bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosinase isoenzymes in mammalian melanocytes
Authors: VALVERDE Paloma , GARCIA‐BORRON Jose C. , JIMENEZ‐CERVANTES Celia , SOLANO Francisco , LOZANO Jose A.
Published: March 2005

In mouse melanoma melanocytes, α‐melanocyte‐stimulating hormone (MSH) stimulates differentiation, melanin synthesis and tyrosinase activity. However, the molecular mechanisms underlying these events have not yet been characterized. We have studied the activation of tyrosinase by MSH. Treatment of B16 melanoma cells with either theophylline, MSH, or its superpotent analog [Ahx4, DPhe7]MSH promotes a larger induction of tyrosine hydroxylase than of dopa oxidase activity in whole cell extracts. This higher activation of tyrosine hydroxylation was found not only in the melanosomal but also in the microsomal fraction; it appears to be dependent on continued transcription and translation since it can be blocked by actinomycin and cycloheximide. The tyrosinase activity of control and theophylline‐treated extracts displayed several kinetic differences, including different Km values for both substrates and requirements for the cofactor L‐dopa. SDS/PAGE, followed by a sensitive specific activity stain, demonstrated that melanosomes of control cells contain one lower‐electrophoretic‐mobility form of tyrosinase, whereas melanosomes of cells treated with either theophylline or MSH display, in addition to the lower‐mobility form, a faster‐migrating activity band. These tyrosinase forms are not interconvertible by proteolysis or deglycosylation. Their nature is discussed as related to the properties of the previously described low‐ and high‐electrophoretic‐mobility tyrosinases (LEMT and HEMT), as well as of the proteins encoded by the c and b loci.
Purification and characterization of endoglucanase C from Clostridium cellulolyticum
Authors: FIEROBE Henri‐Pierre , BAGNARA‐TARDIF Chantal , GAUDIN Christian , GUERLESQUIN Françoise , SAUVE Paul , BELAICH Anne , BELAICH Jean‐Pierre
Published: March 2005

An Escherichia coli clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum. This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa. N‐terminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH‐extremity. The difference in mass indicated that the shorter protein lacks the C‐terminal reiterated domains (20–24‐amino‐acid twice‐repeated sequences). These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles. Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different. Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA. The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention. The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p‐nitrophenyl‐cellobiose to p‐nitrophenyl‐cellopentaose) were also investigated in both forms of CelCCC and CelCCA. It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p‐nitrophenyl‐cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage‐site position and catalytic parameters. CelCCC and CelCCA were found not to act synergistically on the tested substrates.
Activity and spectroscopic properties of bovine liver catalase in sodium bis(2‐ethylhexyl)sulfosuccinate/isooctane reverse micelles
Authors: HABER J. , MAŚALAKIEWICZ P. , RODAKIEWICZ‐NOWAK J. , WALDE P.
Published: March 2005

Spectroscopic properties such as ultraviolet‐visible spectroscopy, circular dichroism, steady‐state fluorescence and the catalatic activity of bovine liver catalase (BLC) have been studied in reverse‐micelles microemulsions formed by sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) in isooctane. Activity measurements were carried out with the highly water soluble hydrogen peroxide as substrate as a function of temperature and pH, at various concentrations of substrate, enzyme, AOT and at different wo values (wo= [H2O]/[AOT]).

The folding of catalase in reverse micelles is not drastically altered, although there are changes in the local surrounding of the prosthetic group. Kinetic measurements have been carried out under first‐order conditions at low substrate concentration. They indicate that the measured substrate decomposition rate is limited by the exchange of substrate molecules between the substrate‐filled and the enzyme‐containing micelles. The specificity constant, ks=kcat/KM, as related to water pool concentrations, ks,wprm, was, at all wo values, considerably lower than ks measured in buffer (ksp). In contrast, overall values of ks in reverse micelles, ks,ovrm, were always 2–4‐times higher than ksb. This apparent superactivity can, however, be ascribed to concentration effects.

No ‘bell‐curve’ to describe the wo dependence of catalase activity in reverse micelles was found, k or ks increasing monotonously up to wo= 50.
The glycosylphosphatidylinositol anchor of the trypomastigote‐specific Tc‐85 glycoprotein from Trypanosoma cruzi
Authors: COUTO Alicia S. , DE LEDERKREMER Rosa M. , COLLI Walter , ALVES M. Julia M.
Published: March 2005

The Tc‐85 glycoprotein, specific for the infective stage of Trypanosoma cruzi, is anchored via glycosylphosphatidylinositol. The protein was purified from parasites, labeled metabolically with palmitic acid, by immunoprecipitation with the H1A10 monoclonal antibody or by affinity column chromatography on wheat germ agglutinin. Antisera to the soluble form of the variant surface glycoprotein of Trypanosoma brucei brucei cross‐reacted with Tc‐85 when the immunoprecipitate was analysed by Western blotting. The reaction was intensified upon previous incubation of the glycoprotein with phosphatidylinositol‐specific phospholipase C. Such recognition was abolished when the cyclic phosphate was opened by mild acid treatment. The lipid cleaved by phospholipase C digestion, was identified as 1‐O‐hexadecylglycerol by reverse‐phase thin‐layer chromatography. The glycan core was deaminated and chemically labeled by reduction with NaB3H4. The labeled glycoprotein was exhaustively treated with pronase and dephosphorylated with 50% HF. Although microheterogeneity of the oligosaccharide moiety was apparent, by thin layer chromatography, a main spot coincident with Man(α1–2) Man(α1–6) Man(α1–4) anhydromannitol was shown, consistent with the conserved core structure of all glycosylphosphatidylinositol anchors analysed to date.
Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan
Authors: ILG Thomas , HARBECKE Dorothee , WIESE Martin , OVERATH Peter
Published: March 2005

