bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and characterization of polyclonal antibodies against a conserved sequence in the catalytic domain of protein kinases
Authors: Hagedorn Curt H. , Tettelbach William H. , Panella Heather L.
Published: July 2002

Using a synthetic oligopeptide (CGGGTPEYLAPEGGK) crosslinked to keyhole limpet hemocyanin we have raised polyclonal rabbit antibodies against a 9 residue homologous region found in the catalytic domain of most protein kinases. These antibodies reacted during Western immunoblotting with cAMP dependent protein kinase catalytic subunit, phosphorylase kinase γ subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPYLAPE and GTPYLPE, respectively. Five other protein kinases did not react with anti‐GTPEYLAPE antibodies during Western immunoblotting. Affinity‐purified antibodies were able to detect as little as 50 ng of cAMP dependent protein kinase and 200 ng of Ca2+/calmodulin dependent protein kinase II. Immunoblotting of A431 cell plasma membrane vesicles indicated the presence of an approximately 55 kDa protein that contains the conserved sequence and is likely to be a protein kinase. Antibodies directed against conserved sequences present in protein kinases, or possibly other enzymes, may be useful in identifying previously uncharacterized enzymes at the protein level.
Neurokinin‐immunoreactivity in human neuroblastomas
Authors: McGregor Gerard P. , Gaedicke Gerhard , Voigt Karlheinz
Published: July 2002

Factors regulating differentiation of the peripheral nervous system (PNS) have been widely studied in neuroblastomas which are tumors of the PNS. Five neuroblastomas were investigated for their content of tachykinin neuropeptides, which arise from two distinct genes which appear differentially expressed in the PNS. Radioimmunoassay and column chromatography revealed large amounts of neurokinin B in three of these tumors and the absence of substance P, neurokinin A, neuropeptide K and neuropeptide γ from all five tumors. This suggests that neuroblastomas can selectively express the preprotachykinin (PPT) II gene and that they may be valuable for investigating the factors involved in the regulation of these two structurally‐related neuropeptide genes.
Proton magnetic resonance spectroscopic analysis of diadenosine 5′,5‴‐polyphosphates
Authors: Mayo Kevin H. , Mvele Omer M. , Puri Rajinder N.
Published: July 2002

Certain diadenosine 5′,5‴‐polyphosphates are potent inhibitors of ADP stimulated platelet aggregation, acting possibly via competitive ADP‐receptor binding. 1H NMR studies of a series of such compounds where the number of phosphate groups between adenosine groups was varied from 2 to 6 were performed to analyze possible preferred solution conformations and to define structure‐activity relations. Relative to mononucleotides ADP and ATP, chemical shifts of adenosine proton resonances in diadenosine polyphosphate analogs are upfield shifted suggesting base stacking. This effect is greatest for AP2A and AP3A. Coupling constants of ribose ring proton resonances support the idea of an anti‐base‐ribose ring conformation, and 3
J
H5'‐P values suggest a preferred gauche H‐C‐O‐P structure. In all cases, NMR parameters for AP2A are near‐limiting values for a static base stacked conformation. Increasing the number of phosphate groups between adenosine moieties tends to weaken this interaction.
The structure of NADH peroxidase from Streptococcus faecalis at 3.3 Å resolution
Authors: Stehle T. , Ahmed S.A. , Claiborne A. , Schulz G.E.
Published: July 2002

NADH peroxidase (EC 1.11.1.1) previously isolated from Streptococcus faecalis 10C1 has been crystallized. The crystal structure has been solved by multiple isomorphous replacement and solvent‐flattening at 3.3 Å (1 Å = 0.1 nm) resolution. The enzyme forms a tetramer consisting of 4 crystallographically related subunits. The monomer chain fold is in general similar to those of glutathione reductase and lipoamide dehydrogenase. FAD binds in the same region and in a similar conformation as in glutathione reductase. The unusual cysteine‐sulfenic acid participating in catalysis is located at the isoalloxazine of FAD.
Single step purification of methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum by specific binding to Blue Sepharose CL‐6B
Authors: Ma K. , Thauer R.K.
Published: July 2002

Methylenetetrahydromethanopterin reductase from metanogenic archaebacteria catalyzes the reversible reduction of N
5,N
10‐methylenetetrahydro‐methanopterin to N
5‐methyltetrahydromethanopterin with reduced coenzyme F420 as electron donor. The enzyme is involved in methane formation from CO2, and in methanoi disproportionation to CO2 and CH4. We report here that the reductase from Methanobacterium thermoautotrophicum specifically binds to Blue Sepharose CL‐6B. Binding was competitive with coenzyme F420 rather than with NAD, NADP, FAD, FMN, AMP, ADP and ATP. The reductase could also be desorbed with salt. Based on this property an affinity Chromatographie procedure for the purification of the enzyme was developed.
On the relation between surface area and partitioning of particulates in two‐polymer aqueous phase systems
Authors: Walter Harry , Fisher Derek , Tilcock Colin
Published: July 2002

Partitioning in dextran‐poly(ethylene glycol) aqueous two‐phase systems is an established method for the separation of biomaterials. Size and surface properties are generally regarded as parameters which contribute to the determination of the materials' partition coefficients, K. While molecular weight or surface area can be one of the determinants of the K value of biomaterials in the size range of macromolecules to very small particulates (e.g. some viruses), partitioning liposomes of identical surface properties and different but distinct sizes indicate that surface areas greater than about 0.2 μm2 do not affect the K value obtained. Analysis of available partitioning data of much larger particulates (i.e. cells) reveals that surface properties per unit area outweigh surface area per se in determining the K value in non‐charge‐sensitive, charge‐sensitive and biospecific affinity phase systems.
Efficient aminoacylation of a yeast tRNAAsp transcript with a 5' extension
Authors: Perret Véronique , Florentz Catherine , Giegé Richard
Published: July 2002

