bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of possible functional domains in sup2 gene product of the yeast Saccharomyces cerevisiae
Authors: Kushnirov Vitaliy V. , Ter-Avanesyan Michail D. , Surguchov Andrei P. , Smirnov Vladimir N. , Inge-Vechtomov Sergey G.
Published: July 2002

Primary structures of yeast sup2 gene and polypeptide product coded by the gene are compared with the current nucleotide and amino acid sequence data base. The amino acid sequence of the sup2 product shows homology to elongation factors from different sources. Especially high homology is found in the regions, corresponding to conservative aminoacyl‐tRNA‐ and GTP‐binding domains, described in elongation factors and other proteins. The data obtained are discussed in relation to the functions of sup2 polypeptide product in protein synthesis.
Characterization of glucacon receptors in Golgi fractions of fetal rat liver
Authors: de Diego Juana G. , Alvarez Elvira , Blázquez Enrique
Published: July 2002

The present study was designed to determine if Golgi fractions from fetal rat liver contain glucagon receptors and to characterize the properties of such receptors. Purification patterns of liver plasma membranes and Golgi fractions from fetal and adult rats were similar, as verified by morphological and biochemical approaches. Glucagon binding was greater in plasma membranes of adult than fetal rats, while in Golgi fractions glucagon binding was similar in both groups. The modifications in glucagon binding reflect changes in glucagon receptors. Glucagon association and glucagon receptor inactivation by liver membranes were similar in the two groups of animals, while glucagon degradation was lower in fetal than in adult rats.
A yeast gene coding for a putative protein kinase homologous to cdc25 suppressing protein kinase
Authors: Ohno Shigeo , Aoshima Miho , Matsumoto Seiji , Yahara Ichiro , Suzuki Koichi
Published: July 2002

A yeast gene termed YKR coding for a putative protein kinase was isolated by using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein (YKR), composed of 380 amino acid residues, shows extensive sequence homology to serine/threonine‐specific protein kinases from various species in the approx. 320 C‐terminal amino acid residues, strongly suggesting that YKR is endowed with a protein kinase activity. The observed homologies to the cdc25 suppressing protein kinase from yeast, the catalytic subunit of mammalian cAMP‐dependent protein kinase, and mammalian protein kinase C were 76, 48 and 37%, respectively. Gene replacement experiments showed that YKR itself is not essential for cell proliferation.
Interaction of melittin with negatively charged phospholipids: Consequences for lipid organization
Authors: Batenburg A.M. , van Esch J.H. , Leunissen-Bijvelt J. , Verkleij A.J. , de Kruijff B.
Published: July 2002

A characterization of the structural alterations induced by melittin in model‐membranes of dioleoylphosphatidic acid and egg phosphatidylglycerol is presented, based on the use of 31P‐NMR, freeze‐fracture electron microscopy and small angle X‐ray scattering. In accordance with earlier findings on the cardiolipinmelittin system, melittin is found to have an inverted phase inducing effect on these negatively charged lipids, in contrast to the influence on zwitterionic phospholipids. In phosphatidic acid this is expressed in the formation of an HII phase; in phosphatidylglycerol a less ordered, non‐lamellar structure with low water content is adopted.
Stereochemistry of the methylmalonyl‐CoA decarboxylation reaction
Authors: Hoffmann Artur , Dimroth Peter
Published: July 2002

The steric course of the decarboxylation of (S)‐methylmalonyl‐CoA to propionyl‐CoA, catalyzed by the biotin‐dependent sodium pump methylmalonyl‐CoA decarboxylase of Veillonella alcalescens was determined. The decarboxylation of (S)‐methylmalonyl‐CoA in 3H2O yielded (R)‐[2‐3H]propionyl‐CoA; and the decarboxylation of (S)‐[2‐3H]methylmalonyl‐CoA in H2O produced (S)‐[2‐3H]propionyl‐CoA. The results demonstrate retention of configuration during the decarboxylation reaction. The substrate stereochemistry of methylmalonyl‐CoA decarboxylase is thus the same as that of all other biotin‐containing enzymes investigated.
Lithium ion reversibly inhibits inducer‐stimulated adipose conversion of 3T3‐L1 cells
Authors: Aratani Yasuaki , Sugimoto Etsuro , Kitagawa Yasuo
Published: July 2002

Adipose conversion of 3T3‐L1 cells by inducers (dexamethasone, 1‐methyl‐3‐isobutylxanthine and insulin) was inhibited by LiCl at concentrations from 2 to 20 mM. The effect of LiCl was reversible and the inhibited cells were converted to adipocytes when stimulated after the removal of LiCl. Inhibition by LiCl of adipose conversion was accompanied with a blockage of the enhanced [3H]thymidine incorporation and cellular proliferation that occurred before the adipocyte phenotype was expressed. Of the cations tested, only Li+ had these effects.
Clostridium perfringens iota toxin ADP‐ribosylates skeletal muscle actin in Arg‐177
Authors: Vandekerckhove Joël , Schering Beate , Bärmann Michael , Aktories Klaus
Published: July 2002

Clostridium perfringens iota toxin ADP‐ribosylates actin. Substrates of C. perfringens toxin are both non‐muscle β/γ‐actin and skeletal muscle actin. This finding suggests that C. perfringens iota ADP‐ribosylates the same amino acid in skeletal muscle and non‐muscle actin as does C. botulinum C2 toxin in non‐muscle actin. Protein chemical analysis involving thermolysin cleavage on [32P]ADP‐ribosylated actin or tryptic digestion followed by a secondary thermolysin cleavage of the radiolabelled fragments showed one major site of ADP‐ribosylation. From its amino acid composition and sequence, the radiolabelled peptide was identified as peptide 175–177, locating the acceptor ADP‐ribosyl amino acid as Arg‐177.
p‐Benzoquinone monoketals, novel degradation products of β‐O‐4 lignin model compounds by Coriolus versicolor and lignin peroxidase of Phanerochaete chrysosporium
Authors: Kawai Shingo , Umezawa Toshiaki , Higuchi Takayoshi
Published: July 2002

