bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of all thirteen polypeptides of bovine heart cytochrome c oxidase from one aliquot of enzyme Characterization of bovine fetal heart cytochrome c oxidase
Authors: Takamiya Shinzaburo , Lindorfer Margaret A. , Capaldi Roderick A.
Published: July 2002

A protocol has been worked out for separating all thirteen different polypeptides in the beef heart cytochrome c oxidase complex from a single aliquot of enzyme. This involves an initial separation of polypeptides by gel filtration on a Biogel P‐60 column in SDS, a step which purifies subunits CIV and CVIII and gives mixtures of CV+CVI, ASA, AED and STA, as well as CVII, CIX and IHQ. These mixtures are then resolved by reverse‐phase high‐performance liquid chromatography. The separation procedures have been applied to fetal heart cytochrome c oxidase of gestation between 100 and 200 days. No differences were found in the N‐terminal sequences of any of the cytoplasmically made subunits or in the entire sequence of CIX between late fetal and adult forms of the enzyme.
The molecular size required varies according to the reaction step round the sodium pump cycle
Authors: Cavieres J.D.
Published: July 2002

Progress along the path of the sodium pump cycle requires a stepwise recruitment of additional subunits for maximal activity. These results show that whereas a particle the size of the αβ protomer presents Na+,K+‐ATPase activity at 10 μM ATP, an additional subunit, perhaps a second α‐chain, is required to obtain the much greater Na+,K+‐ATPase activity resulting from the occupation of low‐affinity ATP sites at physiological ATP concentrations. A non‐phosphorylating ATP analogue, however, will modestly stimulate the Na+,K+‐ATPase activity acting at an alternative low‐affinity site or step on the αβ protomer.
Dexamethasone‐dependent expression of β1–24 corticotropin stimulated adenylate cyclase during adipose conversion of 3T3‐F442A cells
Authors: Feve Bruno , Pairault Jacques
Published: July 2002

When 3T3‐F442A preadipocytes were grown in culture media supplemented with corticosteroid poor fetal calf serum and insulin they differentiated into adipocytes. Glycerophosphate dehydrogenase, a marker of terminal differentiation, developed a 600‐fold increase of activity whereas the adenylate cyclase system remained unresponsive to the synthetic ACTH(1–24) analog. In contrast, 3T3‐F442A adipocytes, differentiated in the presence of dexamethasone, exhibited an adenylate cyclase activity which was stimulated 4‐fold by ACTH(1–24). The stimulation of the adenylate cyclase activity by GTPγS remained unchanged (about 20–25‐fold) suggesting that the G regulatory coupling protein was not functionally modified by dexamethasone. Binding studies with 125I‐ACTH revealed that specific cellular binding could be evidenced in dexamethasone‐treated cells while control adipocytes did not exhibit any specific binding of 125 I‐ACTH. These findings lend support to the hypothesis that the setting off of this ACTH responsiveness in 3T3‐F442A cells is regulated by dexamethasone after cells are committed to adipose differentiation.
Synthesis and turnover rates of four rat liver mitochondrial RNA species
Authors: Cantatore P. , Flagella Z. , Fracasso F. , Lezza A.M.S. , Gadaleta M.N. , de Montalvo A.
Published: July 2002

The synthesis and turnover rates of the two 12 S and 16 S mt rRNAs and of the mt mRNAs for subunits I and III of cytochrome oxidase have been determined by measuring the kinetics of incorporation of [3H]uridine in the mtRNA of rat hepatocytes. All the RNA species examined have approximately the same turnover (t

~ 100 min) and therefore the rate of synthesis, which is about 10‐times higher for the rRNAs, seems to be the factor responsible for the different mt rRNA and mRNA steady‐state levels.
Two distinct DNA ligases from Drosophila melanogaster embryos
Authors: Takahashi Miho , Senshu Mitsuko
Published: July 2002

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'‐phosphoryl and 3'‐hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The K
m for ATP is 2.7 × 10−6 M for DNA ligase I, and 3.0 × 10−5 M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N‐ethylmaleimide. DNA ligase I is active at a low salt concentration (0–30 mM KCl), but DNA ligase II is active at high salt concentrations (50–100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase‐AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637–10645] seem to be the same enzyme.
Processing of Bacillus subtilis succinate dehydrogenase and cytochrome &‐558 polypeptides
Authors: Hederstedt Lars , Bergman Tomas , Jörnvall Hans
Published: July 2002

The DNA sequence of the Bacillus subtilis sdh operon coding for the two succinate dehydrogenase subunits and cytochrome b‐558 (the membrane anchor protein) has recently been established. We have now determined the extent of N‐terminal processing of each polypeptide by radiosequence analysis. At the same time, direct evidence for the correctness of the predicted reading frames has been obtained. The cytochrome showed a ragged N‐terminus, with forms lacking one residue, and is inserted across the membrane without an N‐terminal leader‐peptide. Covalently bound flavin was not detectable in B. subtilis succinate dehydrogenase expressed in Escherichia coli despite normal N‐terminal processing of the apoprotein. This provides an explanation to why the succinate dehydrogenase synthesized in E. coli is not functional and demonstrates that host‐specific factors regulate the coenzyme attachment.
Primary structure of the biotin‐binding site of chicken liver acetyl‐CoA carboxylase
Authors: Takai Toshiyuki , Wada Kenji , Tanabe Tadashi
Published: July 2002

