bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparative proteolysis of sorbitol and alcohol dehydrogenases
Authors: ROUIMI Patrick , LOOMES Kerry , JÖRNVALL Hans 
Published: March 2005

Mammalian alcohol and sorbitol dehydrogenases, with distantly related subunits but different substrates, quaternary structures and zinc contents, were evaluated by comparison of their sensitivities to proteases. Sorbitol dehydrogenase is more sensitive to proteolysis than alcohol dehydrogenase, but both enzymes show limited cleavage with Lys‐specific and Glu‐specific proteases. With the former, the major cleavage in both proteins involves Lys‐Lys‐Pro segments, at positions 247–248 in alcohol dehydrogenase, surface‐positioned after the most distal β‐strand in the coenzyme‐binding domain, and at 61–62 in sorbitol dehydrogenase, at another surface in the catalytic domain. Further cleavages affect these two and a third surface. A non‐surface cleavage was obtained with the Gluspecific protease and sorbitol dehydrogenase, after insufficient protease inhibition before analysis by SDS/PAGE. It probably reflects non‐native conditions, and the fact that this protease is active in strong SDS, necessitating pre‐analytical use of a specific inhibitor.Differences in cleavage patterns between the two proteins do not involve areas corresponding to the dimer interactions in alcohol dehydrogenase. Hence, these areas are likely to be the same in sorbitol dehydrogenase. However, major differences involve regions that flank the surface which has a 20‐residue loop segment in alcohol dehydrogenase that is missing in sorbitol dehydrogenase. This segment, previously highlighted from comparisons, is therefore also emphasized by experimental results with proteolysis. The surface exposed by the missing segment is likely to correlate with the separate quaternary structures and to indicate possible sites for the additional subunit interactions in the sorbitol dehydrogenase tetramer versus the alcohol dehydrogenase dimer.
Purification and properties of N5‐methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum
Authors: GÄRTNER Peter , ECKER Axel , FISCHER Reinhard , LINDER Dietmar , FUCHS Georg , THAUER Rudolf K. 
Published: March 2005

N
5‐Methyltetrahydromethanopterin: coenzyme M meth‐yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy‐conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purified 30‐fold to apparent homogeneity. The purified enzyme had an apparent molecular mass of 670 kDa and was composed of seven different polypeptides of 34 kDa, 28 kDa, 24 kDa, 23 kDa, 21 kDa, 13 kDa, and 12 kDa. The N‐terminal amino acid sequences of these polypeptides were determined. The native 670‐kDa enzyme was found to contain 7.6 mol 5‐hydroxybenzimidazolyl cobamide/mol, 37 mol non‐heme iron/mol and 34 mol acid‐labile sulfur/mol. Cobalt analyses after sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that the corrinoid was bound to the 23‐kDa polypeptide.The apparent molecualr masses of the polypeptides given above were determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis without boiling the samples prior to analysis. When the samples were boiled, as is usally done, the 23‐kDa polypeptide changed its apparent molecular mass to 33 kDa and the 21‐kDa, 24‐kDa, and 28‐kDa polypeptides formed aggregates.The specific activity (apparent Vmax) of the purified methyltransferase preparation was 11.6 μmol · min−1· mg protein−1. The apparent Km for N5‐methyltetrahydromethanopterin was 260 μM and that for coenzyme M was 60 μM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5–2‐fold.
tRNA fluorescent labeling at 3′ end inducing an aminoacyl‐tRNA‐like behavior
Authors: SERVILLO Luigi , BALESTRIERI Ciro , QUAGLIUOLO Lucio , IORIO Eugenio L. , GIOVANE Alfonso 
Published: March 2005

A fluorescent tRNA derivative labeled at 3′‐O position of the ultimate adenosine residue by reaction, under mild conditions, of tRNA with isatoic anhydride [3,1‐benzoxazine‐ 2,4(1H)‐dione] was obtained. The labeling selectivity was determined by several criteria: digestion with RNase, followed by HPLC of the digest, produces only one labeled nucleoside, identified as 3′‐O‐anthraniloyladenosine; the ratio of the absorbance at 260 nm to 332 nm also suggests a 1:1 molar ratio between the nucleic acid and the fluorophore; finally, the incapacity of the labeled tRNA to be charged by the specific aminoacyltransferase further demonstrates the engagement of the 3′‐O position.Although the 3′‐O‐anthraniloyl‐labeled tRNA does not seem to be functionally active, as far as the aminoacyl charging activity is concerned, surprisingly we found that it is able to form the ternary complex with elongation factor Tu (EF‐Tu) and GTP with an affinity consistently higher than uncharged tRNA. From fluorescence anisotropy measurements the ternary complex dissociation constant was estimated as 73 nM for Escherichia coli and 140 nM for yeast anthraniloyl‐tRNAPhe. These results may be interpreted in terms of the particular structure of the anthraniloyl group that makes the labeled tRNA similar to an aminoacyl‐tRNA.
Troponin‐C mutants with increased calcium affinity
Authors: da SILVA Ana Claudia R. , de ARAUJO Alexandre H. B. , HERZBERG Osnat , MOULT John , SORENSON Martha , REINACH Fernando C. 
Published: March 2005

