bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat liver plasma membranes contain a lipid‐dependent protein kinase activity
Authors: Kiss Zoltán , Mhina Veronica
Published: July 2002

The treatment of rat liver plasma membranes with EDTA resulted in substantial release of a protein kinase activity. Using histones as external substrates both phosphatidylserine (PS) and diolein activated the enzyme. The activatory effect of PS but not that of diolein depended on Ca2+. In contrast, the enzyme activity was inhibited by both PS and diolein when the substrate was protamine. The inhibition by PS, unlike by diolein, depended on Ca2+. Data suggest that mobilization of plasma membrane‐bound Ca2+ by physiological stimuli may be accompanied by release of the lipid‐dependent protein kinase into the cytoplasm.
Islet‐activating protein prevents nicotinic acid‐induced GTPase stimulation and GTP but not GTPγS‐induced adenylate cyclase inhibition in rat adipocytes
Authors: Aktories Klaus , Schultz Günter , Jakobs Karl H.
Published: July 2002

The influence of islet‐activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTPγS, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid‐induced stimulation of a high affinity GTPase. The data suggest that a GTP‐hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.
‘Cap 90’, a 90‐kDa Ca2+‐dependent F‐actin‐capping protein from vertebrate brain
Authors: Isenberg G. , Ohnheiser R. , Maruta H.
Published: July 2002

A Ca2+‐dependent actin filament‐capping protein of 90 kDa was purified from bovine brain using a new and rapid isolation procedure. This basically includes affinity purification on DNase‐I agarose. The protein caps the fast‐growing end of actin filaments but has no fragmenting or severing activity. Using Triton X‐100‐extracted cytoskeletons, capping and severing activities of actin‐binding proteins become clearly distinguishable from each other.
The effect of S‐100a and S‐100b proteins and Zn2+ on the assembly of brain microtubule proteins in vitro
Authors: Deinum Johanna , Baudier Jacques , Briving Carin , Rosengren Lars , Wallin Margareta , Gerard Dominique , Haglid Kenneth
Published: July 2002

The homologous proteins S‐100a and S‐100b affect the microtubule system in a distinctly different way in the presence of low molar ratios of Zn2+. Assembly of brain microtubule proteins can be almost completely inhibited and rapid disassembly can be induced by low molar amounts of S‐100b in the presence of low molar ratios [2–4] of Zn2+. Higher molar ratios per S‐100b (>4) potentiate the general Zn2+ effect, promoting the formation of sheets of microtubules. However, the effect of S‐100a is quite different, no inhibition of assembly can be observed and the presence of S‐100a seems to protect the microtubule proteins against the effect of Zn2+ by chelating the Zn2+ and decreasing the free metal‐ion concentration. S‐100a or S‐100b cannot bind to the microtubule polymer‐form, either in the absence or in the presence of Zn2+.
Effect of elongation factor Tu on the conformation of phenylalanyl‐tRNAPhe
Authors: Riehl N. , Giegé R. , Ebel J.P. , Ehresmann B.
Published: July 2002

Structural features of the tRNAPhe molecule upon ternary complex formation with the bacterial elongation factor Tu were investigates. Phosphodiester bonds at positions 18 and 34 were found to be labilized in bound tRNA. Conversely, a higher stability of the phosphodiester links at positions 20, 21 and 36 was detected. Using ethylnitrosourea as a chemical probe a conformational change occurring at phosphate position 53 was observed in complexed tRNA. These results are interpreted by a structural rearrangement of the nucleic acid induced by complex formation.
The regulation of the proton conductance of brown fat mitochondria
Authors: Rial E. , Nicholls D.G.
Published: July 2002

The binding of purine nucleotides to intact brown fat mitochondria is re‐examined. In addition to the previously reported high affinity binding site, a low‐affinity site is found, which requires several minutes to saturate. Only the high affinity site is functional in regulating the proton and halide permeabilities of the mitochondria. The low affinity site can introduce errors in the use of nucleotide binding to quantitate the M
r 32 000 uncoupling protein unique to these mitochondria.
High‐resolution triplet‐minus‐singlet absorbance difference spectrum of photosystem II particles
Authors: den Blanken H.J. , Hoff A.J. , Jongenelis A.P.J.M. , Diner B.A.
Published: July 2002

