rate constant of about 420 s−1, and a second phase with an apparent rate constant of about 10 s−1. A rate constant of similar magnitude to the latter has previously been assigned to a protein‐folding event on the basis of protein fluorescence studies [Booth, P. J., Farooq, A. & Flitsch, S. L. (1996)Biochemistry 35, 5902–5909]. However the results presented here show that this rate constant may be associated with a rearrangement of the mixed detergent/lipid micelles.When the changes in the retinal absorption band are time‐resolved during assembly of bacteriorhodopsin, a retinal–protein intermediate, with an absorption maximum of about 430 nm, has been identified. This absorption maximum lies between that of unbound retinal (at about 380 nm) and the native chromophore (at about 560 nm). A comparison of fluorescence and absorption data, together with previous evidence [Booth, P. J., Flitsch, S. L., Stern, L. J., Greenhalgh, D. A., Kim, P. S., & Khorana, H. G. (1995) Nat. Struct. Biol. 2, 139–143], suggests that the covalent Schiff‐base link to retinal is not formed in the 430‐nm‐absorbing intermediate.
Structural Studies of the Cell‐Envelope Oligosaccharide from the Lipopolysaccharide of Haemophilus Influenzae Strain RM.118‐28
Authors: Risberg Anna , Schweda Elke K. H. , Jansson Per‐Erik 
Published: July 2004

The structure of the oligosaccharide part of the Haemophilus influenzae RM.118‐28 lipopolysaccharide (LPS) has been investigated. The oligosaccharide was obtained from the LPS by mild acid hydrolysis followed by gel‐permeation chromatography, and was studied by methylation analysis, NMR spectroscopy and mass spectrometry. The structure of the major compound, which is a hexasaccharide, is proposed as follows.
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In the structure, Kdo is 3–deoxy‐d‐manno‐octulosonic acid, PEtn is phosphoethanolamine, PCho is phosphocholine and l,d‐Hep is l‐glycero‐d‐manno‐heptose. Electrospray‐ionization mass spectrometry on O‐deacylated LPS obtained after treatment with anhydrous hydrazine gave evidence for the presence of two minor compounds, which show additional substitution of the main structure with phosphate and PEtn, respectively. These substitutions have not been localized.
26‐Hydroxylation of Ecdysteroids is Catalyzed by a Typical Cytochrome P‐450‐Dependent Oxidase and Related to Ecdysteroid Resistance in an Insect Cell Line
Authors: Kayser Hartmut , Wtnkler Tammo , Spindler‐Barth Margarethe 
Published: July 2004

The epithelial cell line from the dipteran Chironomus tentans responds to the insect steroid hormone 20‐hydroxyecdysone and the non‐steroidal analogue tebufenozide by undergoing a morphogenetic and biochemical differentiation program. Long‐term culture in the presence of 20‐hydroxyecdysone has resulted in the selection of subclones that are resistant to the steroid but respond normally to the non‐steroidal analogue. In the present study, several subclones that were resistant to the steroid hormone have been compared with steroid‐sensitive subclones with respect to their capability to metabolize 20‐ hydroxyecdysone. Homogenates of both types of cells, when incubated with 3H‐labelled steroid in the presence of NADPH, produced 20,26‐dihydroxyecdysone, which was further metabolized to two compounds, which behaved less polar than 20‐hydroxyecdysone on reverse‐phase HPLC. Ecdysone, a less‐ active hormone precursor, provided 26‐hydroxyecdysone as the only product. The metabolites were identified by mass spectrometry coupled to HPLC, chromatography with authentic samples, and formation of acetonides. The structure of 20,26‐dihydroxyecydsone was confirmed by 1H‐NMR.The enzyme responsible for the synthesis of 20,26‐dihydroxyecdysone in the Chironomus cell preparations has been characterized as a typical cytochrome P‐450‐dependent monooxygenase. It was a strictly microsomal enzyme, sensitive to inhibition by carbon monoxide and imidazole/triazole‐based fungicides, and required NADPH for maximal activity. NADH could partly replace NADPH. The Michaelis constant (Km) for 20‐hydroxyecdysone was 0.96 μM, and the maximal enzyme velocity (Vmax) was 50 pmol substrate metabolized mg protein−1. min−1. 26‐Hydroxylation of 20‐hydroxyecdysone was inhibited by ecdysone, an alternative substrate, and by inokosterone, a product analogue, to 50% at 1.4 μM and 0.73 μM, respectively. When various subclones were compared with respect to their in vitro rate of 20‐hydroxyecdysone metabolization, those clones known to be resistant to the steroid were ‘high metabolizers’ (>70% relative rate), whereas the sensitive clones were ‘poor metabolizers’ (<30% relative rate). Hence, it is tempting to conclude that ecdysteroid resistance of the Chironomus cell clones is due to metabolic inactivation of the steroid hormone.
Palytoxin‐Induced Channel Formation within the Na+/K+‐ATPase does not Require a Catalytically Active Enzyme
Authors: Scheiner‐Bobis Georgios , Schneider Heike 
Published: July 2004