Binding of two Ca2+ to the regulatory sites I and II of troponin C (TnC) induces a conformational transition believed to be responsible for the activation of muscle contraction. Based on the known crystal structure (2Ca2+ state), a model for the transition to the 4Ca2+ state has been proposed [Herzberg, O., Moult, J. & James, M. N. G. (1986) J. Biol. Chem. 261, 2638–2644]. The proposed conformational transition predicts that during Ca2+ binding a number of nonpolar residues become exposed to the solvent, creating a hydrophobic patch. Such a model implies that mutation of the hydrophobic to polar residues should increase the Ca2+ affinity at the regulatory sites and reduce the Ca2+ concentration necessary for muscle activation. To test this prediction, we have constructed and functionally characterized two troponin‐C mutants (V45T and M48A mutations).Direct calcium‐binding measurements in the mutants demonstrate an increase in the Ca2+ affinity for two low‐affinity sites. Replacement of endogenous troponin C in skinned muscle fibers by TnC with mutations V45T or M48A increased the Ca2+ sensitivity of their tension development. These results show that the model can be used to construct mutants that regulate muscle contraction at lower Ca2+ concentrations. They provide further experimental support for the proposed calcium‐induced conformational change of troponin C and suggest that the predicted transition plays a central role in the activation of the thin filament.
Crude liver membrane fractions and extracellular matrix components as substrata regulate differentially the preservation and inducibility of cytochrome P‐450 isoenzymes in cultured rat hepatocytes
Authors: SAAD Bashar , SCHOLL Florence A. , THOMAS Helmut , SCHAWALDER Hanspeter , STREIT Veronika , WAECHTER Felix , MAIER Peter 
Published: March 2005

The influence of cell‐substrata interactions on the preservation of basal or in‐vivo‐induced micro‐somal cytochrome P‐450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposure to phenobarbital or 3‐methylcholanthrene in vitro, was investigated. Hepatocytes from untreated or phenobarbital‐treated rats were cultured in serum‐free, aprotinin‐supplemented culture medium in 96‐well microtiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal cell functions were characterized by measuring the total protein content and lactate dehydrogenase release. The relative contributions of CYP1A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were determined with ELISA using monoclonal antibodies raised against purified cytochromes P‐450 from rat liver microsomes.The characterization of the CMF revealed that contaminations with mitochondria, nuclei and lysosomes are relatively low. Among these, membranes derived from the endoplasmic reticulum appeared to be the major organelle contaminant of the CMF. The matrix components laminin, fibronectin and collagen type IV were found in appreciable amounts.Hepatocytes from untreated rats, cultured for up to nine days on CMF/COL‐coated plates, retained their relative cytochrome P‐450 contents at 1.5–3‐fold higher levels when compared to cells cultured on COL, fibronectin or laminin. Similarly, hepatocytes from phenobarbital‐treated rats preserved the contents of barbiturate‐inducible CYP2B1/2 and CYP3A proteins best when cultured on CMF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarbital from days 3–6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3–6‐fold. After exposure to 3‐methylcholanthrene, a threefold increase of CYP1A proteins was found in CMF/COL and laminin cultures. These result indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates gene expression of cytochromes P‐450 isoenzymes for up to 9 days and provides a matrix which enables the cells to respond qualitatively similar to the response observed in different zones of the liver. This activity cannot be replaced by single matrix components.
Primary structures of the N‐linked carbohydrate chains from honeybee venom phospholipase A2
Authors: KUBELKA Viktoria , ALTMANN Friedrich , STAUDACHER Erika , TRETTER Verena , MÄRZ Leopold , HÅRD Karl , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. 
Published: March 2005

The N‐linked carbohydrate chains of phospholipase A2 from honeybee (Apis mellifera) were released from glycopeptides with peptide‐N‐glycanase A and reductively aminated with 2‐aminopyridine. The fluorescent derivatives were separated by size‐fractionation and reverse‐phase HPLC, yielding 14 fractions. Structural analysis was accomplished by compositional and methylation analyses, by comparison of the HPLC elution patterns with reference oligosaccharides, by stepwise exoglycosidase digestions which were monitored by HPLC, and, where necessary, by 500‐MHz 1H‐NMR spectroscopy.Ten oligosaccharides consisted of mannose, N‐acetylglucosamine and fucose α1–6 and/or α1–3 linked to the innermost N‐acetylglucosamine. Four compounds, which comprised 10% of the oligosaccharide pool from phospholipase A2, contained a rarely found terminal element with N‐acetylgalactosamine. The structures of the 14 N‐glycans from honeybee phospholipase A2 can be arranged into the following three series:

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Characterization of macrophage proteins bearing the functional leukotriene D4‐binding site of an anti‐cysteinylleukotriene monoclonal antibody
Authors: Van HILTEN Joost A. , BRUNE Kay , BICKEL Daniela , MOLLENHAUER Jürgen 
Published: March 2005

Protein conformations of the putative cysteinylleukotriene (LT) receptor of macrophages were characterized using anti‐idiotypic IgG (AIAb) against an anti‐LT monoclonal Ab (LTmAb). The AIAb nature of two rabbit antisera were demonstrated with titers of up to 1: 1000 against F(ab')2 from the LTmAb (in an enzyme‐linked immunoassay) which also inhibit LTD4 binding to the LTmAb (in a radioimmunoassay), whereas non‐immunized rabbit serum was not reactive. The specific reactivity of Fc‐purified AIAb towards LTmAb was measured by two fractions obtained after passage over columns of Sepharose either coupled with LTmAb (fraction A, representing immunoglobulins not absorbed to LTmAb) or coupled with homologous immunoglobins (fraction B, representing immunoglobulins not absorbed to homologous IgG). The difference in immunoreactivity between both fractions showed that fraction B contains AIAb against a LT‐recognizing domain of the LTmAb (in enzyme‐linked immunoassays coated with LTmAb and homologous IgG) and AIAb against the functional LT‐binding site of LTmAb (in radioimmunoassay).Using the antisera, Western‐blot analysis with peritoneal cell proteins detected signals at 236, 198, 118, 99, 75, 25 and 18kDa. Dithiothreitol‐reduced proteins were detected at 25kDa and 18 kDa. In general, this suggested recognition of a 236‐kDa oligomeric protein composed of subunits with molecular masses of 25kDa and 18 kDa, including intramolecular disulfide bridges all bearing an epitope similar to the LTmAb. From these conformations, an overlay assay with [3H]LTD4 favoured a 75‐kDa protein. Immunohistochemical analysis demonstrated that the recognized proteins may be located at cell membranes, because (a) in an ELISA, enriched plasma membrane preparations from peritoneal cells showed a threefold increase in reactivity to the AIAb, compared to the original cell homogenate; (b) after Western‐blot analysis, the membrane‐enriched protein fraction exhibited stronger protein signals than the microsomal fraction and the original cell homogenate; (c) regions of AIAb binding on the surface of cultured mouse peritoneal macrophages were detected by indirect immunofluorescence. Taken together, this study demonstrated AIAb binding to macrophage membrane‐associated proteins bearing the LTD4‐binding site of LTmAb, which may include identification of the putative LT receptor.
Specific binding of progesterone receptor to progesterone‐responsive elements does not require prior dimerization
Authors: COHEN‐SOLAL Karine , BAILLY Alain , RAUCH Claudine , QUESNE Monique , MILGROM Edwin 
Published: March 2005

Steroid‐hormone receptors undergo, prior to binding to DNA, a hormone‐dependent dimerization. It is generally accepted that this dimerization is indispensable for the high‐affinity binding of hormone receptor to hormone‐responsive elements.Using a progesterone‐receptor mutant with the complete steroid‐binding domain deleted (positions 663–930), with or without the epitope required for binding the monoclonal antibody Let 126, we have shown that this receptor species was unable to undergo dimerization in solution. However, this mutant retained a high affinity (60–70% of the affinity of the wild‐type receptor) for the progesterone‐responsive elements of the mouse‐mammary‐tumor‐virus long‐terminal‐repeat promoter and for a consensus palindromic progesterone‐responsive element, as measured by both DNase‐I protection experiments and gel‐shift experiments. This mutant also increased gene transcription. Thus, at least in the case of the progesterone receptor, prior dimerization is dispensable for receptor binding to regulatory DNA elements and for subsequent transcription activation.
Novel hirudin variants from the leech Hirudinaria manillensis
Authors: SCACHERI Emanuela , NITTI Gianpaolo , VALSASINA Barbara , ORSINI Gaetano , VISCO Carlo , FERRERA Monica , SAWYER Roy T. , SARMIENTOS Paolo 
Published: March 2005

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion‐exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2), and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C‐terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the C‐terminal portion of the molecule which is replaced by aspartic acid.Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin‐like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre‐hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported.The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20‐amino‐acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides.HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.
Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action
Authors: FAURE Grazyna , HARVEY Alan L. , THOMSON Elisabeth , SALIOU Bernard , RADVANYI François , BON Cassian 
Published: March 2005