Leishmania promastigotes, the stage of the parasite characteristic for the sandfly vector, express an abundant glycoconjugate, called lipophosphoglycan, at their surface. Lipophosphoglycan consists of lysoalkyl‐sn‐glycerophosphoinositol linked to a phosphosaccharide core conserved in all species, which is connected to PO4‐6Galβ1,4Manα1 repeats with species‐specific substitutions at the Gal residue; the repeats are capped by conserved and species‐specific oligosaccharides. Most Leishmania species also secrete an acid phosphatase, which, in Leishmania mexicana, is a filamentous complex composed of a phosphorylated glycoprotein and non‐covalently associated proteo‐(high‐molecular‐mass)phosphoglycan.

The secreted acid phosphatase complex was used as an antigen to derive a panel of monoclonal antibodies (mAbs). A total of 25 mAbs (17 novel and 8 previously described) were tested by different techniques for their specificity against lipophosphoglycan and secreted acid phosphatase from several Leishmania species. This comparison and the modification of the antigens by chemical or enzymic treatments allowed a classification of the mAbs into several groups.

First, from 25 mAbs examined, 22 recognize lipophosphoglycan and the enzyme complex of L. mexicana; only three are specific for secreted acid phosphatase. Two of the latter group are also directed against carbohydrate structures, whereas the third mAb recognizes the 100‐kDa polypeptide of the complex. The secreted acid‐phosphatase‐specific class detects antigen in the flagellar pocket of promastigotes while all anti‐lipophosphoglycan mAbs bind to the cell surface.

Second, all 15 anti‐lipophosphoglycan mAbs investigated in detail appear to be directed against the phosphosaccharide repeats or the cap structure rather than the phosphosaccharide core. Two mAbs recognize terminal cap‐structures containing Manα1,2Man residues. Four antibodies are specific for L. mexicana and are probably directed against PO4‐6[Glcβ1,3]Galβ1,4Manα1 repeats while six mAbs react with the unmodified repeats. Two antibodies specific for Leishmania major recognize Galβ1,3‐substituted repeats unique for lipophosphoglycan from this species.

Analysis by immunoblotting indicates that the high‐molecular‐mass proteo‐phosphoglycan of L. mexicana secreted acid phosphatase carries epitopes for all anti‐lipophosphoglycan mAbs suggesting the presence of capped phosphosaccharide repeats while the enzymically active glycoprotein subunit is modified by caps but probably not by repeats. In the case of Leishmania donovani secreted acid phosphatase, the enzymically active polypeptide may be directly modified by repeats.

The mAbs are used to characterize changes in lipophosphoglycan structure, which occur in culture during the transition of promastigotes from the logarithmic to the stationary growth phase. Furthermore, testing the mAbs against seven species demonstrates their potential for serotyping Leishmania.
Different organization of the tRNA‐gene‐associated repetitive element, DRE, in NC4‐derived strains and in other wild‐type Dictyostelium discoideum strains
Authors: MARSCHALEK Rolf , HOFMANN Jörg , SCHUMANN Gerald , BACH Martina , DINGERMANN Theodor
Published: March 2005

The retrotransposon DRE (Dictyostelium repetitive element) was discovered in the course of an extensive study concerning the genomic organization of tRNA genes in the NC4‐derived strains AX2 and AX3 of the cellular slime mold Dictyostelium discoideum. As a striking feature, DRE was found exclusively in a constant orientation and at a constant distance upstream from different tRNA genes. About 150–200 DRE with intact 5′‐terminal‐repeat structures are present in NC4‐derived strains. These strains were termed high‐copy DRE strains (HCD strains) as opposed to low‐copy DRE strains (LCD strains) such as the wild‐type D. discoideum isolates DD61, WS380B, OHIO and V12. LCD strains contain only 3–15 DRE with intact 5′‐terminal‐repeat‐structures. However, in addition to these few intact elements, many 5′‐truncated DRE elements are present in LCD strains. In HCD strains, most DRE show typical structural characteristics of retrotransposons containing terminal repeats at both ends, which seems to be one prerequisite for active transposition. In LCD strains, however, most DRE elements are 5′‐truncated, which is a common feature of eukaryotic LINE elements. Despite their truncated 5′‐ends, DRE in LCD strains retain unique integration specificities, i.e. they are always found position‐specifically and orientation‐specifically integrated in front of tRNA genes, flanked by a 12–16‐bp target‐site duplication.
Transcriptional control of the heme oxygenase gene during mouse spermatogenesis
Authors: KURATA Shun‐ichi , MATSUMOTO Midori , HOSHI Motonori , NAKAJIMA Hiroshi
Published: March 2005