It has been demonstrated that palytoxin binds to and forms a channel within the Na+/K+‐ATPase. To investigate whether palytoxin‐induced channel formation within the sodium pump can occur independently of ATP hydrolysis and phosphorylation of the enzyme, an Asp369→Ala mutant of the α1 subunit of the sheep sodium pump was produced and coexpressed with β subunits in the yeast Saccharomyces cerevisiae. This aspartic acid residue, which during ion transport becomes phosphorylated from ATP, is essential for the function of the sodium pump. Therefore, as expected, microsomes isolated from yeast expressing the mutant sodium pump do not exhibit any ouabain sensitive ATPase activity, whereas in microsomes from yeast expressing the wild‐type sodium pump, 60% of the total ATPase activity is ouabain‐sensitive. Ouabain binds to yeast membranes containing either wild‐type or mutant sodium pumps with similar Bmax (1.45±0.05 versus 1.37±0.02 pmol/mg) and Kd values (27.7±0.91 versus 29.57±0.93 nM), thus indicating that the mutant sodium pumps are expressed in the yeast and that the mutation does not considerably affect the conformation of the enzyme. In the presence of phosphate ouabain binds to microsomes containing the wild‐type sodium pump with a Kd of 3.62±0.34 nM, showing that, although not necessary, phosphoenzyme formation enhances binding of the steroid. Phosphate or ATP, however, inhibit binding of ouabain to microsomes containing the mutant sodium pump with IC50 values of 78±3 μM and 3.0±0.4 μM, respectively.Despite these radical changes in the interactions of the mutant enzyme with ouabain, the interactions with palytoxin are not affected by the mutation. Palytoxin causes K+ efflux from yeast cells expressing the wild‐type or mutant sodium pumps with EC50 values of 3.5±0.4 nM and 6.2±0.9 nM, respectively. Palytoxin‐induced efflux from cells expressing wild‐type or mutant sodium pumps occurs with similar t1/2 values of 20.3±2.1 min and 22.2±3.1 min, respectively. Ouabain inhibits K+ efflux from both cell types with IC50 values of 28±2 μM and 210±15 μM, respectively. Cells expressing the Asp369→Ala mutants have an IC50 7.5‐fold higher than that obtained with cells expressing the wild‐type sodium pumps, possibly because ATP or phosphate present in the cytosol of the yeast cells influence and decrease ouabain binding to the mutant sodium pump. Thus, while ouabain binding and the associated inhibition of ion fluxes is promoted by phosphorylation of the wild‐type enzyme by phosphate or ATP, palytoxin‐induced channel formation is independent of phosphorylation and can be separated from the ATPase function of the sodium pump. Since ion fluxes through the sodium pump protein do not depend on ATP hydrolysis, the results suggest that the ionophores of pumps and ion channels might share common structural features.
Benzoyl‐CoA Reductase (Dearomatizing), A Key Enzyme of Anaerobic Aromatic Metabolism
Authors: Boll Matthias , Albracht Simon S. P. , Fuchs Georg 
Published: July 2004