2‐(4‐Ethoxy‐3‐methoxyphenyl)‐3‐hydroxymethyl‐6,10‐dimethoxy‐1,4‐dioxaspiro[4,5]deca‐6,9‐diene‐8‐one (III) and its isomer IV were identified as catabolites of 4‐ethoxy‐3‐methoxyphenylglycerol‐β‐syringaldehyde ether (I) by the culture of Coriolus versicolor. Compound III was also produced from 4‐ethoxy‐3‐methoxyphenylglycerol‐β‐syringic acid ether (II) by lignin peroxidase of Phanerochaete chrysosporium. An isotopic experiment showed that molecular oxygen was incorporated into the quinone oxygen of III in the degradation of II by lignin peroxidase.
Retention of actin synthesis in liver under conditions that inhibit synthesis of almost all other proteins
Authors: Felipo Vicente , Grisolía Santiago
Published: July 2002

As briefly reported [(1986) Fed. Proc. 45, 1771, Abstr. 1690], rats fed a protein‐free diet for a few days often show a marked inhibition of protein synthesis in liver cytosol. However the synthesis of a protein of molecular mass ~42 kDa is fully retained. We show here on the basis of its molecular mass, number of bands on isoelectric focusing, isoelectric point and immunological reactivity that this protein is actin and also that actin mRNA is not degraded by micrococcal nuclease under conditions which degrade the bulk of other mRNAs.
Insulin‐like growth factor I binding and receptor kinase in red and white muscle
Authors: Zorzano Antonio , James David E. , Ruderman Neil B. , Pilch Paul F.
Published: July 2002

IGF‐I receptors were partially purified from red and white skeletal muscle by lectin‐affinity chromatography and the resultant fraction was depleted of insulin receptors by insulin affinity chromatography. Equilibrium binding of 125I‐IGF‐I to receptor preparations from red and white muscle yielded identical Scatchard plots. The integrity of the IGF‐I receptor preparation in the two fiber types was identical as determined by affinity cross‐linking. The tyrosine kinase activity of the receptor from red muscle was 2–3‐fold more active towards exogenous substrates in both the basal and ligand‐activated states as compared to white muscle. These data show that there is IGF‐I‐dependent kinase activity intrinsic to IGF‐I receptors from skeletal muscle, and suggest that identical cellular factors may regulate the kinase activity of insulin and IGF‐I receptors in a parallel manner in vivo.
Analysis of the electron transfer from Pheo− to QA in PS II membrane fragments from spinach by time resolved 325 nm absorption changes in the picosecond domain
Authors: Eckert H.-J. , Wiese N. , Bernarding J. , Eichler H.-J. , Renger G.
Published: July 2002

Absorption changes at 325 nm (ΔA
325) induced by 15 ps laser flashes (λ = 650 nm) in PS II membrane fragments were measured with picosecond time‐resolution. In samples with the reaction centers (RCs) kept in the open state (P I QA) the signals are characterized by a very fast rise (not resolvable by our equipment) followed by only small changes within our time window of 1.6 ns. In the closed state (P I Q−
A) of the reaction center the signal decays with an average half‐life time of about 250 ps. It is shown that under our excitation conditions (E = 2 × 1014 photons/cm2 per pulse) subtraction of the absorption changes in closed RCs (ΔA
closed
325) from those in open RCs (ΔA
open
325) leads to a difference signal which is dominated by the reduction kinetics of QA. From the rise kinetics of this signal and by comparison with data in the literature it is inferred that QA becomes reduced by direct electron transfer from Pheo− with a time constant of about 350 ± 100 ps.
Direct 19F NMR observation of the conformational selection of optically active rotamers of the antifolate compound fluoronitropyrimethamine bound to the enzyme dihydrofolate reductase
Authors: Tendler Saul J.B. , Griffin Roger J. , Birdsall Berry , Stevens Malcolm F.G. , Roberts Gordon C.K. , Feeney James
Published: July 2002

The molecular basis of the binding of the lipophilic antifolate compound fluoronitropyrimethamine [2,4‐diamino‐5‐(4‐fluoro‐3‐nitrophenyl)‐6‐ethylpyrimidine] to its target enzyme dihydrofolate reductase has been investigated using a combination of 19F NMR spectroscopy and molecular mechanical calculations. 19F NMR reveals the presence of two different conformational states for the fluoronitropyrimethamine‐Lactobacillus casei enzyme complex. MM2 molecular mechanical calculations predict restricted rotation about the C5‐C1′ bond of the ligand and this gives rise to two slowly interconverting rotamers which are an enantiomeric pair. The results of 19F NMR spectroscopy reveal that both these isomers bind to the enzyme, with different affinities. There is no detectable interconversion of the bound rotamers themselves on the NMR timescale. The effect of the addition of co‐enzyme to the sample is to reverse the preference the enzyme has for each rotamer.
Activation energy of the rate constant of P+Q−A absorption decay in reaction centers fromRhodobacter sphaeroides reconstituted with different anthraquinones
Authors: Sebban Pierre
Published: July 2002

The free energy differences between the first stable charge‐separated state, P+Q−
A and the thermally excited state, M, via which P+Q−
A recombines, were measured in the reaction centers fromRhodobacter sphaeroides reconstituted with anthraquinones. We find that the free energy level of M is independent of the in situ redox potential of QA and consequently the free energy level of P+Q−
A. This suggests that M is a state independent of P+Q−
A. This is in support of the identification of M with a relaxed form of P+I−.
Effect of site‐specific mutagenesis of tyrosine‐55, methionine‐110 and histidine‐217 in porcine kidney D‐amino acid oxidase on its catalytic function
Authors: Watanabe Fusao , Fukui Kiyoshi , Momoi Kyoko , Miyake Yoshihiro
Published: July 2002