The triplet state of the primary donor of photosystem II particles prepared from a mutant of Chlamydomonas reinhardtii has been studied at 1.2 K with absorbance‐detected ESR in zero‐magnetic field (ADMR). Two sets of resonances with slightly different zero‐field splitting parameters |D| and |E| were observed, |D| = 285.5, |E| = 38.8 and |D| = 288.8 × 10−4 cm−1, |E| = 42.2 × 10−4 cm−1, respectively. Both sets of |D| and |E| values are close to those found for PT‐700, as are the sublevel decay rates k

x
= 930 ± 40, k

y
= 1088 ± 50 and k

z
= 110 ± 5 s−1. The AMDR‐detected triplet‐minus‐singlet absorbance difference spectrum of PT‐680 is very similar to that of PT‐700 and closely resembles that of covalently connected Chl a dimers in vitro. We conclude that P‐680 is a Chl a dimer whose general structure is similar to that of P‐700.
On the mechanism of tRNATrp aminoacylation catalysed by beef tryptophanyl‐tRNA synthetase using presteady‐state kinetics
Authors: Trezeguet Véronique , Merle Michel , Gandar J.C. , Labouesse Bernard
Published: July 2002

The dimeric tryptophanyl‐tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme [Eur. J. Biochem. (1982) 128, 389–398]. By using quenched‐flow and stopped‐flow methods under presteady‐state conditions, we show that only one enzyme subunit operates at a time in the aminoacylation of tRNATrp and that the transfer reaction is not the rate‐limiting step in the overall aminoacylation process.
Extended superfamily of short alcohol‐polyol‐sugar dehydrogenases: structural similarities between glucose and ribitol dehydrogenases
Authors: Jörnvall Hans , von Bahr-Lindström Hedvig , Jany Klaus-Dieter , Ulmer Wolfgang , Fröschle Marion
Published: July 2002

The recently determined primary structure of glucose dehydrogenase from Bacillus megaterium was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty‐one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc‐containing alcohol dehydrogenases, 25% sorbitol and zinc‐containing alcohol dehydrogenases, 25%; ribitol and non‐zinc‐containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprising short and long alcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non‐zinc‐containing alcohol‐polyol‐glucose dehydrogenases.
Monoclonal antibodies block the trypsin cleavage site on human placental alkaline phosphatase
Authors: Jemmerson Ronald , Stigbrand Torgny
Published: July 2002

Monoclonal antibody
Trypsin cleavage
Restricted proteolysis
Placental alkaline phosphatase
Blocking antibody
A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase
Authors: Sicsic Sames , Durand Philippe , Langrene Sylvie , le Goffic François
Published: July 2002

The use of enzymes requiring a cofactor as substrate in organic synthesis is still a problem since the cofactors are expensive. This study deals with a new approach consisting of using fragments of NAD+. Three fragments of NAD(H) are examined. The activities of NMN+ and NMNH are greatly improved by the addition of adenosine in ethanol oxidation and in cyclohexanone reduction, respectively. Nicotinamide monocucleoside is not active in the ethanol oxidation but the addition of AMP promotes this reaction.
Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221
Authors: Shaw William V. , Brenner Daniel G. , LeGrice Stuart F.J. , Skinner Sarah E. , Hawkins Alastair R.
Published: July 2002

The nucleotide sequence of the inducible chloramphenicol acetyltransferase gene (cat) of Staphylococcus aureus plasmid pC221 has been determined. The deduced primary structure for the 215 residue polypeptide (25.9 kDa) is in agreement with partial amino acid sequence data on the purified protein, previously designated as the type C variant of CAT. In common with the inducible cat elements of pC194 and B. pumilus, the 5' non‐coding region of the cat of pC221 contains an inverted complementary repeat (‘stem‐loop’ or ‘hairpin’) which may sequester the predicted ribosome bonding site of the mRNA. The likely transcription initiation site has been determined in vitro using purified B. subtilis RNA polymerase. Recombinant plasmids carrying the cat of pC221 on a 1156 bp TaqI fragment are expressed inefficiently in Escherichia coli, wherein induction is both poor and orientation‐specific.
Isolation and partial characterization of distinct forms of tyrosine protein kinases from rat spleen
Authors: Brunati Anna Maria , Marchiori Fernando , Pinna Lorenzo A.
Published: July 2002

Three peaks of tyrosine protein kinase activity (TK‐I, TK‐II and TK‐III) can be resolved when the extract of rat spleen particulate fraction is subjected to DEAE‐cellulose gradient chromatography. TK‐I and TK‐II, insensitive to both EGF and insulin, have been further purified by Sephacryl S200 gel filtration and characterized. TK‐I has an apparent m

r
of 65000, by far prefers Mn2+ over Mg2+ as activator, can use GTP besides ATP as phosphate donor and is stimulated 2‐3‐fold by polylysine. TK‐II, whose m

r
approximates 50000, is equally activated by Mg2+ and Mn2+, does not use GTP and is insensitive to polylysine. TK‐I and TK‐II can phosphorylate the synthetic peptide Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly (as well as its derivative with Orn in place of Arg), angiotensin II and poly(Glu, Tyr) 4:1 which exhibits different k
m, values with TK‐I and TK‐II, (100 and 10 μM, respectively). When TK‐I was incubated with [γ‐32P]ATP and MnCl2 a doublet of alkali‐stable radiolabeled bands with molecular masses of 55 and 60 kDa were observed. Under identical conditions TK‐II gives rise to a single alkali‐stable radiolabeled band of 51 kDa, which may represent the autophosphorylation product of TK‐II itself.
Probing the three‐dimensional structures of DNA and RNA oligonucleotides in solution by nuclear Overhauser enhancement measurements
Authors: Clore G.Marius , Gronenborn Angela M.
Published: July 2002