Limited proteolysis of chicken liver acetyl‐CoA carboxylase by staphylococcal serine proteinase yielded a fragment of 31 kDa which contained the biotinyl active site. This polypeptide was purified by preparative polyacrylamide gel electrophoresis and characterized. The complete amino acid sequence of this polypeptide has been deduced from the nucleotide sequence of cloned DNA complementary to the chicken liver acetylCoA carboxylase mRNA. A highly conserved sequence of Met‐Lys‐Met was found in the biotin‐binding site. Appreciable homology was observed among the sequences in close vicinity of the biotin sites of chicken liver acetyl‐CoA carboxylase and other biotin‐dependent carboxylases including biotin carboxyl carrier protein of Escherichia coli acetyl‐CoA carboxylase.
Effect of troponin C on the cooperativity in Ca2+ activation of cardiac muscle
Authors: Gulati Jagdish , Scordilis Stylianos , Babu Arvind
Published: July 2002

This study describes the effects of exchanging native cardiac troponin C (CTnC) from the right ventricular muscle of Syrian hamster for purified skeletal (S) TnC from fast twitch muscles in triggering cardiac contraction. Ca2+ sensitivity of the myocardium became decreased with STnC to 62% of the original value with CTnC. Furthermore, the slope of the pCa‐force curve of cardiac muscle was found to be increased with STnC. The results show that the TnC moiety, as part of the switching mechanism during activation, also regulates thin‐filament cooperativity in muscle. Modifications in both the Ca2+ sensitivity and cooperativity are associated with alterations in the primary structure of TnC.
Characterization of inositol 1,3,4‐trisphosphate phosphorylation in rat liver
Authors: Hansen Carl A. , vom Dahl Stephan , Huddell Benjamin , Williamson John R.
Published: July 2002

Liver homogenates phosphorylated Ins 1,3,4‐P3 to an InsP4 isomer that was distinct from Ins 1,3,4,5‐P4. This InsP4 isomer accumulated in vasopressin stimulated hepatocytes prelabeled with myo‐[3H]inositol with a time course that lagged behind Ins 1,3,4‐P3 formation. The Ins 1,3,4‐P3 kinase responsible for its formation was partially purified from rat liver. The enzyme had a K
m for Ins 1,3,4‐P3 of 0.29 μM, a K
m for ATP of 141 μM and was not affected by changes in free Ca2+ in the physiological range. The relationship of this new InsP4 isomer to the inositol phosphate signaling pathway is discussed.
Ligands to the 2Fe iron‐sulfur center in succinate dehydrogenase
Authors: Ævarsson Arnthór , Hederstedt Lars
Published: July 2002

Membrane‐bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron‐sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtillis succinate dehydrogenase‐defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe‐2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N‐terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin‐containing subunit of the dehydrogenase.
The amino acid sequence of a cereal Bowman‐Birk type trypsin inhibitor from seeds of Jobs' tears (Coix lachryma‐jobi L.)
Authors: Ary Maria B. , Shewry Peter R. , Richardson Michael
Published: July 2002

The major trypsin inhibitor from seeds of Jobs' tears (Coix lachryma‐jobi) was purified by heat treatment, fractional precipitation with (NH4)2SO4, ion‐exchange chromatography on DEAE‐Sepharose, gel‐filtration on Sephadex G‐75 and preparative reverse‐phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S‐carboxymethylated protein by digestion with trypsin, chymotrypsin and the S. aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong homology with a number of Bowman‐Birk inhibitors from legume seeds and similar proteins recently isolated from wheat and rice.
The ATP‐sensitive potassium channel in pancreatic B‐cells is inhibited in physiological bicarbonate buffer
Authors: Carroll Patricia B. , Li Min-Xu , Rojas Eduardo , Atwater Illani
Published: July 2002

The effects of bicarbonate buffer (HCO−
3/CO2) on the activity of the two K+ channels proposed by some to control the pancreatic B‐cell membrane response to glucose were studied. Single K+‐channel records from membrane patches of cultured B‐cells dissociated from adult rat islets exposed to a glucose‐ and bicarbonate‐free medium (Na‐Hepes in place of bicarbonate) exhibit the activity of both the ATP‐sensitive as well as the [Ca2+]i‐activated K+ channels. However, in the presence of bicarbonate‐buffered Krebs solution, the activity of the ATP‐sensitive K+ channel is inhibited leaving the activity of the K+ channel activated by intracellular [Ca2+]i unaffected. In the absence of bicarbonate (Hepes/NaOH in place of bicarbonate), lowering the external pH from 7.4 to 7.0 also has differential effects on the two K+ channels. While the K+ channel sensitive to ATP is inhibited, the K+ channel activated by a rise in [Ca2+]i is not affected. To determine whether the response of the B‐cell in culture to bicarbonate is also present when the B‐cell is functioning within the islet syncytium, the effects of bicarbonate removal on membrane potential of B‐cells from intact mouse islets were compared. These studies showed that glucose‐evoked electrical activity is also blocked in bicarbonate‐free Krebs solution. Furthermore, in the absence of bicarbonate and presence of glucose (11 mM), electrical activity was recovered by lowering the pHo from 7.4 to 7.0. The ATP‐sensitive K+‐channel activity is greatly reduced by physiologically buffered solutions in pancreatic B‐cells in culture. The most likely explanation for the bicarbonate effects is that they are mediated by cytosolic pH changes. Removal of bicarbonate (keeping the external pH at 7.4 with Hepes/NaOH as buffer) would increase the pHi. Since the activity of the [Ca2+]i‐dependent K+ channels is not affected by the removal of the bicarbonate buffer, our patch‐clamp data in cultured B‐cells indicate an involment of [Ca2+]i‐activated K+ channels in the control of the membrane potential. For the B‐cell in the islet, we propose that the burst pattern of electrical activity (Ca2+ entry) is controlled, at least in part, by the [Ca2+]i‐activated K+ channel.
Variability in electrophoretic mobility of Gi‐like proteins: effect of SDS
Authors: Jones JoLynda , Kopf Gregory S. , Schultz Richard M.
Published: July 2002