The activation of the heme oxygenase (HO) gene during development of mouse male germ cells was examined by nuclear run‐off assay. Results showed that the HO gene was activated in male germ cells, especially in spermatogonia and that the mRNA was stored throughout meiosis. In contrast, Western blotting analysis revealed that HO was expressed during spermatogenesis to spermatocytes. As the mouse HO gene is known to have a 12‐o‐tetradecanoyl‐phorbol 13‐acetate(TPA)‐responsive element (TRE) in its upstream region and is activated by TPA in mouse M1 cells, we also examined the activation of the nuclear proto‐oncogenes fos and jun, which are activators of TRE. Both genes were found to be activated with expression of their protein products only in spermatogonia. Moreover activation of fos, jun and HO genes in spermatogonia was strongly inhibited by a c‐kinase inhibitor. These results suggest that the HO gene is activated at the spermatogonia stage by a fos/jun heterodimer complex (AP1) through c‐kinase activation.
Solution conformation of an antibacterial peptide, sarcotoxin IA, as determined by 1H‐NMR
Authors: IWAI Hideo , NAKAJIMA Yuki , NATORI Shunji , ARATA Yoji , SHIMADA Ichio
Published: March 2005

The solution conformation of sarcotoxin IA, which is an antibacterial peptide isolated from Sarcophaga peregrina with a molecular mass of 4 kDa, was determined by NMR spectroscopy and hybrid distance geometry/dynamical simulated annealing calculations. On the basis of 227 experimental constraints, including 185 distance constraints obtained from NOE and 42 constraints associated with 21 hydrogen bonds, a total of 18 converged structures of sarcotoxin IA were obtained. The final 18 converged structures exhibit backbone‐atomic root‐mean‐square differences about the averaged coordinate positions of 0.070±0.027 nm for residues 3–23 and 0.040±0.017 nm for residues 28–38. It has been indicated that sarcotoxin IA consists of two amphiphilic α‐helical regions, i.e. helix I (Leu3–Gln23) and helix II (Ala28–Ala38), with a hinge region (Gly24–Ile27), which connects helix I and helix II. We conclude that these two amphiphilic helical segments of sarcotoxin IA are of importance for the expression of the antibacterial activity.
Accessibility to modification of histidine residues of RecA protein upon DNA and cofactor binding
Authors: TAKAHASHI Masayuki , NORDÉN Bengt
Published: March 2005

The potential role of histidine residues of RecA protein in binding DNA has been investigated by monitoring their accessibility to diethylpyrocarbonate. In the absence of both DNA and cofactor, only one of two histidine residues is modified by the reagent, indicating that the other residue is buried. However, both histidine residues become accessible after addition of cofactor analog adenosine 5′‐O‐(3‐thiotriphosphate) (ATP[S]) indicating a change in the organization of the RecA filament and/or a change in the conformation of protein. The diethylpyrocarbonate‐modified RecA is found to be able to polymerize just as the unmodified protein. The binding of double‐stranded DNA, in the presence of ATP[S], reduces the reactivity of both histidine residues to diethylpyrocarbonate. The binding of single‐stranded DNA (with ATP[S]) has a similar, though smaller, protective effect. However, no significant dissociation of either of the complexes as a result of the modification was observed and a RecA molecule which had been modified in the absence of DNA could still bind DNA. A protection of the histidine residues is also effected by high salt concentration which promotes, just as DNA binding, ATPase and coprotease activity in RecA. The protection of histidine residues to diethylpyrocarbonate upon DNA binding probably relates to a conformational change of RecA and may not be any direct effect of shielding by the DNA. Nonetheless, the domains including the histidine residues could be centers of allosteric effects and are concluded to be close to the DNA binding site.
The α‐mannosyl‐binding lectin from leaves of the orchid twayblade (Listera ovata)
Authors: SAITO Keiko , KOMAE Akiko , KAKUTA Mariko , VAN DAMME Els J. M. , PEUMANS Willy J. , GOLDSTEIN Irwin J. , MISAKI Akira
Published: March 2005

The carbohydrate‐binding specificity of an α‐D‐mannose‐specific lectin isolated from leaves of the orchid twayblade (Listera ovata) was elucidated by quantitative precipitation of mannose‐containing polysaccharides and glycoproteins, hapten inhibition, and affinity chromatography on the immobilized lectin. L. ovata agglutinin (LOA) interacted with various α‐mannans and galactomannans of yeasts, fungi and bacteria, but not with α‐glucans, e.g., dextran and glycogen, as do mannose/glucose‐binding lectins. This lectin, LOA, appears to be highly specific for α1–3 mannosidic linkages. It reacted with a linear α1–3‐mannan (D. P. 15) and, surprisingly, even with a linear α1–3‐mannoheptasaccharide. The LOA/C. tropicalis mannan precipitation reaction was inhibited by α‐linked mannooligosaccharides, in the order, α1–3 > α1–6 > α1–2 linkages; α1–3 [Man]4 and [Man]5 were the best inhibitors among various mannooligosaccharides tested, having 7‐times greater potency than α1–3 [Man]2, and 18‐times that of methyl α‐mannoside. LOA/mannan interaction was also inhibited by periodate‐oxidized and reduced α1–3 [Man]5 which had an inhibitory potency similar to that of α1–3 [Man]3, confirming that LOA also recognizes the internal α1–3‐mannosidic linkages of carbohydrate chains.