DNA and RNA oligonucleotides are ideally suited for high‐resolution X‐ray crystallographic and 1H‐NMR studies. The solution structures of such oligonucleotides can potentially be solved using proton‐proton nuclear Overhauser enhancement measurements to demonstrate the proximity of protons in space and to determine their separation, thereby enabling a comparison of the structure in the crystalline and solution states to be made. In this review we describe (i) the general strategy for the sequential resonance assignments of oligonucleotide 1H‐NMR spectra, the essential prerequisite for further structural work, (ii) the approach to obtaining interproton distances from pre‐steady state nuclear Overhauser enhancement measurements, and (iii) the use of interproton distances in structure determination. This is illustrated by several examples including double‐ and single‐stranded DNA oligonucleotides as well as RNA stem and loop structures.
In vitro reconstitution of messenger ribonucleoprotein particles from globin messenger RNA and cytosol proteins
Authors: Gaedigk R. , Oehler S. , Köhler K. , Setyono B.
Published: July 2002

Deproteinized globin poly(A) + mRNAs reassociate readily in vitro with soluble RNA‐binding proteins of the cytosol; reconstituted messenger ribonucleoprotein complexes are obtained which are very similar to native globin polyribosomal‐mRNP as far as bouyant density in Cs2SO4 and the composition of proteins which can be crosslinked to the mRNA are concerned. Proteins thus identified bind specifically to mRNA and not to ribosomal RNA or any synthetic oligonucleotides, with one exception: a 78‐kDa protein could be cross‐linked to poly(A).
An antisuppressor mutant of Saccharomyces cerevisiae deficient in isopentenylated tRNA has reduced Δ2‐isopentenylpyrophosphate: tRNA‐Δ2‐isopentenyl transferase activity
Authors: Laten Howard M. , Timmons Ruth M. , Suid Sopiah
Published: July 2002

We have previously reported the isolation and initial characterization of a mutation in Saccharomyces cerevisiae, designated mod5‐1, that reduces the capacity of altered tyrosine tRNAs to suppress ochre nonsense mutations. The mutation results in the virtual elimination of the modified tRNA nucleoside, N‐Δ2‐(isopentenyl) adenosine, normally found adjacent to the anticodons of certain tRNA species. We demonstrate here that MOD5 codes for Δ2‐isopentenylpyrophosphate: tRNA‐Δ2‐isopentenyl transferase, or a protein that regulates its synthesis.
The primary structure of transcription factor TFIIIA has 12 consecutive repeats
Authors: Brown Raymond S. , Sander Christian , Argos Patrick
Published: July 2002

Analysis of the amino acid sequence of transcription factor TFIIIA from Xenopuslaevis reveals the presence of 12 repeating structures, each about 30 residues in length. These segments have been aligned and their secondary structure predicted. The repeats each contain two invariant cysteines and two invariant histidines, perhaps to coordinate a zinc cation. Possible nucleic acid interaction modes are discussed.
On the physico‐chemical rationale of the genetic code
Authors: Rèmme Jaanus , Villems Richard
Published: July 2002

We analysed two different and seemingly independent aspects of protein biosynthesis: the primary structure of codons and the reactivity of aminoacyl groups. This analysis revealed that more reactive aminoacyl groups correspond to less stable codon‐anticodon complexes. The possible meaning of such a correlation is discussed in terms of the kinetic proofreading theory.
Calcium‐dependent polyphosphoinositide hydrolysis is associated with exocytosis in vitro
Authors: Whitaker Michael , Aitchison Michael
Published: July 2002

Calcium
Exocytosis
Phosphoinositide
Membrane fusion
Phospholipase C
Egg
Complete characterization of the human IGF‐I nucleotide sequence isolated from a newly constructed adult liver cDNA library
Authors: Le Bouc Y. , Dreyer D. , Jaeger F. , Binoux M. , Sondermeyer P.
Published: July 2002