Two forms of Gi‐like protein are resolved in both somatic cells and mouse gametes when Sigma SDS (95% grade) is used during polyacrylamide gel electrophoresis, whereas only a single species is resolved when Bio‐Rad SDS (electrophoresis grade) is used. These two Gi‐like proteins are likely to reflect two distinct species, since (i) the two species resolved in the presence of Sigma SDS migrate with the same electrophoretic mobility upon re‐electrophoresis in the presence of Sigma SDS and (ii) exchanging Sigma SDS for Bio‐Rad SDS resolves a single species, whereas exchanging Bio‐Rad SDS for Sigma SDS resolves two species.
Selective cleavage of skeletal myosin subfragment‐1 to form a 26 kDa peptide which shows ATP‐sensitive actin binding
Authors: Griffiths A.Jane , Trayer Ian P.
Published: July 2002

A 26 kDa peptide has been cleaved from the C‐terminus of the S1 heavy chain with formic acid. Cleavage occurs in the ‘50 kDa’ domain probably at the Asp‐600‐Pro‐601 bond. This fragment has been renatured in the presence of the A2 light chain and the 26 kDa(A2) complex shown to interact with actin in an ATP‐sensitive manner.
Localization and characterization of the glycosylation site of human pancreatic elastase 1
Authors: Wendorf Petra , Geyer Rudolf , Sziegoleit Andreas , Linder Dietmar
Published: July 2002

Crystalline elastase 1 from human pancreas was digested with trypsin. Two peptides, containing the potential N‐glycosylation sites at Asn‐86 and Asn‐125, were isolated and analyzed by amino acid analysis, sequencing and carbohydrate component analysis. The results demonstrate that only Asn‐86 is glycosylated.
Detection and localization of a new enzyme catalyzing the β‐aryl ether cleavage in the soil bacterium (Pseudomonas paucimobilis SYK‐6)
Authors: Masai Eiji , Katayama Yoshihiro , Nishikawa Seiji , Yamasaki Makari , Morohoshi Noriyuki , Haraguchi Takafusa
Published: July 2002

Cleavage of the arylglycerol‐β‐aryl ether linkage is the most important process in the biological degradation of lignin. We determined the activity of the enzyme cleaving the β‐aryl ether linkage in membranes of Pseudomonas paucimobilis SYK‐6. This enzyme was tightly associated with the cellular membrane and catalyzed the unique and reductive cleavage of compound II but not cleavage of compound I. This enzymatic activity was stimulated by addition of NADH. On the basis of this evidence, we present a model of the specific cellular assimilation of β‐aryl ether by P. paucimobilis SYK‐6.
Charybdotoxin blocks both Ca‐activated K channels and Ca‐independent voltage‐gated K channels in rat brain synaptosomes
Authors: Schneider M.J. , Rogowski R.S. , Krueger B.K. , Blaustein M.P.
Published: July 2002

Charybdotoxin (ChTX), a 4.3 kDa polypeptide toxin from the venom of the scorpion Leiurus quinquestriatus, blocks both a Ca‐activated K channel (IC50 ≈ 15 nM) and a Ca‐independent voltage‐gated K channel (IC50 ≈ 40 nM) in rat brain synaptosomes. These results indicate that in this preparation ChTX is not specific for the Ca‐activated K channel and suggest that there may be structural homology among the toxin‐binding sites on various types of K channels.
Calpain proteolysis of free and bound forms of calponin, a troponin T‐like protein in smooth muscle
Authors: Tsunekawa Shoji , Takahashi Katsuhito , Abe Masahiro , Hiwada Kunio , Ozawa Kazue , Murachi Takashi
Published: July 2002

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction‐associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin‐linked contractile machinery in smooth muscle.
Charybdotoxin blocks dendrotoxin‐sensitive voltage‐activated K+ channels
Authors: Schweitz Hugues , Stansfeld Catherine E. , Bidard Jean-Noël , Fagni Laurent , Maes Pierrette , Lazdunski Michel
Published: July 2002

Charybdotoxin, a short scorpion venom neurotoxin, which was thought to be specific for the blockade of Ca2+‐activated K+ channels also blocks a class of voltage‐sensitive K+ channels that are known to be the target of other peptide neurotoxins from snake and bee venoms such as dendrotoxin and MCD peptide. Charybdotoxin also inhibits 125‐dendrotoxin and 125I‐MCD peptide binding to their receptors. All these effects are observed with an IC50 of about 30 nM.
The Na+‐motive respiration in Escherichia coli
Authors: Avetisyan A.V. , Dibrov P.A. , Skulachev V.P. , Sokolov M.V.
Published: July 2002