Complete resolution of mannan and glycogen from yeast cells, by affinity chromatography on an immobilized LOA column, and retention of several high‐mannose‐glycoproteins suggest this lectin to be a useful tool for purification and structural investigation of α‐mannosyl‐containing polysaccharides and glycoconjugates.
Molecular characterization of flayanone 3β‐hydroxylases
Authors: BRITSCH Lothar , DEDIO Jürgen , SAEDLER Heinz , FORKMANN Gert
Published: March 2005

A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone‐3β‐hydroxylase‐encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3β‐hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non‐heme iron‐(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of threc histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron‐binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3β‐hydroxylases was corroborated by diethyl‐pyrocarbonate modification of the partially purified recombinant Petunia enzyme.
Modulation of tensin and vimentin expression in chick embryo developing cartilage and cultured differentiating chondrocytes
Authors: VAN DE WERKEN Rinke , GENNARI Massimo , TAVELLA Sara , BET Paola , MOLINA Francesco , LIN Shin , CANCEDDA Ranieri , CASTAGNOLA Patrizio
Published: March 2005

It has been proposed that tensin, in association with several other proteins, mediates the microfilament‐integrin link. Here we describe the isolation of clones spanning about 5 kb from the 3′ end of tensin mRNA from cultured chick embryo chondrocyte and embryonic heart cDNA libraries. Tensin expression was investigated in cultured chick embryo cells. It was observed that tensin expression is dependent upon substrate adhesion and it is turned off after 7 days of suspension culture. This process is reversible. Tensin expression is also regulated during cartilage cell differentiation in vivo; at Hamburger and Hamilton stage 39–40, non‐hypertrophic tibial chondrocytes express both RNA and protein while hypertrophic chondrocytes do not. In the culture system the expression of vimentin, a major component of intermediate filaments, showed an opposite behaviour since the suspension culture enhances the accumulation of both vimentin and its mRNAs. Therefore in chick embryo cultured chondrocytes and in vivo, during cartilage development, cell shape changes and/or integrin‐extracellular matrix protein interactions may be involved in the regulation of these two genes coding for cytoskeletal proteins.
Components of glycine reductase from Eubacterium acidaminophilum
Authors: LÜBBERS Meike , ANDREESEN Jan R.
Published: March 2005

The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein Pc of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called ‘atypically small lipoamide dehydrogenase’ or ‘electron transferring flavoprotein’ could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox‐active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.
Broad binding‐site specificity and affinity properties of octamer 1 and brain octamer‐binding proteins
Authors: BENDALL Andrew J. , STURM Richard A. , DANOY Patrick A. C. , MOLLOY Peter L.
Published: March 2005

The ubiquitous Pit‐1‐Oct‐1‐Unc‐1 (POU)‐domain protein octamer 1 (Oct‐1) has been observed to bind specifically to a number of degenerate and dissimilar sequences. We have used antibodies directed against a C‐terminal Oct‐1 peptide to immunoselect binding sequences for HeLa cell Oct‐1 from random‐sequence oligonucleotides and we describe the isolation of binding sequences of considerable heterogeneity. Although our consensus alignment indicated a 9‐bp TATGCAAAT motif with AT‐rich flanking sequences, this binding motif is not immediately obvious in the population of sequences and no clone actually contained this sequence. Screening these Oct‐1‐binding sequences with a mouse whole‐brain extract demonstrated that the neuronal octamer‐binding proteins exhibit similar but distinct DNA sequence specificities. Unlike the reported selection of binding sequences for other transcription factors, the dependence of Oct‐1‐binding affinity upon sequence did not correspond tightly to the degree of conservation at particular positions of the consensus sequence. Our results suggest that either base‐specific hydrogen bonding is not the only major determinant of binding affinity and specificity, or that Oct‐1 binding to some sequences is mechanistically different from its binding to an octamer. These results exemplify the potential to overlook binding sites for some factors by searching gene sequences with a consensus nucleotide sequence.
The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis
Authors: GOLOLOBOV Mikhail Y. , STEPANOV Valentin M. , VOYUSHINA Tatjana L. , ADLERCREUTZ Patrick
Published: March 2005

α‐Chymotrypsin‐catalyzed acyl tranfer was studied using three acyl‐group donors (Mal‐l‐Ala‐l‐Ala‐l‐PheOMe, Bz‐l‐TyrOEt and Ac‐l‐TrpOEt; Mal, maleyl; Bz, benzoyl; OMe, methyl ester; OEt, ethyl ester) and a series of amino‐acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl‐enzyme. The α‐chymotrypsin‐catalyzed transfer of the Mal‐l‐Ala‐l‐Ala‐l‐Phe group to the amides of L‐Phe and L‐Tyr showed a linear dependence of the partition constant, p, on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl‐enzyme–nucleophile complex. The α‐chymotrypsin‐catalyzed transfer of the Bz‐l‐Tyr and Ac‐l‐Trp groups to several amino‐acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl‐enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of α‐chymotrypsin‐catalyzed acyl transfer evident in previous studies.
Proteolytic cleavage of synthetic fragments of vesicle‐associated membrane protein, isoform‐2 by botulinum type B neurotoxin
Authors: SHONE Clifford C. , QUINN Conrad P. , WAIT Robin , HALLIS Bassam , FOOKS Sarah G. , HAMBLETON Peter
Published: March 2005