A full‐size cDNA sequence coding for insulin‐like growth factor I (IGF‐I) was isolated from a human liver library. For the construction of this bank, a new method was developed which anneals dG‐tailed cDNA with a synthetic adaptor 5'‐AATTCCCCCCCCCCC‐3' followed by ligation into the EcoRI site of a lambda immunity‐insertion vector. Based on the sequence analysis of the complete IGF‐I messenger we concluded that the protein is synthesized as a precursor containing a signal peptide of 22 or 25 amino acid residues. In addition, the sequence extended in the 3'‐direction and showed the presence of multiple polyadenylation sites in the IGF‐I message.
Full‐length sequence and expression of the 42 kDa 2–5A synthetase induced by human interferon
Authors: Wathelet M. , Moutschen S. , Cravador A. , DeWit L. , Defilippi P. , Huez G. , Content J.
Published: July 2002

Interferon‐induced 2–5A synthetases are probably involved in some antiviral actions of interferon. In human cells two different mRNAs (1.6, 1.8 kb long) coding for this protein are transcribed from the same gene and are produced by differential splicing. The relationship between the two mRNAs of different size and the active enzyme is not clear, nor is the cellular localization of the latter known. We have cloned a cDNA corresponding to the 1.6 kb RNA. This cDNA was sequenced and its complete coding region was subcloned into pSP64. The resulting plasmid was used to direct the synthesis of micrograms of capped RNA transcript after linearization in the 3'‐non‐coding region. A 39 kDa protein was synthesized when this RNA was translated in rabbit reticulocyte lysate. When this capped RNA was introduced by microinjection into Xenopus oocytes, production of 2–5A synthetase was clearly observed in the cytoplasm and 10–30% of the enzyme accumulated with time in the nucleoplasm. Analysis of cytoplasmic homogenates of these oocytes on a glycerol gradient revealed that the enzyme is fully active in the monomeric form.
Energy‐linked cardiac transport system for glutathione disulfide
Authors: Ishikawa Toshihisa , Zimmer Maria , Sies Helmut
Published: July 2002

The relationship between the rate of glutathione disulfide (GSSG) export and the energy state was studied in isolated perfused rat heart. The intracellular GSSG level was maintained at saturation for transport (7.5 nmol GSSG · min−1 · g heart−1) by continuous perfusion with 20 μM t‐butyl hydroperoxide. GSSG release was substantially restricted upon the addition of inhibitors of mitochondrial respiration such as KCN, antimycin A or rotenone. In contrast, no effect was observed on GSSG release during potassium‐induced cardiac arrest, although changes in oxygen consumption and coronary flow were similar to those observed with KCN. The dependence of the GSSG transport rate on the cytosolic free ATP/ADP ratio reveals that GSSG transport is half‐maximal at (ATP/ADP)free≈ 10. The capacity of GSSG transport was unchanged by infusion of epinephrine, norepinephrine or dibutyryl cyclic AMP.
On the sequence homology of the ribosomal proteins, Escherichia coli S11, yeast rp59 and Chinese hamster S14
Authors: Tanaka Tatsuo , Ishikawa Kiichi , Ogata Kikuo
Published: July 2002

A strong sequence homology was found among the ribosomal proteins of the different species, S11 of Escherichia coli, rp59 of Saccharomyces cerevisiae and S14 of Chinese hamster ovary cell. The significance of this series of highly conserved proteins is discussed.
Protein kinase C activators modulate differentiated thyroid function in vitro
Authors: Ginsberg Jody , Murray Patricia G.
Published: July 2002

Exposure of porcine thyroid cells to the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) leads to inhibition of differentiated thyroid function. We investigated whether this effect is mediated via protein kinase C activation. TPA, phorbol 12,13‐didecanoate and phorbol 12,13‐dibutyrate inhibited TSH‐stimulated iodine organification in porcine thyroid cells by 98, 96 and 45%, respectively. Non‐tumour promoting phorbol esters had no effect. The diacylglycerol analogue, sn‐1,2‐dioctanoylglycerol had similar but quantitatively less activity than TPA. Dibutyryl cAMP could not reverse any inhibition noted. Under conditions that caused significant inhibition of differentiated function, TPA caused translocation of thyroidal protein kinase C from the cytosol to its membrane‐bound form. These data provide evidence that the mechanism of phorbol action on thyroid function in vitro includes activation of protein kinase C.
Fluorescence labeling of an aminoacyl‐tRNA at the 3'‐end and its interaction with elongation factor Tu·GTP
Authors: Joshi R.L. , Faulhammer H.G. , Haenni A.L. , Sprinzl M.
Published: July 2002