A yeast aspartic acid tRNA with a 5' extension of 14 nucleotides was obtained by in vitro transcription with T7 DNA dependent RNA polymerase. This transcript, called extended tRNAAsp transcript, retains its aspartylation capacity with the same K
m and only three times reduced k
cal values as compared to those measured for canonical tRNAAsp. This result indicates that the 5' extension of the amino acid acceptor stem of tRNAAsp does not interfere with recognition by aspartyl‐tRNA synthetase. However, in contrast to the wild‐type tRNAAsp transcript, the 5' extended molecule presents a reduced capacity to be mischarged by arginyl‐tRNA synthetase, suggesting the existence of different structural requirements in aspartyland arginyl‐tRNA synthetases for tRNAAsp recognition.
Effects of dideoxyforskolin on proteoglycan synthesis and structure in embryonic chick chondrocyte cultures
Authors: Hu Lie-Min , Kemp Stephen F. , Peng Chun-Fu , Elders M.Joycelyn , Smith W.Grady
Published: July 2002

1,9‐Dideoxyforskolin inhibits proteoglycan synthesis and xyloside‐initiated glycosaminoglycan (GAG) synthesis in chick embryo chondrocytes. Dideoxyforskolin does not affect the length of xyloside‐initiated GAG chains secreted into the medium but chains from the dense proteoglycan secreted into the medium appear slightly longer. Incorporation of labeled serine into the dense proteoglycan and subsequent digestion with Pronase revealed a dramatic decrease in percent of total radioactivity associated with GAG chains in the proteoglyean from cultures treated with forskolin or dideoxyforskolin. These observations suggest that these diterpenes have a specific inhibitory effect on chain initiation reactions and thus may be useful tools in the study of proteoglycan synthesis and processing.
Overexpression of glucose transporter modulates insulin biosynthesis in insulin producing cell line
Authors: Shibasaki M. , Shibasaki Y. , Asano T. , Kajio H. , Akanuma Y. , Takaku F. , Oka Y.
Published: July 2002

Glucose transporter (GT) has been suggested to be involved in the insulin biosynthesis. However, the functional relationship between GT and insulin biosynthesis is not well understood. In this report, we have generated rat pancreatic B cell lines (RINr) that stably overexpress a cDNA encoding the brain type GT. These cell lines showed 3‐ to 4‐fold increase in insulin mRNA and protein. These results suggest that GT might have some relationship to the insulin biosynthesis in the pancreatic B cells.
γ‐Purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm
Authors: Colilla Francisco J. , Rocher Asuncion , Mendez Enrique
Published: July 2002

Two homologous sulfur‐rich basic polypeptides form wheat endosperm, so‐called γ1‐purothionin and γ2‐purothionin, are described. Purification involves extraction with volatile solvents and ammonium bicarbonate fractionation followed by reversed‐phase high‐performance liquid chromatography. The complete primary structure of these two polypeptides has been determined by automatic degradation of the intact, S‐carboxymethylated γ‐purothionins and peptides obtained by enzymatic cleavage. γ1‐Purothionin and γ2‐purothionin consist of 47 amino acids with an assessed molecular weight of 5239 and 5151 Da, respectively and 8 cysteines organized in 4 disulfide bridges. They present a high degree of homology among themselves (89% of identity) and are the first two thionin‐like polypeptides, so‐called y‐thionins, described from wheat endosperm.
Interaction of histones H1 and H1° with superhelical and linear DNA
Authors: Yaneva Julia , Zlatanova Jordanka , Paneva Elena , Srebreva Ljuba , Tsanev Roumen
Published: July 2002

By using direct competition experiments, the binding of histone H1AB (a mixture of H1A and H1B) and H1° to superhelical and linear DNA forms was studied. Mouse liver H1 isohistones and plasmid pαGD containing part of the 5′ flanking and part of the coding sequence of the mouse α‐globin gene in pUC18 were used as partners in the binding reaction. The competition experiments were performed by direct mixing of the histone with labelled supercoiled DNA (at 125 mM NaCI and at a histone/DNA ratio of 1.0 and addition to the mixture of increasing amounts of cold competitor DNA, either supercoiled or linear. The radioactivity of the complex formed was determined by filter binding. The results show that both histones H1 and H1° possess a strong binding preference for supercoiled DNA forms. Thus, histone H1° resembles the regular somatic set of histone H1 and not the other differentiation‐specific histone H5 studied thus far.
Oriented incorporation of bacteriorhodopsin into the lipid shell of phospholipid‐coated polymer particles
Authors: Rothe Ulrich , Aurich Harald , Engelhardt Harald , Oesterhelt Dieter
Published: July 2002

Phospholipid monolayers covalently fixed to spherical polymer carriers spontaneously form a stable bilayer‐like structure by adsorption of further added phospholipids. Bacteriorhodopsin could be incorporated into the lipid bilayer growing on these particles. From the direction of proton pumping as well as from freeze‐fracture electron micrographs and papain digestion, experimental evidence was obtained that the preferred orientation of bacteriorhodopsin within the lipid coat is ‘inside‐out’ (more than 90%). A ‘right‐side‐out’ incorporation of bacteriorhodopsin could be demonstrated, if the negative charge density of the carrier surface was lowered by chemical modification.
Do the major human glutathione S‐transferases have fatty acid ethyl ester synthase activity?
Authors: Suzuki Takashige , Kovacs Matthew S. , Board Philip G.
Published: July 2002