Recent data suggest that botulinum type‐B neurotoxin is a protease which acts on vesicle‐associated membrane protein, isoform 2 (VAMP‐2). In this report, botulinum type‐B neurotoxin is shown to cleave a synthetic fragment (HV62) of VAMP‐2, corresponding to the bulk of the hydrophillic domain (amino acids 33–94). The neurotoxin acts at a single site between Gln76 and Phe77. Little or no proteolytic activity by botulinum type‐B neurotoxin was observed with peptides containing 7, 10 or 20 amino acids spanning the site of cleavage. The proteolytic action of neurotoxin was strongly inhibited by EDTA and o‐phenanthroline whereas captopril and phosphoramidon were ineffective. A series of model peptide substrates were synthesised in order to define the smallest VAMP‐2 fragment to be cleaved by botulinum type‐B neurotoxin. Data obtained from these substrates suggest that the neurotoxin belongs to a novel class of zinc‐endoprotease; more than 12 amino acid residues are required on both the NH2‐and COOH‐terminal side of the cleavage site for optimal proteolytic activity. The results demonstrate that no other components of cellular vesicles are required for the specific action of the neurotoxin on VAMP‐2. The data further show that the highly specific action of the neurotoxin is not dictated solely by the properties of the amino acid residues at the cleavage site but is also dependent on amino acid sequences distal to its site of action.
Fusion of artificial membranes with mammalian spermatozoa
Authors: ARTS Eugène G. J. M. , KUIKEN Jaap , JAGER Siemen , HOEKSTRA Dick
Published: March 2005

The fusogenic properties of bovine and human spermatozoa membranes were investigated, using phospholipid bilayers (liposomes) as target membranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance‐energy transfer. In addition, fusion was visualized by fluorescence microscopy, using fluorescent lipid vesicles. Cryopreserved bovine sperm fused with liposomes before induction of the acrosome reaction, fluorescence being located in essentially all spermatozoa membrane domains. Fresh bovine and human spermatozoa fused with liposomes only after the induction of the acrosome reaction, as triggered by calcium ionophore A23187 or zonae pellucidae (proteins), while the fluorescence distribution was mainly restricted to the equatorial segment (ES). However, with spermatozoa that had undergone a freeze/thawing cycle, domains other than ES also became labeled. Hence, the redistribution of the lipid probes over the entire membrane occurring during lipid mixing with cryopreserved bovine sperm is probably related to membrane perturbations caused by long‐term cryopreservation. Fusion with liposomes was governed by spermatozoa factors and required the presence of acidic phospholipids like cardiolipin and phosphatidylserine in the liposomal bilayer. Incorporation of the zwitterionic lipid phosphatidylcholine in the vesicles inhibited the fusion reaction. Fusion was pH dependent. The results indicate that the ES is the primary domain of spermatozoa membranes that harbours the fusogenic capacity of sperm. Liposomes appear a valuable tool in further characterizing the properties of this domain, which has been claimed [Yanagimachi, R. (1988) in The physiology of reproduction (Knobil, E. & Neill, J., eds) pp. 135–185, Raven Press, New York] to represent the putative, initial fusion site for the oocyte.
Characterization of the KdpD protein, the sensor kinase of the K+‐translocating Kdp system of Escherichia coli
Authors: VOELKNER Petra , PUPPE Wolfram , ALTENDORF Karlheinz
Published: March 2005

KdpD and KdpE, proteins that control expression of the kdpFABC operon, are members of the class of sensor kinase/response regulator proteins. Using polyclonal antibodies raised against the KdpD protein, we have been able to identify and to localize the chromosome‐encoded KdpD protein in the cytoplasmic membrane of Escherichia coli. Furthermore, it has been possible to detect differences in the expression of the KdpD protein according to the K+ concentration in the growth medium. The phosphorylation capacity of the plasmid‐encoded KdpD protein and the phospho‐transfer to KdpE was investigated. We found that both reactions were strictly dependent on the ionic conditions of the assay medium. Based on optimized conditions, we were able to detect phosphorylation of the chromosome‐encoded KdpD protein. Furthermore, replacement of the conserved histidine (His673), the predicted phosphorylation site in KdpD, by glutamine revealed that phosphorylation of KdpD was no longer possible.
Partial purification and properties of enzymes involved in the processing of a chloroplast import protein from Chlamydomonas reinhardii
Authors: SU Qingxiang , BOSCHETTI Arminio
Published: March 2005