The protonophorous uncoupler carbonyl cyanide m‐chlorophenylhydrazone was found to inhibit the growth rate of E. coli and maintenance of the membrane potential (Δψ) in intact bacterial cells. Both inhibitory effects of the protonophore were strongly decreased by Na+. A favourable action of Na+ could be observed only when cells were grown under conditions of low Δµ̃H, i.e. at alkaline pH (8.6) or in the presence of the photonophore in growth medium. It was abolished by addition of ΔpH‐dissipating agents, i.e. the penetrating weak base diethylamine and the Na+/H+ antiporter monensin, to the incubation medium. In inside‐out subcellular vesicles isolated from cells grown at alkaline pH, NADH oxidation was shown to be coupled to Na+ uptake which was markedly stimulated by protonophore and inhibited by cyanide and HQNO. It is concluded that the growth of E. coli at alkaline pH or in the presence of protonophore gives rise to the induction of a primary respiratory Na+ pump(s).
Lipoxins are major lipoxygenase products of rainbow trout macrophages
Authors: Pettitt Trevor R. , Rowley Andrew F. , Secombes Chris J.
Published: July 2002

Rainbow trout macrophages synthesize lipoxins as major lipoxygenase products entirely from endogenous fatty acids. High‐performance liquid Chromatographic analysis of the supernatants from macrophages challenged with calcium ionophore A23187 revealed a range of lipoxygenase products including mono‐hydroxy fatty acids, leukotrienes B4 and B5 and four major peaks with retention times and UV spectra characteristic of lipoxins (λmax 302 nm). Cochromatography with authentic standards, UV spectroscopy and radiolabelling with (14C]arachidonate and eicosapentaenoate allowed tentative identification of the two largest peaks as lipoxin A4 and A5.
Transfer of sialic acid in α2‐6 linkage to mannose in Manβ1‐4GlcNAc and Manβ1‐4GlcNAcβ1‐4GlcNAc by the action of Galβ1‐4GlcNAc α2‐6‐sialyltransferase
Authors: van Pelt Johannes , Dorland Lambertus , Duran Marinus , Hokke Cornelis H.
Published: July 2002

Sialyltransferase, Galβ1‐4GlcNAcα2‐6; Sialyltransferase; Sialyloligosaccharide; Mannosidosis, β‐
Transforming growth factors (TGFα and TGFβH) stimulate chondroitin sulfate and hyaluronate synthesis in cultured rat liver fat storing cells
Authors: Bachem Max G. , Riess Uwe , Melchior Ralph , Sell Klaus-Martin , Gressner Axel M.
Published: July 2002

Fat storing cell; Fibrogenesis; Glycosaminoglycans; Chondroitin sulfate; Hyaluronic acid; Transforming growth factor
Structure‐function analysis of human interleukin‐6
Authors: Krüttgen Alex , Rose-John Stefan , Möller Carsten , Wroblowski Berthold , Wollmer Axel , Müllberg Jürgen , Hirano Toshio , Kishimoto Tadamitsu , Heinrich Peter C.
Published: July 2002

C‐terminally deleted analogs of human interleukin‐6 (IL‐6) have been constructed at the cDNA level, and after cell‐free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL‐6 resulted in 58% helix, 14% β‐structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL‐6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% β‐structure, and 18% turn and coil.
Effect of the multicatalytic proteinase (prosome) on translational activity in rabbit reticulocyte lysates
Authors: Kuehn Lothar , Dahlmann Burkhardt , Kopp Friedrich
Published: July 2002

In a message‐dependent reticulocyte lysate translation system, incorporation of [3H]leucine into acid‐insoluble protein is increased following selective removal of the multicatalytic proteinase (MCP) with a monospecific antibody. Re‐addition of active proteinase to previously depleted lysates reverses this effect in that the same low levels of translational product are measured as in untreated lysates. Addition of histone‐stimulated MCP further depresses the level of protein product. Conversely, lysates supplemented with inactivated MCP retain the higher level of translational activity which is measured after precipitation of the enzyme with antibody. In these lysates, the effect of the antibody on translational activity is inversely correlated with that on hydrolytic activity towards [14C]methylcasein or N‐succinyl‐Leu‐Leu‐Val‐Tyr‐4‐methyl‐7‐coumarylamide, two substrates of the MCP. These results showing that the MCP is capable of modulating translational activity in vitro, suggest an important role of this molecule in the in vivo translational process.
One of the endothelin gene family, endothelin 3 gene, is expressed in the placenta
Authors: Onda Haruo , Ohkubo Shoichi , Ogi Kazuhiro , Kosaka Takuo , Kimura Chiharu , Matsumoto Hirokazu , Suzuki Nobuhiro , Fujino Masahiko
Published: July 2002

A cDNA encoding human endothelin 3 (ET‐3) precursor was cloned from a cDNA library from the placenta, and its nucleotide and deduced amino acid sequences were determined. This ET‐3 cDNA was found to contain 2.3 kb pairs and encode prepro‐ET‐3 protein consisting of 224 amino acid residues. The putative big‐ET‐3 seems to consist of 42 amino acid residues. Two of the intron insertion sites were determined with information from nucleotide sequences of the cloned genomic ET‐3 gene. This is the first direct evidence that the ET‐3 gene is transcribed and expressed in the placenta.
The effect of amino acid substitutions at position 342 on the secretion of human α1‐antitrypsin from Xenopus oocytes
Authors: Wu Ying , Foreman R.C.
Published: July 2002