Two fatty acid ester (FAEE) synthase isoenzymes purified from human myocardium were reported to be glutathione S‐transferases (GST) [(1989) Proc. Natl. Acad. Sci. USA 86, 4470–4473; and (1989) J. Clin. Invest. 84, 1942–1946]. In the present study, the FAEE synthase activity of several purified and well characterized human GSTs were examined with ethanol and [14C]oleic acid as substrates. Three isoenzymes, GST1, GST2 and GST3 which are members of the evolutionary classes μ, α and π, respectively, were studied and failed to show any significant synthesis of FAEE after 45 min incubation at 37°C, FAEE synthase activity and GST3 activity in human placental extracts can be readily separated by ion exchage chromatography on DEAE cellulose. Thus the results show that FAEE synthase activity is not a feature of the major GSTs found in human tissues. The two FAEE synthase isoenzymes isolated by Bora et al. may have been co‐purified with GST isoenzymes of these FAEE synthases may be members of the GST super family that have low specific activity in conventional GST assays and have not been previously described.
Premature termination of transcription and alternative splicing in the human transacylase (E2) gene of the branched‐chain α‐ketoacid dehydrogenase complex
Authors: Lau Kim S. , Lee Jun , Fisher Charles W. , Cox Rody P. , Chuang David T.
Published: July 2002

We have isolated a human genomic clone hgE2‐14 containing exons 5,6,7 and 8 of the branched‐chain α‐ketoacid dehydrogenase E2 transacylase gene. Sequencing of exon B and its surrounding intronic sequences reveals complete identity with the previously reported truncated E2 cDNA (hE2‐1) sequence between nucleotides 938 and 1521. We have identified consensus splice site junctions flanking exon 8 and also a cryptic 3??? splice site 370 bases upstream from the start of exon 8 in the gene. In addition, two polyadenylation signals located in the hE2‐1 cDNA are also present in the intronic sequence downstream of exon 8 which promote termination of transcription. The data indicate that shortened human liver E2 transcripts undergo alternative splicing to yield mRNA of the hE2‐1 type.
Oxidation/reduction explains heterogeneity of pancreatic somatostatin
Authors: Bersani Maurizio , Johnsen Anders H. , Holst Jens Juul
Published: July 2002

Somatostatin 14 (SS 14) has been isolated from pancreatic extracts, but open gel filtration immunoreactive SS often elutes in two peaks. We isolated both peaks, but upon sequence analysis only authentic SS 14 could be identified. By further gel filtration experiments it turned out that both synthetic and extractable SS appeared homogeneous at neutral pH 7.5, but showed an additional earlier peak in acetic acid. After addition of mercaptoethanol, all of the SS eluted at this earlier position regardless of the pH. We conclude that partial reduction/oxidation of SS explains the heterogeneity.
Activation of protein kinase C inhibits hormonal stimulation of the GTPase activity of Gi in human platelets
Authors: Gutiérrez-Venegas Gloria , García-Sáinz J.Adolfo
Published: July 2002

The effect of 12‐tetradecanoyl phorbol 13‐acetate on the GTPase activity of Gi was investigated. Treatment with TPA did not alter basal GTPase activity of membranes or the simulatory effect of prostaglandin E1 (putatively via Gs). In contrast, the phorbol ester markedly diminished stimulation of GTPase by agents whose receptors are coupled to Gi such as epinephrine (α‐adrenergic action), platelet activating factor or thrombin. Pertussis toxin catalyzed ADP‐ribosylation was also decreased in membranes from TPA‐treated platelets as compared to the controls. It is suggested that the Alteration in the hormonal activation of the GTPase activity of Gi is secondary to a perturbation in the receptor‐Gi interaction.
A new family of small (5 kDa) protein inhibitors of insect α‐amylases from seeds or sorghum (Sorghum bicolor (L) Moench) have sequence homologies with wheat γ‐purothionins
Authors: Bloch Carlos , Richardson Michael
Published: July 2002

Three isoinhibitors of locust and cockroach gut α‐amylases were purified from seeds of sorghum by saline extraction, precipitation with ammonium sulphate, affinity chromatography on Red‐Sepharose and preparative RP‐HPLC on Vydac C???. The complete primary structures were determined by automatic degradation of the intact reduced and S‐alkylated proteins, and by manual DABITC/PITC microsequencing of peptides obtained from enzyme digests. The inhibitors consist of 47 (Slα‐1) or 48 (Slα‐2, STα‐3) amino acids, and are the smallest plant inhibitors of α‐amylase currently known. The sequences of the three isoinhibitors exhibit between 38% and 87% identity among themselves and also have homology (32–81%) with the γ‐purothionins recently isolated from wheat endosperm.
pH‐dependent changes in structure and RNA‐binding activity of casein kinase 2 from Rana temporaria oocytes
Authors: Kandror Konstantin V. , Kapkov Denis V. , Turapov Obolbek A. , Stepanov Aleksandr S.
Published: July 2002

It is demonstrated by filter‐binding assay that casein kinase 2 from Rana temporaria oocytes binds rRNA in vitro with high affinity. Ligand‐blotting shows that rRNA‐binding activity is inherent to α and α′ subunits of the enzyme. Increase of pH from 6.5 to 7.5 has little effect on casein kinase but completely suppresses rRNA‐binding activity of the enzyme. Sedimentation coefficient of casein kinase 2 also defends on pH: at pH 7.5 it is mainly 10 S, and at 6.5 – 18 S. At pH 6.95 the amounts of both forms are equal. The heavy form of casein kinase 2 practically lacks rRNA‐binding activity. Casein kinase 2; RNA‐binding; pH
RNA—RNA and RNA—protein interactions in 30 S ribosomal subunits Association of 16 S rRNA fragments in the presence of ribosomal proteins
Authors: Afonina E.I. , Chichkova N.V. , Bogdanov A.A.
Published: July 2002