A new approach for the fluorescence labeling of an aminoacyl‐tRNA at the 3'‐end is applied to study its interaction with bacterial elongation factor Tu (EF‐Tu) and GTP at equilibrium. The penultimate cytidine residue in yeast tRNATyr‐C‐C‐A was replaced by 2‐thiocytidine (s2C). The resulting tRNATyr‐C‐s2C‐A was aminoacylated and then alkylated at the s2C residue with N‐(iodoacetylaminoethyl)‐5‐naphthylamine‐1‐sulfonic acid (1,5‐I‐AEDANS). A > 100% increase in the intensity of fluorescence emission of the modified Tyr‐tRNATyr‐C‐s2C(AEDANS)‐A was observed upon interaction with EF‐Tu · GTP. A ternary complex dissociation constant of 1.27 × 10−8 M was calculated from this direct interaction. Using such fluorescent aminoacyl‐tRNA the affinity of any unmodified aminoacyl‐tRNA can be determined by competition experiments. By this approach we show here that the affinity of unmodified Tyr‐tRNATyr‐C‐C‐A is identical to that of the modified Tyr‐tRNATyr. This indicates that the fluorescence labeling procedure applied does not alter the affinity of the aminoacyl‐tRNA for EF‐Tu·GTP. The introduction of 2‐thiocytidine into nucleic acids and their labeling with spectroscopic reporter groups may provide a unique means of investigating various types of nucleic acid‐protein interactions.
Identification of Chl‐binding proteins in a PS II preparation from spinach
Authors: Irrgang Klaus-Dieter , Renger Gernot , Vater Joachim
Published: July 2002

Using a mild solubilization procedure we have been able to detect several Chl‐binding proteins by SDS‐urea‐PAGE. Their molecular masses were 46, 42, 34, 32, 29‐24 and approx. 110 kDa (presumably a CPI contamination). The stability of the Chl‐protein complexes depended strongly on experimental conditions. A very faint band at 34‐30 kDa was resolved in visible light (in 40–50% of the experiments). With a more sensitive fluorometric technique (illumination at 366 nm, fluorescence detection in the red) we identified two Chlcontaining bands in the 30 kDa region. Limited proteolysis studies with trypsin revealed that the extent of the enzymatic degradation of some Chl‐binding proteins in vitro is obviously dependent on pH and the presence of Ca2+.
Human protein S cDNA encodes Phe‐16 and Tyr 222 in consensus sequences for the post‐translational processing
Authors: van Amstel Hans K.Ploos , van der Zanden A.Linda , Reitsma Pieter H. , Bertina Rogier M.
Published: July 2002

Partial cDNAs coding for human protein S were isolated from a pUC9 human liver cDNA library. Together, the overlapping clones span a (partial) 5′‐non‐coding region, and the complete protein S coding and 3′‐untranslated regions. The derived amino acid sequence deviates at five positions from two previously reported protein S sequences. Two of these differences (Phe instead of Leu at position — 16 and Tyr instead of Asp at position 222) are found in regions that are important for the post‐translational modification of protein S, the γ‐carboxylation of glutamic acid and the hydroxylation of asparagine, respectively.
Large unilamellar liposomes with low uptake into the reticuloendothelial system
Authors: Allen T.M. , Chonn A.
Published: July 2002

Particulate drug carriers, including liposomes, are rapidly removed from blood by cells of the reticuloendothelial system (RES) with resulting adverse effects on this important host defense system. In order to overcome this and other major disadvantages of liposomes, we have altered liposome composition in an effort to achieve prolonged circulation half‐lives. Gangliosides and sphingomyelin act synergistically to dramatically diminish the rate and extent of uptake of liposomes by macrophages in vivo. The significantly extended circulation times achieved by these modified large unilamellar liposomes overcome an important barrier to the targeting of particulate drug carriers to specific tissues in vivo.
In the uncoupling protein from brown adipose tissue the C‐terminus protrudes to the c‐side of the membrane as shown by tryptic cleavage
Authors: Eckerskorn Christoph , Klingenberg Martin
Published: July 2002

Limited proteolytic digestion of the uncoupling protein (UCP) with trypsin yielded a cleavage product only about 2 kDa smaller than the original UCP (33 kDa). This cleavage can be obtained with the solubilized isolated protein detergent micelle as well as in original brown adipose mitochondria. The cleavage site is identified by C‐terminal sequence to be located near the C‐terminus at lysine 292. This C‐terminus, a 10 residue long peptide, is strongly hydrophilic and can be expected to be localized outside the membrane. In UCP this C‐terminal stretch represents a structural difference to the similarly folded ADP/ATP carrier which does not form a corresponding cleavage product. Comparison of tryptic cleavage of UCP in mitochondria with differently broken outer membrane, in sonic particles of mitochondria, as well as in UCP proteoliposomes, indicate that the C‐terminus is directed versus the cytosolic site of the membrane. Because of the easy susceptibility to trypsin, the cleavage site must be surface‐exposed and the C‐terminal section unusually mobile.
The human genome encodes at least three non‐allellic G proteins with αi‐type subunits
Authors: Suki Wadi N. , Abramowitz Joel , Mattera Rafael , Codina Juan , Birnbaumer Lutz
Published: July 2002