To assess the contributions of Tyr‐55, Met‐110 and His‐217 in porcine kidney D‐amino acid oxidase (EC 1.4.3.3, DAO) to its catalytic function, we constructed three mutant cDNAs coding for the enzymes possessing Phe‐55, Leu‐110 and Leu‐217 by site‐specific mutagenesis. The mutant and wild type cDNAs could be expressed in vitro with similar efficiency. The three mutant enzymes thus synthesized showed catalytic activities comparable to that of the wild type oxidase. It is concluded that Tyr‐55, Met‐110 and His‐217 are not directly involved in the catalytic function.
Increased muscarinic receptor binding in heart membranes by an inhibitor of protein kinase C
Authors: Creazzo Tony L. , Wrenn Robert W.
Published: July 2002

The number of muscarinic acetylcholine receptors (MAChRs), as detected by binding of [3H]quinuclidinyl benzilate (QNB), was investigated under conditions which promote protein phosphorylation. Incubation of a crude heart membrane preparation in the presence of ATP/Mg2+ reduced MAChR number by 50%. Incubation with polymyxin B, an inhibitor of protein kinase C, blocked the efrect of ATP/Mg2+ and increased MAChR number by 74%.
Potassium channels expressed from rat brain cDNA have delayed rectifier properties
Authors: Stühmer Walter , Stocker Martin , Sakmann Bert , Seeburg Peter , Baumann Arnd , Grupe Andrew , Pongs Olaf
Published: July 2002

Injection into Xenopus oocytes of RNA synthesized in vitro using the rat brain cDNA RCK1 as a template or nuclear injection of the cDNA results in the expression of functional potassium channels. These channels exhibit properties similar to those of the non‐inactivating delayed rectifier channel found in mammalian neurons and other excitable cells.
A methyl‐CoM methylreductase system from methanogenic bacterium strain Göl not requiring ATP for activity
Authors: Deppenmeier U. , Blaut M. , Jussofie A. , Gottschalk G.
Published: July 2002

Crude inside‐out vesicles from the methanogenic strain Göl were prepared via protoplasts. These vesicles catalysed methane formation from methyl‐CoM and H2 at a maximal rate of 35 nmol/min · mg protein. Methane formation by the vesicles did not depend on the addition of ATP. This was in contrast to conventionally prepared crude extracts from the same organism or from Methanosarcina barkeri which exhibited strict ATP dependence of methanogenesis. ATP analogues inhibited methanogenesis by extracts to a much higher extent than that by vesicles. Both, particulate and soluble components prepared from the crude vesicles by ultracentrifugation were necessary for ATP‐independent methane formation from methyl‐CoM and H2. Hydrogenase activity was mainly associated with the particulate fraction whereas methyl‐CoM methylreductase could be assigned to the soluble fraction. The detergent sulfobetaine inhibited methane formation from methyl‐CoM without affecting hydrogenase or titanium citrate‐dependent methylreductase activities, indicating that an additional membraneous component is involved in methanogenesis for methyl‐CoM and H2.
Calmodulin‐ and protein phosphorylation‐independent release of catecholamines from PC‐12 cells
Authors: Matthies Heinrich J.G. , Palfrey H.Clive , Miller Richard J.
Published: July 2002

Catecholamine secretion from PC‐12 cells can be triggered by agents that increase intracellular Ca2+ and is enhanced by phorbol esters and agents that elevate intracellular cAMP concentrations. In mutant PC‐12 cells lacking cAMP‐dependent protein kinase (PK‐A) in which protein kinase C (PK‐C) was down‐regulated, Ca2+‐dependent secretion occurred normally but was no longer enhanced by cAMP or phorbol esters. In digitonin‐permeabilized PC‐12 cells that lacked PK‐C and PK‐A, a range of calmodulin (CaM) inhibitors failed to block Ca2+‐triggered catecholamine release. Moreover, Mn2+, a CaM activator, failed to trigger catecholamine release whereas Ba2+, which does not activate CaM, supported secretion. These results indicate that the basic mechanism of stimulus/secretion coupling in PC‐12 cells does not absolutely require a regulated protein phosphorylation‐ or calmodulin‐dependent step.
Histidine 64 is not required for high CO2 hydration activity of human carbonic anhydrase II
Authors: Forsman Cecilia , Behravan Gity , Jonsson Bengt-Harald , Liang Zhi-wei , Lindskog Sven , Ren Xilin , Sandström Jan , Wallgren Katarina
Published: July 2002

To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a ‘proton shuttle group’ during catalysis of CO2‐HCO−
3 interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site‐directed mutagenesis. All these variants turned out to have high CO2 hydration activities. The k
cat values at pH 8.8 and 25°C were only reduced by 1.5–3.5‐fold compared to the unmodified enzyme. These results show that intramolecular proton transfer via His 64 is not a dominating pathway in the catalytic reaction. The variants also catalyze the hydrolysis of 4‐nitrophenyl acetate. The pK
a values for the activity‐controlling group are between 6.8 and 7.0 for all studied forms of the enzyme except the Glu 64 variant which shows a complex pH dependence with the major pK
a shifted to 8.4
Characterization of an invertebrate nicotinic acetylcholine receptor gene: The ard gene of Drosophila melanogaster
Authors: Sawruk Erich , Hermans-Borgmeyer Irm , Betz Heinrich , Gundelfinger Eckart D.
Published: July 2002