The E. coli 16 S rRNA with single‐site breaks centered at position 777 or 785 was obtained by R Nase H site‐specific cleavage of rRNA. Spontaneous dissociation of the cleaved 16 S rRNA into fragments occurred under ‘native’ conditions. The reassociation of the 16 S rRNA fragments was possible only in the presence of ribosomal proteins. The combination of S4 and S16(S17) ribosomal proteins interacting mainly with the 5′‐end domain of 16 S rRNA was sufficient for reassociation of the fragments. The 30 S subunits with fragmented RNA at ca. 777 region retained some poly(U)‐directed protein synthetic activity.
Study of mammalian selenocysteyl‐tRNA synthesis with [75Se]HSe−
Authors: Mizutani Takaharu , Kurata Hiroshi , Yamada Kenichiro
Published: July 2002

The mechanisms of the synthesis of mammalian selenocysteyl‐(Scy)‐tRNA were studied using [75Se]H2Se. H2Se was prepared from [75Se]selenite, glutathione, NADPH and glutathione reductase, and was purified by chromatography. It was confirmed that this H2Se was a Se donor in the reaction of the synthesis of Scy‐tRNA. [75Se]Scy, liberated from aminoacyl‐tRNA, was analyzed by TLC on silica gel an subsequent autoradiography. The activity of Scy‐tRNA synthesis was found in the supernatant at 105 000 × g of the murine liver extract, but not in the precipitate. The supernatant was chromatographed on DEAE‐cellulose, and the activity was eluted at a concentration of 0.17 M KCl. This position is at the front shoulder of the peak of seryl‐tRNA synthetase which was eluted at 0.20 M KCl. Major serine tRNAIGA is not a substrate on which to synthesize Scy‐tRNA, but natural opal suppressor serine tRNA is. On a chromatographic pattern of a Scy‐tRNA preparation on Sephacryl S‐200, the radioactivity of 75Se was eluted at the tRNA peak. This showed that Scy bound to tRNA. The active protein fraction from DEAE‐cellulose did not contain tRNA kinase, therefore Scy‐tRNA must be directly synthesized from seryl‐tRNA, not through phosphoseryl‐tRNA. This mechanism is similar to that seen in Escherichia coli [1991, J. Biol. Chem. 266, 6324].
Identification of a rat liver dipeptidyl aminopeptidase IV with a liver plasma membrane glycoprotein (gp110) A study using dipeptidyl aminopeptidase IV‐deficient rats
Authors: Iwaki-Egawa Sachiko , Watanabe Yasuhiro , Fujimoto Yukio
Published: July 2002

A rat liver plasma membrane glycoprotein, gp110, was compared with dipeptidyl aminopeptidase IV (DAP IV) by using Wistar rats (DAP IV‐positive rats) and Fischer 344 rats (DAP IV‐negative rats). Fischer rats also lacked gp110 and gp110 of Wistar rats had DAP IV activity. Furthermore, we showed that the C‐terminal sequence of gp110 was Ser‐Leu‐Arg, which was the same as the C‐terminal amino acid sequence deduced from the nucleotide sequence of the cDNA of DAP IV. According to these results, we concluded that gp110 was identical with DAP IV.
Significant correlations between atmospheric spectra according to Baumer and the in vitro incorporation of [3H]thymidine into the nuclear DNA of C6‐glioma cells
Authors: Vogl Siegfried , Hoffmann Georg , Stöpfel Barbara , Baumer Hans , Kempski Oliver , Ruhenstroth-Bauer Gerhard
Published: July 2002

Significant correlations between certain spectra of atmospherics (spherics) according to Baumer (a.t.B), i.e. naturally occurring electro‐magnetic impulses in the range of 4–50 kHz, and several diseases or biological parameters have been published earlier. Now we show that there exists a highly significant negative correlation (r= −0.61, P>0.004) between the occurrence of 28 kHz impulses (a.t.B.) and the in vitro incorporation of thymidine into the nuclear DNA of C6‐glioma cells. The positive correlation with the 10 kHz impulses (a.t.B.) (r=0.39), however, is statistically not significant (P>0.055).
Molecular cloning and mammalian expression of human β2‐glycoprotein I cDNA
Authors: Kristensen Torsten , Schousboe Inger , Boel Esper , Mulvihill Eileen M. , Hansen Rita Rosendahl , Møller Karin Bach , Møller Niels Peter Hundahl , Sottrup-Jensen Lars
Published: July 2002

Human β2‐glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19‐residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible
Nuclear localization of budgerigar fledgling disease virus capsid protein VP2 is conferred by residues 308–317
Authors: Rihs Hans-Peter , Peter Reiner , Hobom Gerd
Published: July 2002

The capsid protein VP2 of budgerigar fledgling disease virus (BFDV) contains two sequences (residues 309–315 and 334–340) which are homologous to the prototypic nuclear localization sequence (NLS) of the simian virus 40 T‐antigen. Using recombinant potential NLS‐β‐galactosidase fusion proteins we identified amino acid residues 308–317 (VPKRKRKLPT) to be the NLS of BFDV capsid proteins VP2 and VP3. Microfluorometry studies show that the BFDV‐VP2 signal is considerably more efficient in nuclear transport kinetics, than the NLS of SV40‐VP2, corresponding to amino acid residues 317–326 (PNKKKRKLSR).
Possible involvement of GTP‐binding proteins in growth regulation of human epidermoid carcinoma cell line A431
Authors: Masuda Nobuhisa , Ui Michio
Published: July 2002