Two stromal peptidases (SPP‐1 and SPP‐2) were partially purified from chloroplasts of Chlamydomonas reinhardii. They specifically processed in vitro the precursor of the small subunit of ribulose‐1,5‐bisphosphate carboxylase (pSS), which had been synthesized by using the cloned rbcS‐2 gene of Chlamydomonas. SPP‐1 shortened pSS to an intermediate‐sized form (iSS), while SPP‐2 cut pSS and iSS to the mature small subunit SS. N‐terminal amino acid sequencing demonstrated that the reaction product obtained with SPP‐2 had an N‐terminus identical to natural SS, and that iSS derived from pSS by hydrolysis at the amino side of the methionine located within the transit sequence. By gel filtration, apparent molecular masses of 340 kDa and 90 kDa were determined for SPP‐1 and SPP‐2, respectively. The comparison of these molecular masses with the protein patterns obtained by SDS/PAGE of the partially purified enzymes suggested that at least SPP‐1 was a multimeric protein. The enzymes differed also in their pH optima of about 8 (SPP‐1) and 9 (SPP‐2) and in their sensitivity to different inhibitors. However, both enzymes seem to be serine proteases as they were completely blocked by N‐α‐tosyl‐l‐lysinechloromethane or tosylphenylalaninechloromethane, respectively. Competition experiments, using either mature SS or a synthetic hexadecapeptide with 15 amino acids similar to the C‐terminal end of the transit sequence of pSS, indicated that SPP‐2 had some affinities not only to the transit sequence of pSS, but especially to sequences in the mature protein part. We conclude that SPP‐2 in Chlamydomonas is the enzyme involved in import of pSS into chloroplasts and responsible for its processing by a one‐step mechanism.
A new brain‐specific 14‐kDa protein is a phosphoprotein
Authors: NAKAJO Shigeo , TSUKADA Kazuhiko , OMATA Kumiko , NAKAMURA Yasuharu , NAKAYA Kazuyasu
Published: March 2005

We previously reported a new brain‐specific protein with a molecular mass of 14 kDa, specifically present in synapses around neurons but not in glial cells [Nakajo, S., Omata, K., Aiuchi, T., Shibayama, T., Okahashi, I., Ochiai, H., Nakai, Y., Nakaya, K. & Nakamura, Y. (1990) J. Neurochem. 55, 2031–2038]. In the present study, we determined the primary structure of this protein, found that it is phosphorylated in vitro and in vivo, and designated it phosphoneuroprotein 14 (PNP 14). The protein is a single polypeptide with 134 amino acid residues (molecular mass = 14122Da), and it contains a hydrophobic region at the center of the molecule. The carboxy‐terminal region has all seven proline residues, and is rich in glutamic acid, which contribute to the acidic property of the protein. The amino‐terminal region possesses four unique repetitive motifs, Glu(Ser)‐Lys‐Thr‐Lys‐Glu(Gln)‐Gly(Gln)‐Val(Ala). When a cytosolic fraction prepared from rat cerebral cortex was incubated with [γ−32P]ATP, 32P was incorporated into PNP 14. Phosphorylated PNP 14 was immuno‐precipitated from rat brain synaptosomes labeled metabolically with [32P]orthophosphate. Injection of [32P]orthophosphate into the third ventricle of rat brain resulted in incorporation of radioactive phosphate into PNP 14. We have also found that Ca2+, calmodulin‐dependent protein kinase II phosphorylates serine residue(s) of PNP 14 in vitro. The results suggest that PNP 14 may be important to neuronal cells, but not to glial cells, and that its physiological functions may be controlled by the phosphorylation reaction.
Expression of human cytochrome c, oxidase subunits during fetal development
Authors: BONNE Giséle , SEIBEL Peter , POSSEKEL Stefanie , MARSAC Cécile , KADENBACH Bernhard
Published: March 2005

Expression of human cytochrome c oxidase (COX) subunits was examined at fetal (20–28 weeks) and adult state by Northern blot hybridization with mRNA from liver, heart, skeletal muscle, and intestine. The data were related to COX and citrate synthase activities and to immunodetected COX subunits (II/III, IV, VIIaH). In liver little changes of COX transcripts are observed from fetal to adult state. In contrast, in heart and skeletal muscle all transcripts of COX subunits increase between 2–20‐fold, when related to the amount of 28S rRNA. In fetal heart and skeletal muscle the relative amounts of the liver‐type transcripts of subunit VIa were 30% and 25% from total VIa transcripts (VIaL + VIaH), respectively, but decrease to only 2–5% at adult state. The liver‐type transcripts of subunit VIIa occur to 50% in fetal heart and skeletal muscle, which remained unchanged in adult heart and decrease to 5–8% in adult skeletal muscle. The results clearly indicate a switch of gene expression in heart and skeletal muscle during development, from the liver type to the heart/muscle type of subunit VIa (and partly VIIa).
Mutational analysis of phospholipase C‐β2
Authors: SCHNABEL Petra , CAMPS Montserrat , CAROZZI Amanda , PARKER Peter J. , GIERSCHIK Peter
Published: March 2005