The ard gene encodes a neuronal nicotinic acetylcholine receptor (AChR) protein from Drosophila (ARD protein). Cytogenetically this gene maps at position 64B/C on the left arm of the 3rd chromosome. Five introns interrupt the protein coding region of the gene, and one is found upstream of the translation start site. The ard gene thus contains less introns than vertebrate muscle AChR genes, but, with one exception, the positions of the resident introns are precisely conserved. Implications for the evolution of AChR genes are discussed.
Identification of a new gene in the chloroplast genome encoding a low‐molecular‐mass polypeptide of photosystem II complex
Authors: Murata Norio , Miyao Mitsue , Hayashida Nobuaki , Hidaka Tadashi , Sugiura Masahiro
Published: July 2002

Low‐molecular‐mass polypeptides in spinach photosystem II membranes were separated by SDS‐polyacrylamide gel electrophoresis. The partial amino acid sequence of one of the polypeptides was determined. Comparison of this sequence with the entire nucleotide sequence of the tobacco chloroplast genome revealed that this polypeptide is encoded in the chloroplast genome. The gene for the polypeptide, designated as psbK, is located between the genes for tRNASer and tRNAGln in the large single‐copy region and oriented in the direction opposite to the tRNA genes. The amino acid sequence deduced from the gene indicates that the translation product consists of 98 amino acid residues and its 62nd residue corresponds to the amino‐terminus of the mature polypeptide. The putative polypeptide consists of 37 amino acid residues with a molecular mass of 4285 Da and has a single membrane‐spanning segment. Northern blot hybridization analysis revealed that psbK was transcribed in the chloroplast.
Energy transfer within the isolated B875 light‐harvesting pigment‐protein complex of Rhodobacter sphaeroides at 77 K studied by picosecond absorption spectroscopy
Authors: Bergström H. , Westerhuis W.H.J. , Sundström V. , van Grondelle R. , Niederman R.A. , Gillbro T.
Published: July 2002

The energy transfer dynamics at 77 K within detergent solubilized and purified preparations of the B875 pigment‐protein complex of Rhodobacter sphaeroides have been investigated by picosecond absorption spectroscopy. Isotropic absorption recovery and decay of induced absorption anisotropy provide clear evidence that B875 is inhomogeneous in these preparations. We interpret the results as fast (τ = 15 ± 5 ps) energy transfer from the major BChl 875 pigments to a minor pigment pool, B896. The excited state of B896 decays with a time constant of 650 ± 50 ps. We suggest that B896 is intrinsic to B875 complexes and exists in a highly organized state, close to the reaction center. In the intact membrane, B896 may concentrate excitation energy in the vicinity of the reaction center special pair, thereby increasing the efficiency of the final energy transfer step.
A third human CALC (pseudo)gene on chromosome 11
Authors: Höppener J.W.M. , Steenbergh P.H. , Zandberg J. , Adema G.J. , van Kessel A.H.M.Geurts , Lips C.J.M. , Jansz H.S.
Published: July 2002

A genomic locus in man (CALC‐III) containing nucleotide sequences highly homologous to both exon 2 and exon 3 of the CALC‐I and ‐II genes, is described in this paper. The CALC‐I gene produces calcitonin (CT) (encoded by exon 4) or calcitonin gene‐related peptide (CGRP) (encoded by exon 5) in a tissue‐specific fashion. The CALC‐II gene produces a second human CGRP, but probably not a second CT. The CALC‐III gene does not seem to encode a CT‐ or CGRP‐related polypeptide hormone and is probably a pseudogene. Like the other two CALC genes, the CALC‐III gene is located on human chromosome 11.
Involvement of bicarbonate in the protonation of the secondary quinone electron acceptor of photosystem II via the non‐haem iron of the quinone‐iron acceptor complex
Authors: van Rensen Jack J.S. , Tonk Willem J.M. , de Bruijn Steef M.
Published: July 2002

The mechanism of the bicarbonate effect was investigated by monitoring flash‐induced pH changes. In control chloroplasts the proton yields exhibit a binary oscillation with a period of four. In CO2‐depleted chloroplasts the binary oscillation disappears and only the period four pattern remains, which can be described by proton liberation in the water‐oxidizing system. It is concluded that bicarbonate is involved in the protonation of Q2−
B. The affinity of bicarbonate to its binding site is much lower in the presence of dithionite. It is suggested that bicarbonate exerts its influence through being a ligand for the non‐haem iron between QA and QB.
Epoxidation of cis and trans Δ9‐unsaturated lauric acids by a cytochrome P‐450‐dependent system from higher plant microsomes
Authors: Salaün Jean-Pierre , Weissbart Daniel , Durst Francis , Pflieger Philippe , Mioskowski Charles
Published: July 2002

A new epoxygenase, differing in substrate and stereospecificity from those previously described in mammals, bacteria and Vicia faba, has been characterized in higher plants. A microsomal fraction from Jerusalem artichoke tubers which catalyses in‐chain monohydroxylation of lauric acid has now been found to convert synthetic 9‐dodecenoic acid (9‐DDNA) to 9,10‐epoxylauric acid. Epoxidation of the cis and trans isomers proceeds at similar rates affording the corresponding cis and trans epoxides. This reaction is dependent upon O2 and NADPH and does not occur with boiled microsomes. Lauric acid, carbon monoxide and polyclonal antibody against NADPH‐cytochrome P‐450 reductase from Jerusalem artichoke inhibit epoxide formation. Compared to freshly sliced tuber tissues, epoxidation activity was strongly enhanced in tissues aged in 20 mM aminopyrine solution. The possibility that 9, 10‐epoxidation of 9‐DDNA is catalyzed by the cytochrome P‐450 isoenzyme involved in lauric acid in‐chain hydroxylation in this plant was investigated.
Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli
Authors: Karlsson B.Göran , Pascher Torbjörn , Nordling Margareta , Arvidsson Rolf H.A. , Lundberg Lennart G.
Published: July 2002