A431 cells, a human epidermoid carcinoma cell line, express an unusually large number of cell surface receptors for the epidermal growth factor (EGF). The growth rate of A431 cells was estimated by measuring [3H]thymidine incorporation at the logarithmic growth phase. The growth of the cells in protein‐free medium was partially inhibited by exposure of the cells to pertussis toxin, islet‐activating protein (IAP). The growth in both serum‐containing and protein‐free medium was inhibited by high concentrations of EGF, and these inhibitions were partially reversed by treatment of the cells with IAP. The effects of IAP were well correlated with the degree of ADP‐ribosylation of a membrane 40‐kDa protein. Thus, IAP sensitive G‐proteins appear to be involved in the signal transduction of both positive and negative regulation of A431 cell growth. The possibility is also discussed that phosphatidylinositol turnover may participate in growth regulation.
Use of a monoclonal antibody (APE‐1) directed against the conserved GTPEYLAPE motif present in protein kinases as an affinity purification reagent
Authors: Hagedorn Curt H.
Published: July 2002

cDNA sequence studies have predicted the existence of protein kinases that have not been characterized at the protein level. This laboratory has previously shown that polyclonal rabbit antibodies directed against a highly conserved sequence motif, GTPEYLAPE, present in the catalytic domain of many protein kinases will react during immunoblots with several protein kinases containing the same or homologous motifs. In this report I describe the development of a mouse monoclonal antibody, designated APE‐1, that reacts with the GTPEYLAPE motif. In addition, the use of this monoclonal antibody in the affinity purification of kinase activities from rabbit reticulocyte lysate is described. This approach may provide a general method for the purification of previously uncharacterized protein kinases that share regions which are homologous to the GTPEYLAPE motif. Refinements in this method may permit the rapid purification of nonabundant and/or rapidly inactivated protein kinases that have not been isolated using other approaches.
Photooxidation of high‐potential (c559, c556) and low‐potential (c552) hemes in the cytochrome subunit of Rhodopseudomonas viridis reaction center
Authors: Nabedryk E. , Berthomieu C. , Verméglio A. , Breton J.
Published: July 2002

The photooxidation of c
559, c
556, and c
552 hemes in Rhodopseudomonas viridis cytochrome has been characterized by light‐induced FTIR difference spectroscopy. Apart from the common features at 1659 cm−1 and 1561/1551 cm−1 which could arise from one (or possibly two) peptide bond(s), no evidence for major structural rearrangement of the polypeptide backbone was observed. A significant difference with respect to redox‐induced FTIR spectra of cytochrome c is the absence of the Tyr marker at 1514/1518 cm−1 in Rps. viridis cytochrome, indicating that the localized shift of a Tyr side chain observed between ferro‐ and ferri‐cytochrome c does not occur in Rps. viridis cytochrome.
Demonstration of L1‐related mRNAs in rat brain using DNA oligonucleotide probes
Authors: Gaardsvoll Henrik , Andersson Anna-Maria , Bock Elisabeth
Published: July 2002

Only one copy of the cell adhesion molecule L1 gene is present in the mouse genome, and only one mRNA of 6 kilobases (kb) is expressed in mouse brain [1987, Neurosci. Lett. 82, 89–94]. We have constructed 5 synthetic oligonucleotide probes covering different parts of the published mouse L1 cDNA sequence. Using these probes 3 distinct mRNAs of 9.0, 7.0 and 6.0 kb in rat brain could be demonstrated. Hybridizations performed at different stringency conditions indicated that the 9.0 and 7.0 kb mRNAs were highly related to the L1 mRNA of 6.0 kb expressed in rat brain. The 7.0 kb mRNA is possibly coding for a rat homologue of chicken Nr‐CAM, whereas the 9.0 kb mRNA may code for a new member of the L1 family.
An interferon‐induced protein with release factor activity is a tryptophanyl‐tRNA synthetase
Authors: Bange Franz-Ch. , Flohr Thomas , Buwitt Ute , Böttger Erik C.
Published: July 2002

Interferon gamma induces expression of a protein termed IFP 53 according to its molecular weight of 53 kDa. IFP 53 shows significant sequence homology to rabbit peptide chain release factor as well as to bovine tryptophanyl‐tRNA synthetase. IFP 53 has been shown to possess release factor activity for the UGA stop codon. We demonstrate here, by using a recombinant IFP 53 fusion protein, that IFP 53 tryptophanylates tRNA. These data indicate that IFP 53 is a protein with two activities: peptide chain termination and aminoacylation.
Identification of reactive tyrosine residues in cysteine‐reactive dehydrogenases Differences between liver sorbitol, liver alcohol and Drosophila alcohol dehydrogenases
Authors: Prozorovski Vladimir , Krook Maria , Atrian Silvia , Gonzàlez-Duarte Roser , Jörnvall Hans
Published: July 2002

Modification of tyrosine residues with tetranitromethane and reversible sulphite protection of cysteine residues were tested on three dehydrogenases of two families. In liver alcohol dehydrogenase no Tyr residue is appreciably labelled, while in the homologous sorbitol dehydrogenase Tyr‐109 is specifically labelled; the difference corresponds to a segment correlating with subunit interactions and the different quaternary structures of the proteins. In Drosophila alcohol dehydrogenase. Tyr modification is multiple, and the results show the presence of two different states of Cys residues, reactive in the presence and absence of cupric ions, respectively. Super‐activation with cyanide was also noticed after S‐sulphocysteine protection. The results demonstrate the possibility of identification of specific Tyr residues in proteins with reversibly protected Cys residues.
A novel non‐peptide endothelin antagonist isolated from bayberry, Myrica cerifera
Authors: Fujimoto Masafumi , Mihara Shin-ichi , Nakajima Shigeyuki , Ueda Motohiko , Nakamura Miharu , Sakurai Ken-suke
Published: July 2002