The amino acid sequence and composition of α‐subunits of signal transducing G proteins of the same kind appear to vary by no more than 2% from species to species. Here we isolated a human liver cDNA using an oligonucleotide complementary to the sequences encoding the pertussis toxin (PTX) ADP‐ribosylation site of the α‐subunit of the rat brain G protein called Gi. Its open reading frame characterizes it as an αi‐type cDNA—as opposed to αo‐type—but predicts an amino acid composition that differs by 7% and 14%, respectively, from two other human αi‐type molecules. Together with human brain αi (type‐1) and human monocyte αi (type‐2), the new human liver αi cDNA (type‐3) forms part of a family of αi molecules. Type‐3 αi cDNA hybridizes to a ∼ 3.6 kilobase long mRNA and type‐2 αi cDNA hybridizes to an mRNA species of ∼ 2.7 kilobases. This indicates that the human genome has at least three non‐allellic genes encoding non‐αo‐type PTX substrates and provides structural evidence for the hypothesis that distinct effector systems are regulated by similar but nevertheless distinct PTX substrates.
Increase of urate formation by stimulation of sympathetic hepatic nerves, circulating noradrenaline and glucagon in the perfused rat liver
Authors: Püschel Gerhard P. , Nath Annegret , Jungermann Kurt
Published: July 2002

In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the α1‐blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied.
Role of tryptophan 54 in the binding of E. coli single‐stranded DNA‐binding protein to single‐stranded polynucleotides
Authors: Khamis Mustafa I. , Casas-Finet Jose R. , Maki August H. , Murphy Janet B. , Chase John W.
Published: July 2002

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single‐stranded polynucleotides with both E. coli single‐stranded DNA‐binding protein and an E. coli ssb gene product in which Trp‐54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp‐54 in stabilizing the protein‐nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.
Expression of the T4 molecule (AIDS virus receptor) by human brain‐derived cells
Authors: Dewhurst Stephen , Stevenson Mario , Volsky David J.
Published: July 2002

Three human cell lines of astrocytic origin were evaluated for expression of a human T‐lymphocyte surface glycoprotein, T4, which also serves as a cellular receptor for the human immunodeficiency virus (AIDS virus, HIV). T4 antigen was detected on the cell surface of 2 of these cell lines using monoclonal OKT‐4 antibody and flow cytometry. Gene transcripts encoding the T4 molecule were detected by a ribonuclease protection assay in surface T4‐positive and ‐negative cells. Our results suggest that astrocytes may serve as targets for HIV infection in the brain.
1H resonances of proximal histidine in CO complexes of hemoglobins provide a sensitive probe of coordination geometry
Authors: Dalvit Claudio , Tennant Linda , Wright Peter E.
Published: July 2002

A straightforward strategy for assignment of the CϵH, CδH and NδH proton resonances of the proximal histidine ligand in diamagnetic complexes of monomeric hemoglobins and myoglobins is reported. These resonances are subject to large ring current shifts and are highly sensitive to coordination geometry. There are no significant differences between the CO complexes of myoglobin, leghemoglobin or hemoglobin α‐subunits in proximal His coordination geometry or hydrogen bonding to the backbone at Leu F4. Ring current calculations show that the His F8 coordination geometry in the CO complexes of myoglobin and hemoglobin α‐subunits is very similar in crystal and solution.
Primary and secondary structure of the 18 S ribosomal RNA of the insect species Tenebrio molitor
Authors: Hendriks Lydia , De Baere Raymond , Van Broeckhoven Christine , De Wachter Rupert
Published: July 2002

The sequence of the 18 S rRNA of Tenebrio molitor is reported. A detailed secondary structure model for eukaryotic small subunit rRNAs is proposed. The model comprises 48 universal helices that eukaryotic and prokaryotic small subunit rRNAs have in common, plus a number of helices in areas of variable secondary structure. For the central area of the model, an alternative structure is possible, applicable only to eukaryotic small subunit rRNAs. Possibly, small subunit rRNA switched to this alternative conformation after the eukaryotic branch had been established in evolution. Another possibility is that the two conformers represent a dynamic structural switch functioning during the translational activity of the eukaryotic ribosome.
Escherichia coli hemolysin permeabilizes small unilamellar vesicles loaded with calcein by a single‐hit mechanism
Authors: Menestrina Gianfranco
Published: July 2002