Members of the β isozyme subfamily of the phosphoinositide‐specific phospholipases C (PLCβ) have recently been shown to be stimulated by both guanine‐nucleotide‐binding protein α and βγ subunits. The α subunits of the Gq class activate PLCβ isozymes in the order of PLCβ1≥ PLCβ3≫ PLCβ2, which is different from the order of PLCβ3 > PLCβ2 > PLCβ1 for βγ subunit stimulation. The C‐terminal region of PLCβ1, in particular the sequence between Thr903 and Leu1142, has been shown to be involved in interacting with activated αq subunits and to contain a region required for efficient membrane association of PLCβ1 [Park, D., Jhon, D.‐Y., Lee, C.‐W., Ryu, S. H. & Rhee, S. G. (1993) J. Biol. Chem. 268, 3710–3714, and Wu, D., Jiang, H., Katz, A. & Simon, M. I. (1993) J. Biol. Chem. 268, 3704–3709]. To examine the structure‐function relationships of a PLCβ isozyme highly sensitive to βγ subunit stimulation, we have altered the cDNA of PLCβ2 by site‐directed mutagenesis and have examined the effects of these structural alterations on the functional properties of the mutant polypeptides. The results show that the C‐terminal region of PLCβ2 downstream of Phe818, which corresponds to Tyr816 of PLCβ1, contains a region essential for membrane association, but is required neither for the interaction of PLCβ2 with Ca2+ and the phospholipid substrate, nor for βγ subunit stimulation of PLCβ2. These data suggest that PLCβ isozymes are activated by αq and βγ subunits via distinct domains.
Biosynthesis of vitamin B12 in anaerobic bacteria
Authors: RENZ Paul , ENDRES Birgit , KURZ Birgit , MARQUART Jörg
Published: March 2005

Eubacterium limosum transformed [2‐13C]5‐hydroxybenzimidazole not only into [2‐13C]5‐hy‐droxybenzimidazolylcobamide, but also into [2‐13C]5‐methoxy‐6‐methylbenzimidazolylcobamide and into [2‐13C]5,6‐dimethylbenzimidazolylcobamide (vitamin B12). [2‐13C]5‐Hydroxy‐6‐methyl‐benzimidazole was used by this bacterium to form [2‐13C]5‐hydroxy‐6‐methylbenzimidazolyl‐cobamide, [2‐13C]5‐methoxy‐6‐methylbenzimidazolylcobamide and [2‐13C]5,6‐dimethylbenzimid‐azolylcobamide. The 1H‐NMR spectrum of the 5,6‐dimethylbenzimidazole isolated from the 13Clabeled vitamin B12 preparations revealed that the externally added bases had been transformed into the vitamin B12 base almost without dilution of the label. This suggests that 5‐hydroxybenzimidazole and 5‐hydroxy‐6‐methylbenzimidazole are precursors of 5,6‐dimethylbenzimidazole.

On the basis of these results, a hypothetical scheme for the biosynthesis of 5,6‐dimethylbenzimidazole via 5‐hydroxybenzimidazole and 5‐hydroxy‐6‐methylbenzimidazole is discussed. This scheme can also explain the formation of the other benzimidazole bases found in natural vitamin B12 analogs.
Protein glycosylation
Authors: LIS Halina , SHARON Nathan
Published: March 2005

During the last decade, there have been enormous advances in our knowledge of glycoproteins and the stage has been set for the biotechnological production of many of them for therapeutic use. These advances are reviewed, with special emphasis on the structure and function of the glycoproteins (excluding the proteoglycans). Current methods for structural analysis of glycoproteins are surveyed, as are novel carbohydrate–peptide linking groups, and mono‐ and oligo‐saccharide constituents found in these macromolecules. The possible roles of the carbohydrate units in modulating the physicochemical and biological properties of the parent proteins are discussed, and evidence is presented on their roles as recognition determinants between molecules and cells, or cell and cells. Finally, examples are given of changes that occur in the carbohydrates of soluble and cell‐surface glycoproteins during differentiation, growth and malignancy, which further highlight the important role of these substances in health and disease.
Molecular analysis of chicken embryo SPARC (osteonectin)
Authors: BASSUK James A. , IRUELA‐ARISPE M. Luisa , LANE Timothy F. , BENSON Janice M. , BERG Richard A. , SAGE E. Helene
Published: March 2005

SPARC is a secreted glycoprotein that modulates cell shape and cell‐matrix interactions. Levels of SPARC are increased at sites of somitogenesis, osteogenesis, and angiogenesis in the embryo and during wound repair in the adult. We have cloned and characterized SPARC from chicken embryo. A 2.2‐kbp cDNA, obtained by a novel use of the polymerase chain reaction, was determined to encode a 298‐residue protein that is 85% identical to human SPARC. Antigenic sites in particular appear to be highly conserved, as antibodies against C‐terminal sequences of murine and bovine SPARC reacted with a 41–43 kDa protein in chicken embryo extracts. Chicken SPARC can be defined by four sequence signatures: (a) a conserved spacing of 11 cysteine residues in domain II, (b) the pentapeptide KKGHK in domain II, which is contained within a larger region of 31 identical residues, (c) a 100% conserved region of 10 residues in domain III, and (d) a C‐terminal, calcium‐binding EF‐hand motif. SPARC mRNAs in the 10‐day‐old chicken embryo are represented by three sizes of 1.8, 2.2 and 3.0 kb. The relative steady‐state levels for the 2.2‐kb mRNA were determined as aorta ≥ skeletal muscle > calvarium > vertebra > anterior limb > kidney > heart > brain > skin and lung ≥ liver. The relative abundance of the 1.8‐kb and 2.2‐kb mRNAs varied among tissues and indicated that differential processing of SPARC mRNAs might occur. All three RNA species were detected by a cDNA probe for the N‐terminal part of the coding region. Thus, the three mRNA species appear to arise from differential 3′ splicing and/or polyadenylation. Collective evidence demonstrates that SPARC has been well‐conserved during vertebrate evolution, a finding that indicates a fundamental role for this protein in development.
Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis
Authors: MACKAY Elaine A. , OVERNELL Julian , DUNBAR Bryan , DAVIDSON Ian , HUNZIKER Peter E. , KÄGI Jeremias H. R. , FOTHERGILL John E.
Published: March 2005