The structural gene for the blue copper protein azurin from Pseudomonas aeruginosa has been subcloned in different expression plasmid vectors. The highest yield of expression was obtained when the gene with its native ribosome‐binding site was placed downstream of the lac promoter in plasmid pUC18. The protein is exported to the periplasmic space in Escherichia coli and the amount corresponds to 27% of the total protein content in the periplasmic space. The preprotein is cleaved correctly according to N‐terminal sequencing of the purified protein. Azurin has been purified in large amounts and is spectroscopically indistinguishable from the protein purified from P. aeruginosa.
One of two subunits of masking protein in latent TGF‐β is a part of pro‐TGF‐β
Authors: Okada Fumio , Yamaguchi Kazuo , Ichihara Akira , Nakamura Toshikazu
Published: July 2002

A high molecular mass latent form of transforming growth factor type‐β (TGF‐β) was purified to homogeneity from rat platelets by a seven‐step procedure involving group‐specific affinity chromatographies on Red‐Toyopearl and zinc chelating‐Sepharose. The purified latent TGF‐β was a complex of TGF‐β (25 kDA) and the binding protein previously named masking protein (∼400 kDA) [(1986) Biochem. Biophys. Res. Commun. 141, 176–184]. Analysis of the peptide structure by gel electrophoresis showed that the masking protein consisted of two subunits of 39 kDA and 105–120 kDA linked by disulfide bonds. N‐terminal amino‐acid sequencing of the 39 kDA subunit indicated that this subunit was identical to the N‐terminal part of the TGF‐β precursor.
The isolation and reconstitution of the ADP/ATP carrier from wild‐type Saccharomyces cerevisiae Identification of primarily one type (AAC‐2)
Authors: Knirsch Martin , Gawaz Meinrad P. , Klingenberg Martin
Published: July 2002

Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT‐AAC complex, the free and active AAC for reconstitution, or the SDS‐denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC‐1 and AAC‐2 recently found in yeast, due to the exclusive occurrence of a methionine (M‐115) residue at the end of the first domain in AAC‐2. Thus the AAC isolated from wild‐type yeast is primarily or exclusively AAC‐2. The isolated AAC is active in ADP/ATP exchange in reconsitituted liposomes with a V
max of 1100 μmol/min per g protein and K
m = 15 μM for ADP, and a V
max of 900 μmol/min per g protein and K
m = 9 μM for ATP.
Time‐resolved ultrafast blue‐shifted fluorescence from pea chloroplasts
Authors: McCauley Steven W. , Bittersmann Edith , Holzwarth Alfred R.
Published: July 2002

The antenna chlorophylls in higher plants can produce fluorescence that is blue‐shifted (anti‐Stokes‐shifted) relative to the exciting light. Global analysis of the time‐resolved fluorescence spectra resolves an ultrafast fluorescence emission component with a lifetime of about 15 ps which is attributed to exciton equilibration in the antenna. These results support the hypothesis that the overall exciton decay kinetics in PS I and PS II is trap‐limited as suggested by us earlier.
Complete primary structure of a calcium‐dependent serine proteinase capable of degrading extracellular matrix proteins
Authors: Kinoshita Hirohisa , Sakiyama Hisako , Tokunaga Kastuo , Imajoh-Ohmi Shinobu , Hamada Yoshio , Isono Kaichi , Sakiyama Shigeru
Published: July 2002

A novel calcium‐dependent serine proteinase (CASP) secreted from malignant hamster embryo fibroblast Ni 12C2 degrades extracellular matrix proteins. A complementary DNA encoding CASP has been isolated with the use of oligonucleotide probes synthesized based on partial amino acid sequences of CASP. The complete amino acid sequence of CASP revealed that it has a serine active site at the C‐terminal side. Glu rich and proEGF homologous sites are found at the N‐terminal site suggesting that it is structurally similar to blood coagulation factors such as IX, X and an anti‐coagulation factor, protein C.
Vinblastine sensitivity of leukaemic lymphoblasts modulated by serum lipid
Authors: Holmes Kerry T. , Dyne Marlen , Williams Philip G. , May George L. , Tripp Edith , Wright Lesley C. , Mountford Carolyn E.
Published: July 2002

The high‐resolution proton magnetic resonance spectrum of leukaemic lymphoblasts is characteristic of neutral lipid in an isotropic environment. When such lymphoblasts are selected for resistance to the anticancer drug vinblastine, the intensity of this spectrum increases with increasing drug resistance. A reversal of this trend can be achieved by growing cells in delipidated serum, whereby lipid spectrum and drug resistance are diminished. However, both can be restored by subsequent regrowth in normal medium. Thus, although detectable genetic changes accompany the development of vinblastine resistance, the expression of these changes can be modulated by environmental lipid.
Nitrile hydratase of Rhodococcus sp. N‐774 Purification and amino acid sequences
Authors: Endo Takakazu , Watanabe Ichiro
Published: July 2002

The nitrile hydratase of Rhodococcus sp. N‐774 was purified and crystallized. The enzyme is composed of two different subunits (molecular masses: subunit α, 28 500 Da; subunit β, 29 000 Da). The amino‐terminal amino acid sequence of each subunit was determined. There is no sequence homology between the two subunits, suggesting that the peptides originate from different cistrons. The activity of the purified enzyme did not decrease during incubation in the dark, whereas it gradually decreased in intact cells.
‘Perforated patch recording’ allows long‐term monitoring of metabolite‐induced electrical activity and voltage‐dependent Ca2+ currents in pancreatic islet B cells
Authors: Falke Lee C. , Gillis Kevin D. , Pressel David M. , Misler Stanley
Published: July 2002