A potent non‐peptide ET receptor antagonist, myrireron caffeoyl ester (50–235), was isolated from the bayberry, Myrica cerifera. This compound selectively antagonized specific binding of [125I]ET‐1, but not of [125I]ET‐3, to rat cardiac membranes, ET‐1‐induced increase in the intracellular free calcium concentration in Swiss 3T3 fibroblasts, and ET‐1‐induced contraction of rat aortic strips. Thus, 50–235 is the first non‐peptide ETA receptor antagonist. This compound can be useful for studying the physiological role of endothelin and exploring its role in various diseases.
In vivo activation of the yeast plasma membrane ATPase during nitrogen starvation Identification of the regulatory domain that controls activation
Authors: Benito Begoña , Portillo Francisco , Lagunas Rosario
Published: July 2002

Yeast plasma membrane ATPase is activated during nitrogen starvation when a fermentable substrate is present. This activation is due to changes in the V
max and it is irreversible, independent of protein synthesis and apparently triggered by a decrease in the intracellular pH. It is shown that the ATPase regulatory domain implicated in the activation by fermentable carbon sources is also implicated in activation by nitrogen starvation and by external acidification.
Primary structure required for the inhibition of smooth muscle myosin light chain kinase
Authors: Ikebe Mitsuo , Reardon Sheila , Fay Fredric S.
Published: July 2002

Myosin light chain kinase (MLCK) contains the autoinhibitor sequence right next to the N‐terminus side of the calmodulin binding region. In this paper, the structural requirement of the inhibition of MLCK activity was studied using synthetic peptide analogs. Peptides Ala‐783‐Lys‐799 and Ala‐783‐Arg‐798 inhibited calmodulin independent MLCK at the same potency as the peptide Ala‐783–Gly‐804. Deletion of Arg‐797‐Lys‐799 or substitution of these residues to Ala markedly increased the K
i while the substitution of Lys‐792 and Lys‐793 to Ala and the deletion of Lys‐784‐Lys‐785 did not affect the inhibitory activity of the peptides. The results suggest that Arg‐797‐Arg‐798 are especially important for the inhibitory activity among other basic residues in the autoinhibitory region.
Occupancy of a C2‐C2 type ‘Zinc‐finger’ protein domain by copper Direct observation by electrospray ionization mass spectrometry
Authors: Hutchens T.William , Allen Mark H. , Li Chee Ming , Yip Tai-Tung
Published: July 2002

The metal ion specificity of most ‘zinc‐finger’ metal binding domains is unknown. The human estrogen receptor protein contains two different C2‐C2 type ‘zinc‐finger’ sequences within its DNA‐binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71‐residue ERDBD (K180‐M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA‐binding domain to bind Cu and (ii) document a twofold difference in the Zn‐ and Cu‐binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc‐finger domains both in vitro and in vivo.
Interferon‐mediated intracellular signalling Modulation of different phospholipase activities in Burkitt lymphoma cells
Authors: Cataldi A. , Rana R. , di Primio R. , Bareggi R. , Lisio R. , Toto N. , Miscia S.
Published: July 2002

The effect of interferon‐α on Daudi lymphoma cells either sensitive or resistant to the action of this cytokine has been analysed in terms of phospholipase C (PLC) and D (PLD) activities. Results have shown a combined modulation of PIP2‐specific phospholipase C and phospholipase D. In particular, a decreased activity of PIP2‐specific PLC has been found, concomitant to a PLD‐mediated phosphatidylcholine hydrolysis, suggesting that the intracellular signaling activated by interferon in Daudi cells involves a phospholipase D/phosphohydrolase pathway.
Deletion of subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex specifically impairs electron transfer at the ubiquinol oxidase site (center P) in the bc1 complex
Authors: Graham Laurie A. , Phillips John D. , Trumpower Bernard L.
Published: July 2002

Deletion of QCR9, the nuclear gene encoding subunit 9 of the mitochondrial cytochrome bc
1 complex in Saccharomyces cerevisiae, results in inactivation of the bc
1 complex and inabillity of the yeast to grow on non‐fermentable carbon sources. The loss of bc
1 complex activity is due to loss of electron transfer activity at the ubiquinol oxidase site (center P) in the complex. Electron transfer at the ubiquinone reductase site (center N), is unaffected by the loss of subunit 9, but the extent of cytochrome b reduction is diminished. This is the first instance in which a supernumerary polypeptide, lacking a redox prosthetic group, has been shown to be required for an electron transfer reaction within the cytochrome bc
1 complex.
In vitro biosynthesis of lactase in preweaning and adult rabbit
Authors: Rossi Mauro , Maiuri Luigi , Russomanno Concetta , Auricchio Salvatore
Published: July 2002

Lactase is synthesized is a high‐mannose large precursor (200 kDa) which is subsequently complex‐glycosylated (215 kDa) and split into the 150 kDa mature form. The regulatory mechanisms responsible for the decline of activity at weaning are not yet known. We have set up in vitro cultures of intestinal mucosa from suckling and adult rabbit and found that suckling and adult animals synthesize the same four forms of lactase‐phlorizin hydrolase (LPH) but with a different distribution. In the proximal adult small intestine there is very little 180 kDa form, which is most probably a product of the 215 kDa complex‐glycosylated precursor. The 180 kDa form comprises a greater percentage of total LPH in the middle of the small intestine in adult and particularly in sukling rabbits. In the latter tissue this form is apparently more stable than in the adult tissue. Posttranscriptional control of lactase synthesis is therefore different in the various parts of the adult small intestine, and it is different in the suckling as compared to adult tissue.
Enflurane is a potent inhibitor of high conductance Ca2+‐activated K+' channels of Chara australis
Authors: Antkowiak Bernd , Kirschfeld Kuno
Published: July 2002