Escherichia coli hemolysin produces small unilamellar lipid vesicles permeable to the fluorescent dye calcein by forming pores through their membrane. The process of permeabilization proceeds as a pseudo first‐order reaction, indicating that the toxin is active as a monomer; consistently no evidence for cooperativity has been found in a dose‐response titration. The rate of interaction increases on lowering the pH of the solution and by introducing negatively charged lipids into the vesicles. The overall pore formation mechanism resembles that of other toxins of bacterial origin such as colicins, diphtheria, tetanus and botulinum toxin.
Automated Sanger dideoxy sequencing reaction protocol
Authors: Zimmermann J. , Voss H. , Schwager C. , Stegemann J. , Ansorge W.
Published: July 2002

The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps. An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions. The system is set up in a short time for routine operation and it is faster and more reliable than a human operator. It is flexible and allows variations and optimization of the standard procedure. Disposable microtiter plates at a controlled temperature are used. In one reaction cycle (about 50 min) up to 48 templates are processed. Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot. The protocol is applicable to fluorescent as well as to radioactive labeling.
Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA
Authors: Sarih Leila , Araya Alejandro , Litvak Simon
Published: July 2002

Bovine tRNATrp can be partially hybridized to the avian myeloblastosis virus (AMV) 35 S RNA at 37°C, in the presence of AMV RNA‐dependent DNA polymerase (reverse transcriptase). This template‐primer complex is active in the synthesis of viral cDNA. The size of the cDNA products synthesized in the in vitro reconstituted AMV system was determined by urea‐polyacrylamide gel electrophoresis using a tRNA labelled at the 3′‐end by yeast tRNA nucleotidyl transferase. The synthesized cDNA has a size of about 100 nucleotides and was shown by Southern blotting to be complementary to a specific sequence of the 5′‐end of the retroviral genome. These results indicate that reverse transcriptase is able to anneal the exogenous primer tRNA at the ‘primer‐binding site’ near the 5′‐end of the long terminal repeat (LTR) of AMV RNA.
Pyruvate carboxylase from Pseudomonas citronellolis: Shape of the enzyme, and localization of its prosthetic biotin group by electron microscopic affinity labeling
Authors: Fuchs Jutta , Johannssen Walther , Rohde Manfred , Mayer Frank
Published: July 2002

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with α4β4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per αβ unit, i.e. four per enzyme particle) are located close to the inter‐unit junctions of pairs of αβ units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.
Studies on the biosynthesis of surfactin, a lipopeptide antibiotic from Bacillus subtilis ATCC 21332
Authors: Kluge Britta , Vater Joachim , Salnikow Johann , Eckart Klaus
Published: July 2002

The biosynthesis of the lipopeptide antibiotic surfactin was studied in whole cells of Bacillus subtilis ATCC 21332 which incorporate 14C‐labeled precursor amino acids directly into the product. [14C]Acetate appeared in the fatty acid portion of surfactin and was also partially converted into leucine. An enzyme was isolated and partially purified from a cell‐free extract of the bacillus which catalyzes ATP‐Pi‐exchange reactions which are mediated by the amino acid components of surfactin. This activation pattern is consistent with a peptide synthesizing multienzyme which activates its substrate amino acids simultaneously as reactive aminoacyl phosphates.
Phosphorylation of the 90 kDa heat shock protein in heat shocked HeLa cell lysates
Authors: Legagneux Vincent , Dubois Marie-Françoise , Morange Michel , Bensaude Olivier
Published: July 2002

The 90 kDa heat shock protein (hsp 90) is a major phosphorylated protein under normal growth conditions. However, it does not incorporate detectable levels of phosphate by incubation of control HeLa cell lysates with [γ‐32P]ATP in vitro. In this paper we show that strong phosphorylation of hsp 90 occurs in lysates prepared from heat shocked HeLa cells. Possible involvement of the eukaryotic initiation factor 2 kinase of the heme‐controlled repressor of translation is discussed.
Complete cDNA encoding a putative phospholipase C from transformed human lymphocytes
Authors: Ohta Shigeo , Matsui Akira , Nazawa Yoshinori , Kagawa Yasuo
Published: July 2002

Phosphoinositide‐specific phospholipase C (PLC) is a crucial enzyme in transmembrane signaling. A cDNA encoding the putative phospholipase C was cloned from human lymphocytes that were transformed by infection with Epstein‐Barr virus. The deduced amino acid sequence of the cDNA accounted for 146.1 kDa of the molecular mass of the complete enzyme and showed 50.2% sequence similarity to bovine brain PLC. This cDNA contained regions, the sequences of which were similar to those of some tyrosine kinase‐related oncogene products. Northern blotting demonstrated that the mRNA for this PLC is expressed in human promyelocytic leukemia cells (HL‐60). Since the other cloned cDNAs for PLCs could not hybridize with the RNA from this cell, it is strongly suggested that the gene obtained here encodes an additional isozyme of PLC in blood cells.
Interleukin 1 and tumour necrosis factor increase phosphorylation of fibroblast proteins
Authors: Kaur P. , Saklatvala J.
Published: July 2002