We describe the application of ‘perforated patch recording’ using the pore‐forming antibiotic nystatin, to monitor the electrical activity and underlying ionic currents of rat and human pancreatic islet B cells. We demonstrate that glucose‐induced electrical activity is seen even in single B cells during current‐clamp recordings lasting hours ‘L‐type’ Ca2+‐channel currents can also be monitored over this period of time. This technique may prove useful in examining hormone and neurotransmitter modulation of electrical activity in B cells, while minimizing the effects of cytoplasmic ‘wash‐out’.
Participation of acid phospholipids in protein translocation across the bacterial cytoplasmic membrane
Authors: Nesmeyanova Marina A. , Bogdanov Michail V.
Published: July 2002

Recent observations confirm the participation of acid phospholipids in protein translocation. The hypothesis proposed coupled protein translocation with transmembrane movement of acid phospholipids, their metabolism as a precursor of cell envelope components and recycling. These factors ensure the unidirectional vector value of the secretion, restoration of the membrane site competent for protein translocation and its self‐organization.
Flavin adenine dinucleotide causes oligomerization of acetohydroxyacid synthase from black Mexican sweet corn cells
Authors: Singh Bijay K. , Schmitt Gail K.
Published: July 2002

Acetohydroxyacid synthase activity is stabilized and stimulated by flavin adenine dinucleotide. Flavin adenine dinucleotide was found to cause aggregation of acetohydroxyacid synthase from the dimeric to a tetrameric form. The different aggregation states of the enzyme have differential sensitivities to inhibition by branched chain amino acids as well as by imazapyr, an imidazolinone herbicide. These observations indicate that flavin adenine dinucleotide is of structural as well as of functional importance for the plant acetohydroxyacid synthase enzyme.
Suramin, an anti‐cancer drug, inhibits protein kinase C and induces differentiation in neuroblastoma cell clone NB2A
Authors: Hensey Carmel E. , Boscoboinik Daniel , Azzi Angelo
Published: July 2002

Protein kinase C purified from rat brain was found to be inhibited by suramin, a substance used originally in the therapy of antitrypanosomic infections and more recently proposed as antineoplastic agent. The inhibition of suramin was competitive with one of the substrates of the enzyme, ATP with a K
i of 10 μM. At concentrations adequate to inhibit the isolated enzyme, suramin was shown to slow the rate of proliferation of neuroblastoma NB2A cells in vitro and to induce their differentiation as evidenced by typical morphological changes.
Cation channels by subunit III of the channel portion of the chloroplast H+‐ATPase
Authors: Schönknecht G. , Althoff G. , Apley E. , Wagner R. , Junge W.
Published: July 2002

The chloroplast H+‐ATPase (CF0CF1) was isolated and reconstituted into lipid vesicles by dialysis technique. Vesicles were fused by dehydration/ rehydration to obtain cell‐size liposomes, which were studied by patch‐clamp techniques. Single‐channel activity was observed with several conductance levels in the range of some 10 pS (100 mM KCl). In contrast to intact CF0, which conducts protons, only (even at pH 8), these channels were permeable for potassium and sodium. Venturicidin, which blocks proton flow through intact CF0, here greatly decreased the single‐channel open probability. Subunit III of CF0, alone, yielded cation channels resembling the former. Our tentative interpretation is, that dearrangement or fragmentation of CF0 caused the potassium and sodium permeability, which, however, is suppressed in intact CF0.
RNA‐binding protein‐related sequence in a malaria antigen, clustered‐asparagine‐rich protein
Authors: Kuma Keiichi , Iwabe Naoyuki , Kawanishi Yuichi , Miyata Takashi
Published: July 2002

Members of the RNA‐binding protein superfamily contain RNA binding domains of about 90 amino acids with a highly conserved motif ‘GFGF’. Using the conserved motif with some variations G‐(F/Y)‐(G/A)‐(F/Y)‐(V/I)‐X‐(F/Y) as a probe, we screened protein sequences carrying identical amino acids in an NBRF‐protein database. It has been found that the C‐terminal portion of clustered asparagine‐rich protein (CARP), a malaria antigen from Plasmodium falciparum, shows an unexpected sequence similarity with the RNA‐binding protein superfamily for the C‐terminal half of the RNA‐binding domain. Dot matrix comparisons and alignment of these sequences as well as a statistical test have revealed highly significant sequence similarities. From these analyses, we conclude that the malaria antigen CARP belongs to a large family of the RNA‐binding proteins. An evolutionary implication of the sequence similarity was also discussed.
Mannose‐6‐phosphate stimulates proliferation of neuronal precursor cells
Authors: Nielsen Finn C. , Gammeltoft Steen
Published: July 2002

The mitogenic signal function of mannose‐6‐phosphate (Man‐6‐P)/insulin‐like growth factor II (IGF‐II) receptors was studied in neuronal precursor cells from developing rat brain (E15). About 30% of the cellular Man‐6‐P/IGF‐II receptors were present on the cell surface. Man‐6‐P and IGF‐II stimulated DNA synthesis twofold and their effects were additive. Antibody 3637 to the Man‐6‐P/IGF‐II receptor blocked the response to Man‐6‐P but not that to IGF‐II. Other phosphorylated hexoses were also active. Fructose‐1‐phosphate was equally potent with Man‐6‐P, whereas glucose‐6‐phosphate was 5 times less potent. We conclude that Man‐6‐P‐containing proteins and IGF‐II act as mitogens in developing brain by interaction with the Man‐6‐P/IGF‐II receptor and the IGF‐I receptor, respectively.
cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells
Authors: Kumatori Atsushi , Tanaka Keiji , Tamura Tomohiro , Fujiwara Tsutomu , Ichihara Akira , Tokunaga Fuminori , Onikura Aya , Iwanaga Sadaaki
Published: July 2002