The volatile anaesthetic, enflurane, is commonly used in surgery for inducing the state of general anaesthesia. It is assumed, that general anaesthetics act on ion channels, but little is known of how they do so and what kinds of channels are sensitive. We found, that enflurane inhibits a large conductance Ca2+‐activated K+ channel of the green alga, Chara australis. Effects occur at clinically relevant concentrations and are fully reversible. The actions of enflurane are distinct from those of charybdotoxin and tetraethylammonium, which are well known blockers of this channel type. Kinetic analysis of single‐channel data demonstrates multiple effects of enflurane on the channel protein.
Isolation and characterisation of a functional αβ heterodimer from the ATP synthase of Rhodospirillum rubrum
Authors: Andralojc P.J. , Harris D.A.
Published: July 2002

An αβ heterodimer of the F1‐ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each αβ heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg2+. The dimer can be reversibly dissociated by removal of Mg2+‐ions. The αβ heterodimer restores both ATP‐synthetic and hydrolytic activities to LiCl‐treated chromatophores, saturation being achieved at approximately 2 mmol αβ · mol BChl−1. The heterodimer itself hydrolyses Mg‐ATP with an activity distinct from RF1, being unaffected by azide or sulphite ions. The V
max and K
m (ATP) for this Mg2+‐dependent activity were 110 ± 10 nmol · min−1 mg protein−1 and 100 ± 30 μM, respectively. The K
m did not differ significantly from that of RF1.
Effect of thiamin on cordycepin sensitivity in Saccharomyces cerevisiae
Authors: Iwashima A. , Kawasaki Y. , Nosaka K. , Nishimura H.
Published: July 2002

The sensitivity of Saccharomyces cerevisiae to the antibiotic cordycepin (3′‐deoxyadenosine) was found to be decreased by the addition of thiamin to the growth medium. A thiamin transport mutant of S. cerevisiae was also found to be resistant to the growth inhibitory effect of cordycepin. Not only the thiamin uptake but also adenosine uptake by this mutant cell was markedly reduced compared to those by the parent yeast cells. This strongly suggested that cordycepin, an analog of adenosine, is virtually taken up by the thiamin transport system of growing yeast cells; thus the drug sensitivity is decreased by the presence of thiamin in the growth medium.
Enhanced IgG‐ and complement‐independent phagocytosis of sulfatide‐enriched human erythrocytes by human monocytes
Authors: Serra Maria Vittoria , Mannu Franca , Matera Aida , Turrini Franco , Arese Paolo
Published: July 2002

Phagocytosis by adherent human monocytes of human erythrocytes (RBC), sulfatide‐enriched by incubation with 10−12 to 10−9 M cerebroside sulfate, was enhanced approx. 6‐fold. Increased phagocytosis was observed only in RBC opsonized with fresh plasma, and not in non‐opsonized or serum‐opsonized RBC. Increased phagocytosis was immunoglobulin‐ and complement independent. Thrombospondin and von Willebrand factor, present in plasma but not in serum, and binding selectively to sulfatides, are likely mediators of the enhanced phagocytosis.
Ligand‐activated ion channels may share common gating mechanisms with the Shaker potassium channel
Authors: Lee Chyuan-Yih
Published: July 2002

This paper proposes a detailed gating mechanism for the N‐methyl‐d‐aspartate (NMDA) channel. In the NMDARI subunit, the signal of agonist binding may be carried from Y456 to W590 through an electron transport chain, including W480 which could be the glycine modulatory site. The channel's opening may arise from repulsion between negatively charged W590s, analogous to W435s or the Shaker K+ channel. The cyclic nucleotide‐gated channels may be activated by a similar mechanism, but the opening of nicotinic acetylcholine receptor (nAChR) channels is likely to be initiated by the formation of tyrosine radicals. The role of disulfide‐bonded cysteines in the redox modulation can also be explained.
POU‐specific domain of Oct‐2 factor confers ‘octamer’ motif DNA binding specificity on heterologous Antennapedia homeodomain
Authors: Brugnera Enrico , Xu Licen , Schaffner Walter , Arnosti David N.
Published: July 2002

The bipartite DNA binding domain of the POU family of transcription factors contains a ‘POU‐specific’ domain unique to this class of factors and a ‘POU homeodomain’ homologous to other homeodomains. We compared DNA binding of the Oct‐2 factor POU domain and the Antennapedia (Antp) homeodomain with a chimeric Oct‐2/Antp protein in which the distantly related Antp homeodomain was substituted for the Oct‐2 POU homeodomain. The Oct‐2/Antp chimeric protein bound both the octamer and the Antp sites efficiently, indicating that DNA binding specificity is contributed by both components of the POU domain.
Insight into topological and functional relationships of cytochrome c oxidase subunit I of Saccharomyces cerevisiae by means of intragenic complementation
Authors: Meunier Brigitte , Coster Françoise , Lemarre Philippe , Colson Anne-Marie
Published: July 2002

In yeast, revertants were selected from four respiratory deficient mutants carrying mutations in the cytochrome c oxidase subunit I gene. Intragenic second site mutations revealed amino acids which are functionally complementary to the original mutated position and may be in topological interaction with it. The results provide additional data in favour of the model proposed for the structure of the binuclear centre in proton‐motive oxidases.
Cloning of a novel putative G‐protein‐coupled receptor (NLR) which is expressed in neuronal and lymphatic tissue
Authors: Kouba Monika , Vanetti Mirko , Wang Xioamin , Schäfer Martin , Höllt Volker
Published: July 2002