Cadmium‐induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10 kDa and 20 kDa. The 10‐kDa group was resolved by anion‐exchange chromatography into four fractions while the 20‐kDa group was resolved into three fractions using this method. After metal removal and S‐methylation of the cysteine residues using methyl‐p‐nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20‐kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72‐amino‐acid proteins of the 10‐kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT‐10‐IV isoform and other metallothioneins. The mussel MT‐10‐IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non‐molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.
Effect of lysine ionization on the structure and electrochemical behaviour of the Met44→Lys mutant of the blue‐copper protein azurin from Pseudomonas aeruginosa
Authors: VAN DE KAMP Mart , CANTERS Gerard W. , ANDREW Colin R. , SANDERS‐LOEHR Joann , BENDER Christopher J. , PEISACH Jack
Published: March 2005

The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5–8, the optical absorption, resonance Raman, EPR and electron spin‐echo envelope modulation spectroscopic properties of wild‐type and Met44‐→Lys (M44K) azurin are very similar, indicating that the Cu‐site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pKa 9–10) is accompanied by changes in the optical‐absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR g∥ value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self‐exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild‐type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation.
Effects of glycosylation on protein structure and dynamics in ribonuclease B and some of its individual glycoforms
Authors: JOAO Heidi C. , DWEK Raymond A.
Published: March 2005

In an attempt to elucidate the role of carbohydrates on protein structure and dynamics in glycoproteins, ribonuclease B (RNase B), containing a single glycosylation site at Asn34, has been investigated and compared with the enzyme in the unglycosylated form (RNase A). RNase B consists of five glycoforms: Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2and Man9GlcNAc2(Man5–9GlcNAc2). The individual glycoforms Man1GlcNAc2(synthetic) and Man5GlcNAc2of RNase B have been studied to ascertain any specific effects of the different oligosaccharides. NMR measurement of amide‐proton/deuterium exchange rates shows that glycosylation of the enzyme leads to the protection of amide‐proton resonances from solvent exchange for a large number of residues, both in the vicinity of the glycosylation site (e.g. residues 29–34 and 35) and remote from it (e.g. residues 57–61 and 75–76). In addition, residues 10–13, 32, 34 and 35, which are observed to be protected from exchange as a result of glycosylation in the individual glycoforms Man1GlcNAc2‐RNase and Man5GlcNAc2‐RNase (when compared with RNase A) are less protected in RNase B. This additional protection in the glycoforms Man1GlcNAc2‐RNase and Man5GlcNAc2‐RNase may arise from steric hindrance between the oligosaccharide and protein reducing solvent accessibility. The rates of solvent exchange of amide protons for residues 10–13, 32, 34 and 35 are dependent on the oligosaccharide moiety. The average amide‐proton/deuterium exchange rate in Man6–9GlcNAc2‐ribonucleases for residues 10–13 and 35 is approximately three times greater than Man5GlcNAc2‐ribonuclease, while for residues 32 and 34 it is approximately 7–11 times greater. CD analysis of RNase A and RNase B revealed the carbohydrate moiety to have a small stabilizing effect (approximately 5 kJ/mol) on the protein.
Gene targeting in human somatic cells
Authors: PORTER Andrew C. G. , ITZHAKI Jane E.
Published: March 2005

The role, if any, of the human interferon‐inducible 6–16 gene in the establishment of a cellular antiviral state is unknown. To address this problem, and as part of a wider investigation of homologous recombination (HR) and its applications in somatic cells, we have been using HR to disrupt the 6–16 gene in human cell lines [Itzhaki, J. E. & Porter, A. C. G. (1991) Nucleic Acids Res. 19, 3835–3842.] We describe here the design and use of insertion and replacement‐type targeting constructs based on a promoterless bacterial gpt gene that is activated by HR with the 6–16 gene. In HeLa cells, both targeting constructs underwent extrachromosomal HR with a cotransfected plasmid carrying the 6–16 gene. In a previously targeted clone derived from the fibrosarcoma cell line HT1080, the replacement construct underwent HR with either the modified or the unmodified 6–16 allele. The latter events generated doubly disrupted (6–16–/–) clones that failed to express any detectable 6–16 messenger RNA in response to interferon. Plaque assays of infected 6–16–/– cells showed that expression of the 6–16 gene was not required for the induction by interferon of an antiviral state against encephalomyocarditis virus, semliki forest virus or cocal virus.