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence ofC9 consists of 261 amino acid residues with a calculated molecular weight of 29496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.
Structural analysis of the regulatory region of the human corticotropin releasing hormone gene
Authors: Vamvakopoulos Nikos C. , Karl Michael , Mayol Vanessa , Gomez Teresa , Stratakis Constantine A. , Margioris Andrew , Chrousos George P.
Published: July 2002

A DNA fragment containing the human corticotropin releasing hormone (CRH) gene, along with 9 kb of upstream and 4 kb of downstream sequences, was isolated from a human genomic DNA library. Nucleotide sequence analysis of the proximal 918 nucleotides 5' flanking the putative major mRNA start site of the human gene and comparison to the 866 nucleotide long homologous ovine sequence, revealed that this region of the CRH gene consists of two distinct areas with different degrees of homology, varying from 72% to 94%. The putative functional features of the human sequence were identified. Many, but not all, features were conserved in the ovine sequence. The highly conserved nature of the regulatory region of this gene makes it a good candidate for tracing possible related genetic defects of the hypothalamic‐pituitary‐adrenal (HPA) axis.
Characterization of receptors for glucagon‐like peptide‐1(7–36) amide on rat lung membranes
Authors: Richter Gerd , Göke Rüdiger , Göke Burkhard , Arnold Rudolf
Published: July 2002

Specific binding of 125I‐labelled GLP‐1(7–36)amide to rat lung membranes was dependent upon time and temperature and was proportional to membrane protein concentration. Binding was inhibited in a concentration‐dependent manner by unlabelled GLP‐l(7–36)amide consistent with the presence of a single class of binding sites with a dissociation constant (K
d) of 1.67 ± 0.29 . GLP‐1(1–36)amide was 260 times less potent in inhibiting the binding of 125I‐labelled GLP‐1(7 36)amide to lung membranes (k
d of 448 ± 93 ). Vasoactive intestinal polypeptide and peptide‐histidine‐isolcucine also displaced 125I‐labelled GLP‐1(7–36)amide from the receptor concentration‐dependently; the k
d was 4.31 ± 0.8 and 7.93 ± 4.79 . respectively. Guaninc nucleotides (GTP‐γ‐S, GDP‐β‐S) decreased the binding of 125I‐labelled GLP‐1(7–36)amide to rat lung membranes as was found for GLP‐1(7–36)amide receptors in RINm5F cells which were also shown to be coupled to the adenylate cyclase system.
Electron microscopy of the reconstituted complexes of the F1‐ATPase with various subunit constitution revealed the location of the γ subunit in the central cavity of the molecule
Authors: Fujiyama Yuichiro , Yokoyama Ken , Yoshida Masasuke , Wakabayashi Takeyuki
Published: July 2002

The subunit structure of the F1‐ATPase from the thermophilic bacterium PS3 was probed by comparing electron microscopic images of the subunit complexes reconstituted to contain different subunit compositions. The following structural features were observed. (1) The α3β3 complex has a hexagonal, apparently symmetrical arrangement of masses with a central cavity. (2) The projections of the α3β3δ complexes are similar to those of the α3β3 complexes. (3) In contrast, the α3β3λ complex has an additional mass in the centre which is similar to that found in the native enzyme (α3β3γδϵ). From these observations, it is concluded that the central mass in the F1‐ATPase is comprised mostly of the γ subunit.
Differential expression of the neural cell adhesion molecule NCAM 140 in human pituitary tumors
Authors: Aletsee-Ufrecht Maria C. , Langley Keith , Gratzl Otmar , Gratzl Manfred
Published: July 2002

We have analyzed the expression of the intracellular marker protein neuron specific enolase (NSE), synaptophysin (SYN) and of the cell surface marker NCAM (neural cell adhesion molecule) in both normal human hypophysis and in pituitary adenomas in order to explore their potential use as diagnostic tools. All adenomas (4 prolactinomas, 3 growth hormone (GH) producing adenomas and 4 inactive adenomas) showed SYN and NSE immunoreactivity on tissue sections and this was confirmed by immunoblots. NCAM 140 (an isoform of NCAM with molecular mass 140 kDa) was detected by immunoblotting in normal human adenohypophysis, in all GH adenomas, and in three out of four inactive adenomas, but not in prolactinomas. Using highly sensitive techniques, NCAM immunoreactivity was observed by electron microscopy in all adenomas. These data indicate that NCAM 140 is a constituent of the cell surface of endocrine cells in both normal human adenohypophysis and its tumors. Since prolactinomas express very low levels of NCAM 140 compared to other hypophyseal tumors its virtual absence could be used for differential diagnosis. A combined analysis of NCAM, SYN and NSE could be useful to characterize inactive adenomas which are not immunoreactive for pituitary hormones and which may contain no or only low levels of the alpha chain of the glycoprotein hormones.
Monoclonal antibodies against the human lymphocyte differentiation antigen CD 76 bind to gangliosides
Authors: Kniep Bernhard , Mühlradt Peter F. , Dörken Bernd , Moldenhauer Gerhard , Vilella R. , Schwartz-Albiez Reinhard
Published: July 2002

Two monoclonal antibodies, HD 66 and CRIS‐4, by which the new CD 76 B‐cell‐associated cluster was defined, bound to several gangliosides (sialic acid containing glycolipids) of different polarity. One of the gangliosides recognized by HD 66 could be identified as NeuAcα2‐6Galβl‐4GlcNAcβl‐3Galβl‐4Glc‐βl‐l'Cer. This antigen was enzymatically synthesized. Sialidase treatment of the ganglioside antigens abolished binding of HD 66 and CRIS‐4.
Monoclonal antibodies against the presynaptic calcium channel antagonist ω‐conotoxin GVI A from cone snail poison
Authors: Tombaccini D. , Adeyemo O.M. , Pollard H.B. , Feuerstein G.
Published: July 2002