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase‐A2 subunit (CB) and a non‐enzymic, non‐toxic subunit (CA). Crotoxin complex (CACB) dissociates upon interaction with membranes: CB binds while CA does not. Moreover, CA enhances the toxicity of CB by preventing its nonspecific adsorption.Several crotoxin isoforms have been identified. Multiple variants of each subunit give different crotoxin complexes that can be subdivided into two classes: those of high toxicity and low enzymic activity and those of moderate toxicity and a high phospholipase‐A2 activity.In this study, we demonstrate that the more‐toxic isoforms block neuromuscular transmission of chick biventer cervicis preparations more efficiently than weakly toxic isoforms. The less‐toxic crotoxin complexes have the same Km and Vmax as CB alone. In contrast, the more‐toxic isoforms are enzymically less active than CB. These differences correlate with the stability of the complexes: less‐toxic isoforms are less stable (Kd∼25 nM) and dissociate rapidly (half‐life about 1 min), whereas the more‐toxic isoforms are more stable (Kd∼4.5 nM) and dissociate more slowly (half‐life 10–20 min). The rate of interaction of crotoxin complexes with vesicles of negatively charged phospholipids paralleled the rate of dissociation of the complexes in the absence of vesicles.The differences of pharmacological and biochemical properties of crotoxin isoforms indicate that the stability of crotoxin complexes plays a major role in the synergistic action of crotoxin subunits: a stronger association between the two crotoxin subunits would account for their slower dissociation rate, a weaker enzymic activity, a slower interaction with phosphatidylglycerol vesicles, a faster blockade of neuromuscular transmission and a higher lethal potency.
Co‐reconstitution of plasma membrane H+‐ATPase from yeast and bacteriorhodopsin into liposomes
Authors: WACH Achim , DENCHER Norbert A. , GRÄBER Peter 
Published: March 2005

The H+‐ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The enzyme was reconstituted with bacteriorhodopsin into asolectin liposomes by detergent dialysis at a molar ratio of 1 H+‐ATPase to 50 bacteriorhodopsins. The overall orientation of the proteins is such that proton pumping to the vesicle interior occurs upon illumination and after addition of ATP. All liposomes which contain H+‐ATPase also contain bacteriorhodopsin.The rate of ATP hydrolysis was measured as function of pH in the dark and during illumination of the proteoliposomes. The pH dependency can be described by the protonation of a monovalent group from the outside with an apparent pK of 7.3 and the deprotonation of a monovalent group at the inside with an apparent pK of 3.7. Inside and outside refer to the orientation of the H+‐ATPase in the liposomes which is opposite to that occurring in vivo. It is concluded that the first step in the reaction cycle is the binding of a proton from the cytosol which is followed by ATP binding, ATP hydrolysis on the enzyme and the release of ADP and phosphate, and finally the proton is released from the enzyme into the external medium.
Evolution of pro‐protamine P2 genes in primates
Authors: RETIEF J. D. , DIXON G. H. 
Published: March 2005

Protamines P1 and P2 form a family of small basic peptides that represent the major sperm proteins in placental mammals. In human and mouse protamine P2 is one of the most abundant sperm proteins. The protamine P2 gene codes for a P2 precursor, pro‐P2 which is later processed by proteolytic cleavages in its N‐terminal region to form the mature P2 protamines. We have used polymerase chain amplification to directly sequence the pro‐P2 genes of the five major primate families: red howler (Alouatta seniculus) is a New World monkey (Cebidae); the two macaque species, Macaca mulatta and M. nemistrina are Old World monkeys (Cercopithecidae), the gibbon, Hylobates lar, represents one branch of the apes (Hylobatidae); the orangutan, Pongo pygmaeus, gorilla, Gorilla gorilla and two species of chimpanzee Pan paniscus and Pan troglodytes represent a second ape family (Pongidae). These pro‐P2 genes are compared with that of human [Domenjoud, L., Nussbaum, G., Adham, I. M., Greeske, G. & Engel, W. (1990) Genomics 8, 127–133].The overall size and organization of the genes are conserved within the group. The mean length of pro‐P2 is 101 residues, with an increase to 102 in M. nemistrina and a decrease to 99 residues in red howler (A. seniculus). In gorilla and red howler one of two 79‐bp tandem repeats that occurs 3′ of the gene is deleted. Of the 101 deduced amino acids examined, an amino acid change occurs in one or more primates at 45 positions. Considering only the most recently diverged group, the human/gorilla/chimpanzee clade, this represents a very high mutation rate of 0.99 changes/100 sites in 106 years. This rapid mutation rate is characteristic of both members of the protamine gene family, P1 and P2. Consideration of the variable nature of the sequences at the multiple sites of proteolysis during the processing of the pro‐P2 indicates either that there are several processing enzymes of differing specificities, or more likely that the folded structure of the pro‐P2 limits accessibility of a non‐specific protease to certain exposed sites.