A glutamic acid to lysine change in the Z variant of human α1‐antitrypsin is associated with a failure to secrete the protein from synthesising cells. The block in export of the protein may be caused either by the loss of an acidic residue or the introduction of a basic one at this point in the polypeptide chain. Site‐directed mutagenesis has been used to construct novel α1‐antitrypsin mutants which show that the side chain interactions from Glu‐342 are not obligatory for protein export and it is rather the introduction of a basic residue at this point which produces the intracellular accumulation of the protein.
Immunologically cross‐reactive and redox‐competent cytochrome b6/f‐complexes in the chlorophyll‐free plasma membrane of cyanobacteria
Authors: Kraushaar Harald , Hager Silvia , Wastyn Marnik , Peschek Günter A.
Published: July 2002

Plasma and thylakoid membranes were separated and purified from cell‐free extracts of the cyanobacteria Anacystis nidulans, Synechocystis 6714, Anabaena variabilis and Nostoc sp. strain Mac. Immunoblots of the membrane proteins using antisera raised against subunits I–IV of the chloroplast b
6/f‐complex gave evidence for the presence of a homologous complex in both plasma and thylakoid membranes from the four species of cyanobacteria investigated. Both plasma and thylakoid membranes catalyzed the electron transfer from (exogenous) plastoquinol‐9 and NADH to horse heart ferricytochrome c. However, while with plasma membranes these reactions were severely inhibited by low concentrations of antimycin A and rotenone, respectively, the inhibitors were without major effect on thylakoid membranes. The results will be discussed in terms of a possible similarity (analogy and/or homology?) of cyanobacterial plasma membranes to the inner mitochondrial membrane.
Structural studies on three flavin‐interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria
Authors: Furushima Rieko , Kita Kiyoshi , Takamiya Shinzaburo , Konishi Kiyoshi , Aoki Takashi , Oya Hiroshi
Published: July 2002

The flavoprotein (Fp) subunit of mitochondrial complex II contains covalently bound FAD as a prosthetic group. In this study, the primary structure of the flavin‐bound tryptic peptide from the Fp subunit of Ascaris complex II was determined and found to be highly similar to those of the corresponding flavin‐binding regions of bovine heart and bacterial Fp subunits. Furthermore, the Ascaris Fp subunit was shown to contain two regions exhibiting striking sequence similarity to the segments that have been predicted to interact noncovalently with the AMP moiety of FAD in bacterial Fp subunits. The conservation of these two regions also in themitochondrial Fp subunit suggests their functional importance.
Complete down‐regulation of low‐density lipoprotein receptor activity in human liver parenchymal cells by β‐very‐low‐density lipoprotein
Authors: Kamps Jan A.A.M. , Kuiper Johan , Kar Kruijt J. , van Berkel Theo J.C.
Published: July 2002

The effect of LDL and β‐VLDL on the expression of the LDL receptor is studied in cultured human parenchymal cells. The high affinity binding of [125I]LDL to cultured human parenchymal cells was down regulated to 37.3±2.9% and 24.0±2.6% of the control value, after preincubation with LDL or β‐VLDL for 22 h, respectively. When LDL receptor synthesis was blocked at 22 h a residual receptor activity of 29% is noticed, indicating a half‐life of LDL receptors in human parenchymal cells of 12 h. It is concluded that LDL receptor expression on human liver parenchymal cells is subject to complete down‐regulation by β‐VLDL, which may be held responsible for the cholesterol‐rich diet induced down‐regulation of LDL receptors, in vivo.
Modification of the stability of phospholipase A2 by charge engineering
Authors: Pickersgill Richard W. , Sumner Ian G. , Collins Margaret E. , Warwicker Jim , Perry Barry , Bhat K.Mahalingeshwara , Goodenough Peter W.
Published: July 2002

Electrostatic interactions play an important role in stabilizing the folded conformation of globular proteins. Here we predict the change in stability of charge engineered mutants, construct these mutants and compare the predicted change in stability with that observed. The change in stability was correctly predicted for two of the three mutants and the factors responsible for the discrepancy between observation and prediction for the third mutant are discussed.
Ouabain‐ and Ca2+‐sensitive ATPase activity of chimeric Na‐ and Ca‐pump molecules
Authors: Luckie Douglas B. , Boyd Kimberly L. , Takeyasu Kunio
Published: July 2002

Chimeric ion‐pumps, consisting of the N‐terminal of the α1‐subunit of the ouabain‐sensitive chicken Na+, K+‐ATPase and the C‐terminal of the sarcoplasmic reticulum Ca2+‐ATPase, were expressed in ouabain‐insensitive mouse L cells. These chimeric molecules exhibited ouabain‐sensitive ATPase activity very similar to that of the wild‐type chicken Na+ K+‐ATPase. This ATPase activity could be stimulated by adding Ca2+ to the assay system. These results suggest that the sites for ouabain‐inhibition are restricted to the N‐terminal of the Na‐pump, and the C‐terminal of the Ca‐pump interacts with Ca3+.
MAP2‐mediated binding of chromaffin granules to microtubules
Authors: Severin F.F. , Shanina N.A. , Kuznetsov S.A. , Gelfand V.I.
Published: July 2002