An enzyme was recently described, benzoyl‐CoA reductase (dearomatizing), which catalyses the ATP‐driven reduction of the aromatic ring of benzoyl‐CoA yielding a non‐aromatic CoA thioester, ADP and phosphate [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921–933]. The 170‐kDa enzyme consists of four different subunits and contains approximately 12 Fe and acid‐labile sulfur/mol. Benzoyl‐CoA reductase exhibits ATPase activity in the absence of substrate. It is shown that only the reduced form of this iron‐sulfur protein has ATPase activity. ATPase activity is reversibly lost when the enzyme is oxidized by thionine; reduction of the enzyme fully restores ATPase and ring‐reduction activity. 2 mol ATP are hydrolyzed/2 mol electrons transferred in the course of the reaction. The product ADP acts as competitive inhibitor (Ki= 1.1 mM) for ATP in benzoyl‐CoA reduction; ADP inhibits ATPase activity to the same extent as ring‐reduction activity. EPR investigation of the dithionite‐reduced enzyme suggested the presence of two separate [2Fe‐2S] clusters and two interacting [4Fe‐4S] clusters. Addition of MgATP to the reduced enzyme resulted in a new isotropic signal at g= 5.15 and a weak signal at g= 12; in controls with MgADP only a minor signal at g= 5.15 was observed. The positions, shapes and temperature dependencies of these MgATP‐induced signals are indicative for excited states of a S= 7/2 spin multiplet. The [2Fe‐2S] signals were not affected by ATP, but one of the [4Fe‐4S] clusters became slowly oxidized. Addition of both benzoyl‐CoA and MgATP resulted in a major oxidation of the iron‐sulfur clusters accompanied by the appearance of some minor signals of unknown origin in the g= 2.037 ‐ 1.96 region. Neither the benzoyl‐CoA plus MgATP‐oxidized nor the thionine‐oxidized enzyme showed the ATP‐dependent formation of the high‐spin signals of the reduced enzyme. At present we hypothesize that the S= 7/2 signal is due to an ATP‐induced change of one of the [4Fe‐4S] clusters.The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates. MgATP‐driven formation of this excited state of the reduced enzyme rather than transfer of electrons from the reduced enzyme to the aromatic substrate appears to be the rate‐limiting step in the catalytic cycle. We suggest that the excited state is required to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible.
Evidence for a Cysteine‐Histidine Thioether Bridge in Functional Units of Molluscan Haemocyanins and Location of the Disulfide Bridges in Functional Units d and g of the βc‐Haemocyanin of Helix Pomatia
Authors: Gielens Constant , DE Geest Natalie , Xin Xue‐Qin , Devreese Bart , Van Beeumen Jozef , PrÉAux Gisèle 
Published: July 2004

In functional units d and g from the βc‐haemocyanin of the gastropod Helix pomatia, amino acid analysis in the presence of dimethyl sulfoxide showed the occurrence of seven cysteine residues. Titration with 5,5′‐dithiobis(2‐nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis and partial sequencing of the cysteine‐containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the remaining cysteine residue is linked by a thioether bridge to a histidine residue located two positions further in the sequence (H. pomatia d Cys60 His62; H. pomatia g Cys66 His68). This residue corresponds to one of the three histidine residues considered to be involved in the coordination of the copper A atom of the dinuclear copper group of the functional units.The presence of the thioether bond was further evidenced by an absorption band at 255 nm and by molecular mass determinations with electrospray mass spectrometry on a peptic peptide from H. pomatia g and on peptides obtained by proteolysis of carboxymethylated H. pomatia d with trypsin and pronase. Upon sequence analysis the cysteine‐histidine thioether bridge was cleaved into didehydroalanine (polymers) and 2‐thiolhistidine.A peptide with a cysteine‐histidine thioether bridge was isolated from pepsinolysates of functional units c, e, f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. officinalis and O. vulgaris, A cysteine‐histidine thioether bridge, which was reported previously for tyrosinase of Neurospora crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.
Purification and Characterization of an Alcohol: N,N‐dimethyl‐4‐nitrosoaniline Oxidoreductase from the Methanogen Methanosarcina Barkeri DSM 804 Strain Fusaro
Authors: Daussmann Thomas , Aivasidis Alexander , Wandrey Christian 
Published: July 2004