A novel G‐protein‐coupled receptor was isolated from mouse and rat neuronal and lymphatic tissues. The amino acid sequence of the rat receptor (rNLR) shows an overall homology of 80% to a recently cloned receptor from Burkitt's lymphoma cells (BLR1) which is exclusively expressed in lymphatic tissues [(1992) Eur. J. Immunol. 22, 2795]. Much less homology between rNLR and BLR1 was observed at the N‐terminus (about 40%), whereas rNLR and the mouse homologue mNLR show 92% amino acid identity. Northern blot analysis of NLR revealed a predominant 5.5 kb mRNA species in various brain regions and neuronal cell lines, whereas in the spleen a 3 kb transcript is predominant. This distribution suggests a role of NLR in the nervous and immune systems.
Bilirubin attenuates radical‐mediated damage to serum albumin
Authors: Neužil Jiří , Stocker Roland
Published: July 2002

Oxidative damage to biological macromolecules has been implicated in a number of diseases. Much interest has focused on how non‐proteinaceous, low‐molecular weight antioxidants prevent oxidative damage to lipids, while comparatively little is known about protein antioxidation. Here we show that bilirubin (BR), the end‐product of heme catabolism, when bound to bovine serum albumin (BSA), is oxidised by hydroxyl ('OH), hydroperoxyl (HO2), and Superoxide anion (Oxxx
2) radicals to so far mostly uncharacterised products. The initial oxidation rates of BSA‐bound BR decreased in the order OH ⪢ HO 2 ⪢ O−.
2 BR protected its carrier protein from oxidative damage inflicted by OH radicals. This protective action included a reduction in the OH‐mediated cleavage of BSA, conversion of Trp into kynurenine and formation of ‘bityrosine‐specific’ fluorescence. BR also strongly inhibited OH‐mediated formation of protein carbonyls, whereas ascorbate and Trolox (a water‐soluble analogue of vitamin E) were much less effective. These results support an antioxidant‐protective function of BR and point towards significant differences in the efficacies of various antioxidants in the prevention of oxidative damage to lipids and proteins.
Targeting of recombinant Na+/glucose cotransporter (SGLT1) to the apical membrane
Authors: Kong Cheng-Te , Varde Anjali , Lever Julia E.
Published: July 2002

A full‐length Na+/glucose cotransporter cDNA (SGLT1) from rabbit intestine was subcloned into the pMAMneo mammalian expression vector and transfected by Ca2+ precipitation into Madin‐Darby canine kidney (MDCK) cells. Stable MDCK transfectants isolated after clonal isolation and selection in G418 exhibited dexamethasone‐inducible Na+/glucose cotransport activity under regulation of the MMTV promoter of the vector. Transfectants expressed the recombinant 75 kDa Na+/glucose cotransporter subunit as shown by Western blot, and SGLT1 mRNA as shown by Northern blot, but these were undetectable in untransfected MDCK cells. Over 93% of total recombinant transport activity was targeted to the apical membrane. This indicates that the primary amino acid sequence of SGLT1 contains the information necessary to target this transporter to the apical membrane.
Association constants of anti‐hapten monoclonal IgGI with mouse FcγRII in the presence and absence of hapten
Authors: Sumner Martina T.
Published: July 2002

The binding of five different mouse monoclonal anti‐dinitrophenyl (DNP) IgGI monomers, in the presence and absence of DNP‐glycine, to FcγRII on the mouse macrophage‐like cell line J774 has been investigated. Membrane association constants were estimated using a competitive radioimmunoassay in which the ability of the IgGl monomers to block the binding of 125I‐labelled Fabs of the anti‐FcγRII monoclonal antibody 2.4G2 was monitored. The IgGl‐FcγRII association constants ranged from (8 ± 1) × 104 M−1 to (6 ± 2) × 105 M−1. No large differences in the IgGl‐FcγRII association constants were measured for different IgG1 antibodies or for any single IgG1 antibody in the presence and absence of DNP‐glycine.
Stoichiometry of a Cl−‐translocating ATPase
Authors: Gerencser George A.
Published: July 2002

It has been determined that Mg2+ stimulates phosphorylation while Cl− stimulates dephosphorylation of the chloride pump during its reaction sequence. The Stoichiometry of ATP hydrolyzed to Cl− transported during a single cycle of the reaction sequence was ascertained. Intracellular concentrations of ATP, ADP and inorganic phosphate were determined and, coupled with an estimate of the standard free energy of hydrolysis for ATP, the operant free energy for ATP hydrolysis was calculated. Because the operating free energy of the pump is approximately one‐half the energy obtained from the total free energy of ATP hydrolysis, the only possible integral stoichiometries are one or, at the most, two net charges transported per cycle per ATP hydrolyzed.
Characterisation of an epitope recognised by a monoclonal antibody against horse alcohol dehydrogenase using peptides synthesised on solid support
Authors: Leone N.A. , Whitehouse D.B. , Swallow D.M. , Wallace G.R. , Adinolfi A.
Published: July 2002

Immunological analysis, using the Pepscan technique, of the tetradecapeptide, Pro344‐Glu357 (PLITHVLPFEKINE), from horse liver alcohol dehydrogenase has identified a five amino acid sequence, HVLPF, which binds a monoclonal antibody. The epitope seems to be rather flexible with only two of the amino adds. Pro and Phe, having the characteristics of contact residues. However, the presence of the adjacent glutamic acid residue as part of the Pepscan peptide has a dramatic negative neighbourhood effect and inhibits binding. This highlights the potential risk of missing an epitope altogether when using the Pepscan procedure for epitope mapping.