We have examined the interaction of chromaffin granules from bovine adrenal modulla with microtubules. Chromaffin granules were mixed with microtubules made of phosphocellulose‐purified tubulin, and pelleted through a 1.6 M sucrose cushion at 12000 × g for 10 min. Both components (granules and microtubules) were pelleted when added together but not separately. This result indicates that granules form a heavy complex with the microtubules. Such a complex was visualized by an electron microscopy of the granule/microtubule mixture. Treatment of the granules with trypsin abolished their ability to interact with the microtubules. The binding of the granules to the microtubules: (i) was not sensitive to ATP: and (ii) was completely inhibited by the cleavage of C‐terminal peptides of α‐ and β‐subunits of tubulin with subtilisin. These relationships suggest that the granule binding is mediated by one of the structural microtubule‐associated proteins rather than by microtubule‐dependent translocators. For identification of protein(s) mediating the binding, the granules were solubilized with Triton X‐100, soluble proteins were mixed with the microtubules, and microtubules with bound proteins were pelleted through a glycerol cushion. At least one granule protein interacting with the microtubules was found in the pellet. This protein was identified as MAP2 according to its electrophoretic mobility and reactivity with a MAP2 antibody. Affinity chromatography of solubilized proteins on a column containing taxol‐stabilized microtubules also revealed MAP2 as a protein of chromaffin granules interacting with the microtubules.
Different expression time of the 105‐kDa protein and 90‐kDa heat‐shock protein in rat testis
Authors: Itoh Hideaki , Tashima Yohtalou
Published: July 2002

To understand the physiological functions of the 105‐kDa protein which is testis‐specific and HSP90 (90‐kDa heat‐shock protein) related protein. the appearance of it in the testis has been followed during the development of rat. On immunoblotting analysis. the 105‐kDa protein did not appear until after the age of five weeks, while HSP90 could be detected at three weeks. In the spermatozoa. the 105‐kDa protein was much abundant but not in the LC‐540 cells (a cell line from Leydig cell tumor in rat testis) cytosol. This finding has attracted much attention to the relationship between this protein and sperm functions.
Cloning and sequencing the urocanase gene (hutU) from Pseudomonas putida
Authors: Fessenmaier Martin , Frank Rainer , Retey János , Schubert Carsten
Published: July 2002

A clone harbouring the entire urocanase gene (hutU) was obtained from a genomic library of Pseudomonas putida using oligonucleotide probes synthesised on the basis of known flanking sequences. One subunit of urocanase consists of 556 amino acids and has a molecular mass of 60 771 Da.
The phosphate recognition site of Escherichia coli maltodextrin phosphorylase
Authors: Schinzel R. , Drueckes P.
Published: July 2002

The role of two positively charged amino acid residues located at the active site of Escherichia coli maltodextrin phosphorylase was investigated by site‐directed mutagenesis, Substitution of Lys539 by an arginine caused a 600‐fold reduction, substitution of Arg534 by a glutamine caused an even larger 7000‐fold reduction of the catalytic rate while substrate binding remained essentially unaffected. Since the Arg534→Gln exchange reduces the catalytic rate near to inactivity and even the conservative Lys534→Arg exchange caused a marked decrease of activity, the central functional role of both positively charged residues in phosphorylase catalysis anticipated by the crystallographic analysis of the corresponding amino acid residues Arg569 and Lys574 in the catalytic site of phosphorylase b was confirmed.
Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins
Authors: Koivula Teija , Palva Ilkka , Hemilä Harri
Published: July 2002

SecY is an integral membrane protein which participates in the translocation of proteins through the bacterial cell membrane. We have cloned the secY gene of Lactococcus lactis, and found its deduced protein sequence, 439 amino acids long, to be similar in length to the previously determined SecY proteins of Escherichia coli, Bacillus subtilis and Mycoplasma capricolum. Comparison of the L. lactis SecY to the 3 other SecY proteins revealed 90 conserved amino acid residues (21%). Nearly half of the conserved residues are clustered in 2 of the 10 transmembrane segments, and in 2 of the 6 cytoplasmic regions. Some of the conserved regions are apparently responsible for the interactions of SecY with signal sequences, and the proteins SecE and SecA.
Internal deletions of amino acids 29‐42 of human interleukin‐6 (IL‐6) differentially affect bioactivity and folding
Authors: Arcone Rosaria , Fontaine Veronique , Coto Iolanda , Content Jean , Ciliberto Gennaro
Published: July 2002

Internal deletions of the human interleukin‐6 (IL‐6) cDNA have been generated in the region encoding residues 29 to 42. Mutant proteins were produced by in vitro transcription—translation or in Escherichia coli and tested for their biological activity using the hybridoma growth factor (HGF) assay or a transcriptional activation assay on human hepatoma cells. The folding of the mutants was also checked by immunoprecipitation with conformation‐specific monoclonal antibodies. The results show that only residues 29 to 34 are crucial for IL‐6 activity and that the first two amino acids are probably involved in the definition of the IL‐6 active site.
Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase
Authors: Mittler Ron , Zilinskas Barbara A.
Published: July 2002

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.
Electrophysiological activity of the C‐peptide of the Locusta insulin‐related peptide Effect on the membrane conductance of Locusta neurones in vitro
Authors: Bermudez Isabel , Beadle David J. , Trifilieff Elisabeth , Luu Bang , Hietter Helene
Published: July 2002