Cell‐free extracts of Methanosarcina barkeri DSM 804 showed alcohol dehydrogenase activity under aerobic conditions when N,N‐dimethyl‐4‐nitrosoaniline(NDMA) was used as an artificial electron acceptor. The NDMA‐dependent alcohol dehydrogenase (NDMA‐ADH) was purified to approximate homogeneity by column chromatography. It is most probably a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass estimated by gel filtration being about 87 kDa. The purified protein had an isoelectric point of 4.3. It possesses a tightly but noncovalently bound NADP(H) cofactor. Each subunit contains 1 mol NADP(H)/mol, about 2 mol Zn2+/mol and significant amounts of magnesium.The purified enzyme preferably oxidized primary alcohols (including benzyl alcohol). NDMA‐ADH from M. barkeri also catalyzed the stoichiometric dismutation of aldehydes, especially higher aliphatic aldehydes, to form equimolar amounts of the corresponding alcohol and acid without addition of an electron carrier. The enzyme did not catalyze the dehydrogenation of methanol or the disproportionation of formaldehyde and therefore is not directly involved in methanogenesis.An alignment of the N‐terminal amino acid sequence of the enzyme with the sequences of other alcohol dehydrogenases from methanogenic and nonmethanogenic bacteria indicated no significant identity. Nevertheless there was a quite interesting sequence similarity in the first 30 N‐terminal amino acids to plant cinnamyl alcohol dehydrogenase. NDMA‐ADH from M. barkeri is a novel type of alcohol dehydrogenase in methanogenic bacteria.
Immunoreactive α‐Melanotropin as an Autocrine Effector in Human Melanoma Cells
Authors: Loir Béatrice , Bouchard Brigitte , Morandini Renato , Marmol Véronique DEL , Deraemaecker Rika , Garcia‐Borron José Carlos , Ghanem Ghanem 
Published: July 2004

Melanotropin is a peptide having several functions, including the stimulation of melanogenesis and the modulation of proliferation of melanocytes and melanoma cells. It acts through binding to high‐affinity receptors of the melanocortin‐1 subtype, exclusively expressed in cells of the melanocytic lineage.Elevated levels of immunoreactive α‐melanotropin were previously reported in melanoma cell lines, tumours and plasma from patients with melanoma. Here, we show that this high ectopic production of melanotropin is restricted to melanoma and non‐pituitary tumours with the same neuroectodermic origin.The occurrence of a melanotropin‐specific autocrine loop was further investigated in human melanoma cells. Immunoreactive α‐melanotropin was spontaneously released from a melanoma cell line (HBL) expressing melanotropin receptors on the cell surface. This release was significantly increased in the presence of melanotropin‐related peptides such as corticotropin‐(4–10) peptide and β‐melanotropin, competing for binding to the melanotropin receptor and was directly correlated to the displacement potential of these peptides. Both spontaneous and induced releases of immunoreactive α‐melanotropin could be blocked at low temperatures, suggesting the involvement of intracellular protein movement in the release mechanism.The release of immunoreactive α‐melanotropin was not significant in melanoma cells expressing very low levels of melanotropin receptors (IGR3) or in non‐melanoma cells (SCC1). However, upon expression of the melanocortin‐1 receptor cDNA into IGR3 cells, spontaneous and competition‐induced releases of immunoreactive α‐melanotropin were both increased and also blocked at low temperatures. This observation further underlines a role for the melanotropin receptor in the release of immunoreactive α‐melanotropin. These experiments indicate that an autocrine loop between the melanocortin‐1 receptor and immunoreactive α‐melanotropin may be functional in human melanoma cells.
A Reciprocal Allosteric Mechanism for Efficient Transfer of Labile Intermediates between Active Sites in CAD, the Mammalian Pyrimidine‐Biosynthetic Multienzyme Polypeptide
Authors: Irvine Heather S. , Shaw Seán M. , Paton Ann , Carrey Elizabeth A. 
Published: July 2004

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240‐kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate.Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition‐state analogue phosphonacetyl‐L‐aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5′‐phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell.We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition‐state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.