Monoclonal antibodies have been prepared against co‐conotoxin GVI A, a peptide isolated from marine snails of the genus Conus (Conus geographus and Conus magus). This toxin is a blocker of select presynaptic Ca2+ channels in the central nervous system. Antigenic ω‐conotoxin GVI A was synthesized as a covalent conjugate with bovine serum albumin and injected s.c. An ELISA assay combined with a competitive inhibition assay was used to select and characterize monoclonal antibodies able to recognize and bind the free toxin. Several of the antibodies were found to block ω‐conotoxin GVI A inhibition of 45Ca transport into rat brain synaptosomes and to block ω‐conotoxin GVI A binding to membranes from the same preparation. The antibodies recognize native, synthetic toxin, and are useful for analysis of toxin in biological fluids.
Retinola induced changes in cAMP‐dependent protein kinase activity detected by a new minigel assay
Authors: Hohmann Philip , Greene Robert S.
Published: July 2002

A new method for the resolution of protein kinases in nondenaturing minigels was used to analyze the type of PKA induced by retinyl acetate in transformed mouse 10T cells. Protein kinases in 0.5 μl aliquots of cell extracts were resolved on nondenaturing gradient Phastgels and assayed for PKA activity in situ using a specific peptide substrate and [32P]ATP. Retinyl acetate caused a decrease in type I and an increase in type II PKA isoforms after about 36 h in culture. The system provides a quick and sensitive means for analyzing the activity of PKA isoforms from small numbers of cells.
High‐efficiency expression of rat protein kinase C‐γ in baculovirus‐infected insect cells
Authors: Fiebich Bernd , Hug Hubert , Marmé Dieter
Published: July 2002

We have expressed rat protein kinase C‐γ in insect cells using a baculovirus vector. The yield of expressed protein kinase C‐γ is about 4% of total protein. The recombinant protein shows a prominent band at about 80 kDa on SDS‐polyacrylamide gels, which can be identified as protein kinase C‐γ by Western blotting using monoclonal antibodies against protein kinase C‐γ. Upon incubation with [γ‐32P]ATP and in the presence of Ca2+ phosphatidylserine and diacylglycerol this protein autophosphorylates. Its enzyme activity shows the characteristic properties of mammalian protein kinase C.
Bacteriorhodopsin in ice
Authors: Heberle Joachim , Dencher Norbert A.
Published: July 2002

The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and in ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H+‐release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice‐like structure.
BiP expression is not increased by the accumulation of PiZ α1‐antitrypsin in the endoplasmic reticulum
Authors: Cresteil Danièle , Ciccarelli Ernesto , Soni Théophile , Alonso Maria A. , Jacobs Paul , Bollen Alex , Alvarez Fernando
Published: July 2002

PiZ, a mutant human α1‐antitrypsin, is associated with liver and pulmonary disease and is characterized by defective secretion and accumulation of the protein in the endoplasmic reticulum. We tested the hypothesis that BiP (a protein that binds newly synthesized protein in the endoplasmic reticulum, prevents secretion of incorrectly folded protein, and solubilizes protein aggregates), could play a part in the retention of PiZ α1‐antitrypsin in the endoplasmic reticulum. Subcellular fractions from PiM (normal) and PiZ livers were prepared and analyzed by immunoblotting. No increase of BiP was detected in the PiZ liver. In addition, when total RNA from the same livers were analyzed by slot and Northern blot hybridization, no difference was found in the level of BiP mRNA between PiM and PiZ livers. Similar results were found in clones of CHO and MDCK cells transfected with PiM of PiZ α1‐antitrypsin cDNAs. These results indicate that BiP does not play a part in the retention of PiZ α1‐antitrypsin and suggest that PiZ protein is not misfolded.
The petFI gene encoding ferredoxin I is located close to the str operon on the cyanelle genome of Cyanophora paradoxa
Authors: Neumann-Spallart C. , Brandtner M. , Kraus M. , Jakowitsch J. , Bayer M.G. , Maier T.L. , Schenk H.E.A. , Löffelhardt W.
Published: July 2002

The petFI gene encoding ferredoxin I was localized in the large single copy region of cyanelle DNA by heterologous hybridization. Sequence analysis revealed an ORF of 99 amino acids (including the N‐tenninal processed methionine) at a position 477 bp from the 3' end of tufA but on the opposite strand. The 25 ammo‐terminal residues well corresponded to partial sequences obtained with purified cyanelle ferredoxin. The assignment of yet another gene that is not found on the genomes of chlorophyll β‐type plastids to cyanelle DNA again corroborates the special position of cyanelles serving as a model for plastid evolution from endocytobiotic cyanobacteria.
Processing of yeast exoglucanase (β‐glucosidase) in a KEX2‐dependent manner
Authors: Basco Ricardo D. , Giménez-Gallego Guillermo , Larriba Germán
Published: July 2002

We have detected proteolytic processing of a form of exoglucanase representative of the endoplasmic reticulum (form A). This processing did not take place when form A was obtained from protoplasts lysed in the presence of either EDTA or leupeptin, two wel‐characterized inhibitors of KEX2 endoprotease from Saccharomyces cerevisiae. Sequencing of the amino terminus of an A‐like form of enzyme secreted by a kex2 mutant indicated the presence of 4 amino acids, with a pair of basic residues (Lys‐Arg) at their carboxyl side, preceding the amino terminus of the wild‐type external exoglucanase.