The C‐peptide of Locusta insulin‐related peptide, which is a 50 residue peptide originally isolated from the corpora cardiaca of the insect Locusta migratoria and to which we refer as 5‐kDa peptide, has been synthesised chemically by the solid‐phase metho, using a BOC strategy. Since this peptide contains in its sequence a potential monobasic cleavage site, we also synthesised its 1–38 residue‐related fragment, named 4‐kDa peptide, although we have no hints of its natural occurrence in the corpora cardiaca. Electrophysiological studies have shown that both the 5‐kDa and 4‐kDa peptides depolarise the membrane and increase the membrane conductance of neurones freshly isolated from the thoracic ganglia of Locusta. Under voltage‐clamp conditions, the current underlying these effects was inwardly directed and could be resolved into 2 components. One component, I(5‐kDa)1, activated at potentials more hyperpolarised than −50 mV, peaked at about −75 mV and was blocked by the potassium channel blockers cesium and rubidium. The second component, I(5‐kDa)2 was activated at potentials more depolarised than −50 mV, increased with depolarisation and was not blocked by cesium and rubidium. The effects of the 5‐kDa and 4‐kDa peptides on the membrane potential and membrane conductance of Locusta neurones suggest that these peptides may have a physiological role in the central nervous system of insects.
The complete primary structure of bovine stefin B
Authors: Križaj Igor , Turk Boris , Turk Vito
Published: July 2002

A new stefin B‐type low‐M
r, CPI was isolated from bovine thymus and subjected to structural analysis. The inhibitor consisted of 98 amino acids and its M
r was calculated to be 11,178. The NH2‐terminal amino acid residue was blocked. The sequence was determined by automated sequencing of peptides derived by cleavage with cyanogen bromide and fragments of the inhibitor resulting from enzymatic digestion with β‐trypsin and Staphylococcus aureus V‐8 proteinase. The NH2‐terminal blocking group was established with mass spectrometry. The inhibitor exhibits considerable sequence homology with inhibitors from the stefin family. Furthermore, a highly conserved QVVAG region within the stefin family is for the first time replaced by the QLVAG sequence.
Mammalian expression of the human sex steroid‐binding protein of plasma (SBP or SHBG) and testis (ABP) Characterization of the recombinant protein
Authors: Hagen Frederick S. , Arguelles Cesar , Sui Li-ming , Zhang Wei , Seidel Paul R. , Conroy Stephen C. , Petra Philip H.
Published: July 2002

A full‐length 1,209 bp cDNA encoding the human sex steroid‐binding protein of plasma (SBP or SHBG) and testis (ABP) was constructed and expressed in BHK‐21 cells. The sequence agrees with the published gene and protein sequences. The cells were found to secrete SBP following transfection and G418r selection. The recombinant protein binds 5α‐dihydrotestosterone with a K
d of 0.28 nM. It also binds testosterone and 17β‐estradiol but not progesterone, estrone or cortisol revealing a steroid‐binding specificity identical to that of human SBP, SDS‐PAGE patterns are less complex than human SBP and show a monomeric molecular weight of about 43 kDa.
Characterisation of the expression and post‐translational processing of the preprotachykinin‐I gene and the regulated release of tachykinins by the RINm5F cell‐line
Authors: McGregor Gerard P. , Hartel Robert , Fehmann Hans-Christoph , Lankat-Buttgereit Brigitte , Göke Burkhard , Göke Rüdiger , Voigt Karlheinz
Published: July 2002

Normal transcription and postranslational processing of the preprotachykinin (PPT)‐I gene and regulated release of substance P and neurokinin A by the rat pancreatic endocrine cell‐line, RINm5F, has been demonstrated, using radioimmunoassays (RlAs), reversed‐phase (rp)HPLC and Northern blot analysis. This is the first stable cell‐line found to express the PPT‐I gene and provides an opportunity for investigating PPT‐I gene expression and tachykinin biosynthesis. RIN5mF cells are a model for the pancreatic beta‐cell, which is not known to exhibit PPT‐I gene expression which may, therefore, be a feature of the transformed state of these cells. These data may imply that the tachykinins are important in pancreatic islet embryogenesis.
Formation of a covalent Hg‐Cys‐bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine‐switch mechanism
Authors: Bläser Jörg , Triebel Susanne , Reinke Heinz , Tschesche Harald
Published: July 2002

A common method for the activation of mammalian matalloproteinases is the use of mercurial compounds. Activation of PMNL procollagenase by soluble mercurials takes place as a three‐step mechanism with a final intermolecular loss of the PRCGVPD autoinhibitor region. In this study covalently bound mercury in the form of mercurial agarose was chosen to probe activation of PMNL procollagenase. Activation was not achieved, since the final intermolecular cleavage with removal of the PRCGVPD motif could not take place. An intermediate form of the enzyme was bound to the column. Its N‐terminal sequence determination proved cleavage of the Asp64‐Met65 peptide bond leaving the cysteine of the propeptide domain for covalent attachment to the mercurial agarose. This gives further evidence of a cysteine‐switch mechanism involving Cys71.
Secondary structure of human granulocyte colony‐stimulating factor derived from NMR spectroscopy
Authors: Zink Thomas , Ross Alfred , Ambrosius Dorothee , Rudolph Rainer , Holak Tad A.
Published: July 2002

Recombinant 13N‐. 13C‐labeled human granulocyte colony‐stimulating factor (rh‐metG‐CSF) has been studied by 2D and 3D NMR using uniformly labeled protein as well is residue‐specific 13N‐labeled samples. Assignment of the 1H, 15N backbone, and 60% 1H sidechain resonances has enabled the determination of the secondary structure of the protein. The secondary structure is dominated by α‐helical regions with four stretches of helices between residues 11–41, 71–95, 102–124 and 144–170.
Expression and characterization of human D4 dopamine receptors in baculovirus‐infected insect cells
Authors: Mills Ann , Allet Bernard , Bernard Alain , Chabert Christian , Brandt Emmanuelle , Cavegn Catherine , Chollet Angelika , Kawashima Eric
Published: July 2002

The human D4 dopamine receptor has been genetically engineered for expression in insect cells using the baculovirus system. A D4 cDNA gene fusion construct [(1991) Nature 350, 610‐614] was synthetically modified to remove two introns from the coding region, and expressed in S. frugiperda (Sf9) cells as a fusion with a short sequence from the polyhedrin protein. Binding assays with [3H]spiperone indicated high levels of D4 receptor binding 90 h after infection and a pharmacological profile identical to that reported for D4 receptors expressed in COS‐7 cells using the cDNA gene hybrid. We also show that the agonist binding affinity of D4 receptors expressed in Sf9 cells can be shifted by GTP‐γ‐S, indicating coupling to G‐proteins.
Human neuroblastoma cells produce the NF‐kB‐like HIV‐1 transcription activator during differentiation
Authors: Kurata Shun-ichi , Wakabayashi Teruko , Ito Yasuhiro , Miwa Naoto , Ueno Ryuji , Marunouchi Tohru , Kurata Nori
Published: July 2002

Human neuroblastoma GOTO and IMR32 cells differentiate into neural cells on treatment with 5‐BrdU. Here we report that in association with differentiation induced by 5‐BrdU, the transcription of HIV‐1 was activated in both neuroblastoma cell lines. An NF‐kB‐like factor was also found to be produced in association with the neural cell differentiation of these cells. These results suggest that, during differentiation of neural cells, an NF‐kB‐like factor was produced, and that when the neural cells are infected with HIV‐1 the production of NF‐kB results in activation of transcription of HIV‐1. Thus this NF‐kB‐like factor may be involved in the mechanism causing AIDS dementia.
In vitro biosynthesis of lactase in suckling and adult rabbits
Authors: Rossi Mauro , Maiuri Luigi , Salvati Virginia M. , Russomanno Concetta , Capparelli Rosanna , Auricchio Salvatore
Published: July 2002

Steady state forms, levels and the in vitro biosynthesis of lactase‐phlorizin hydrolase (LPH) proteins have been studied in proximal and middle intestine of suckling and adult rabbits. In most adult tissues the lactase activity and the LPH protein content were low and the synthesis rate of the 200 kDa lactase precursor was reduced in comparison to suckling tissues. In a few tissues with low enzymatic activity the LPH protein content was relatively high, and high lactase synthesis occurred. In addition, the ratio (labeled lactase)/(lactase protein) was lower in the middle jejunum of the adult rabbit than in the proximal region. Both decreased synthesis of LPH precursor and increased turnover or inactivation of the enzyme may cause the decline of the lactase activity.
Rat endopeptidase‐24.18 α subunit is secreted into the culture medium as a zymogen when expressed by COS‐1 cells
Authors: Corbeil Denis , Milhiet Pierre-Emmanuel , Simon Valérie , Ingram Jean , Kenny A.John , Boileau Guy , Crine Philippe
Published: July 2002

Endopeptidase‐24.18 (EC 3.4.24.18, E‐24.18) is an oligomeric Zn‐ectoenzyme. The α and β submits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C‐terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E‐24.18 α subunit and to test the functionality of the astacin‐like domain in the α subunit when expressed alone, COS‐1 cells were transfected with a cloned cDNA for rat α subunit. Despite the presence of its putative transmembrane domain, the α subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the α subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu‐157 in the active site by Val. Taken together our results suggest that the α subunit of Endopeptidase‐24.18 contains a latent astacin‐like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.
Modulation of α1 β1, α2β1, and α3β1 integrin heterodimers during human neuroblastoma cell differentiation
Authors: Rozzo C. , Ratti P. , Ponzoni M. , Cornaglia-Ferraris P.
Published: July 2002

Two human neuroblastoma cell lines, LAN‐5 and GI‐CA‐N, have been analyzed for their capability to adhere to different extracellular matrix (ECM) components. The GI‐CA‐N cells adhered to all the tested substrates: laminin (LN), type I and type IV collagen (Coll I, Coll IV), vitronectin (VN), and fibronectin (FN). Conversely LAN‐5 cells weakly attached to FN and VN, whilst adhesion on LN and Coll I and IV was strong and induced a rapid elongation of cell processes. By means of RT‐PCR and immunoprecipitation we showed that the integrin pattern of these two lines was different and could explain their diversity in adhesion capability. Both cell lines express a large amount of the β1 integrin subunit, associated with different α chains, probably responsible for their adhesion to some ECM proteins. After treatment of LAN‐5 cells with biological differentiating agents, such as γ‐interferon, alone or in combination with tumour necrosis factor‐α (TNF‐α), or retinoic acid, the levels of α1β1, α2β1, and α3β1 integrin expression were enhanced, while the amount of α, remained constant. In contrast, treatment of LAN‐5 cells with TNF‐α, that did not induce any maturation, or starvation in 2% foetal calf serum, that inhibited cell proliferation without affecting neural differentiation, did not induce any change in the integrin assessment. Messenger‐RNAs for the two α6 isoforms, A and B, were present in both cell lines. However, in LAN‐5 cells, the protein product was neither detectable nor inducible by differentiation. Our results confirm the specific modulation of the α1β1 integrin expression in human neuronal development, and show, for the first time, the involvement of α2β1 and α3β1, heterodimers in this maturational process.