Interleukin 1 (IL1) or tumour necrosis factor (TNF) stimulated phosphorylation of a triad of 27 kDa phosphoproteins (pI 6.0, 5.7 and 5.5) in human dermal fibroblasts. The change was dependent on the dose of cytokine in the range 0.1–20 ng, was detectable between 3 and 5 min after stimulation and was maximal by 10 min. The proteins were found in the cytosol after subcellular fractionation. The 32P was removed from them by alkali, indicating the presence of phosphoserine and/or phosphothreonine. The results suggest that early changes in serine/threonine protein kinase activity may be involved in responses of fibroblasts to IL1 and TNF.
A novel crystallin from octopus lens
Authors: Chiou S.-H.
Published: July 2002

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS‐gel electrophoresis. Octopod and decapod lenses share one common major squid‐type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly‐characterized crystallin (termed Ω‐crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N‐terminal sequence analysis indicated that the Ω‐crystallin is N‐terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S‐transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.
Mitochondrial targeting sequences why ‘non‐amphiphilic’ peptides may still be amphiphilic
Authors: Gavel Ylva , Nilsson Lennart , von Heijne Gunnar
Published: July 2002

The notion that mitochondrial targeting peptides form amphiphilic α‐helices with one apolar and one polar, positively charged face is controversial, since some experimental results seem to imply that non‐amphiphilic targeting peptides can also function as import signals. However, the standard methods used to assess the amphiphilicity of a peptide may be misleading, since they do not take the flexibility of the amino acid side chains into account. To demonstrate this, we have developed a new method for calculating the amphiphilicity of helical peptides.
A new possible binding site for bacteriochlorophyll b in a light‐harvesting polypeptide of the bacterium Ectothiorhodospira halochloris
Authors: Wagner-Huber Regula , Brunisholz René A. , Bissig Iwan , Frank Gerhard , Zuber Herbert
Published: July 2002

Whole cells from Ectothiorhodospira halochloris were extracted with an organic solvent mixture. At least five small hydrophobic polypeptides representing most probably the light harvesting polypeptides were purified by gel filtration and consecutive FPLC‐RP chromatography. The complete amino acid sequence of a 7.4 kDa polypeptide was determined. The polypeptide shows a three domain structure, indicative of an integral membrane protein, similar to the structure of the light‐harvesting polypeptides from purple non‐sulfur bacteria. Sequence homologies to the β‐LHPs of purple bacteria range from 23. 1° to 36.4°. The conserved intramembrane located histidine residue of the antenna polypeptides of purple non‐sulfur bacteria, assigned as the possible binding site for bacteriochlorophyll, was found to be replaced by asparagine.
Activation of chloride channels in the plasmalemma of Nitella syncarpa by inositol 1,4,5‐trisphosphate
Authors: Zherelova O.M.
Published: July 2002

The introduction of 1.5 × 10−8 − 10−6 M inositol 1,4,5‐trisphosphate (IP3) into the bathing solution of Nitella syncarpa cells induced: (i) an increase in amplitude of the inward Ca2+ current; (ii) the appearance of an inward Cl− current; (iii) a shift in the threshold for activation of the Cl− channels on hyperpolarization of the membrane to more positive values. These facts suggest that IP3 participates in the causing of changes in the intracellular free Ca2+ concentration and, hence, in the regulation of ionic channels in Nitella syncarpa plasmalemma.
Polarographic observation of substrate‐level phosphorylation and its stimulation by acetylcholine
Authors: Kondrashova M.N. , Doliba N.M.
Published: July 2002

Substrate‐level phosphorylation was observed under the conditions optimal for this process and opposite to those for oxidative phosphorylation. Polarographic registration of Ca2+ stimulated α‐ketoglutarate oxidation and self‐inhibition of uncoupled α‐ketoglutarate (KG) oxidation was used. Acetylcholine (ACh) administration stimulated KG oxidation and substrate‐level phosphorylation in isolated mitochondria. These effects are stronger in tissues with a higher level of endogenous acetylcholine, such as guinea pig liver vs rat liver and pancreas vs liver. The specific stimulation of KG oxidation by ACh is related to a decrease of succinate oxidation and is contrary to the specific stimulating effect of adrenaline on succinate oxidation. Therefore the existence of reciprocal hormone‐substrate‐nucleotide systems is suggested. The described set of conditions optimal for substrate‐level phosphorylation observation by polarographic registration of respiration is as convenient as the ADP test for the investigation of oxidative phosphorylation.
Hypothyroidism in rats decreases mitochondrial inner membrane cation permeability
Authors: Hafner Roderick P. , Leake Michael J. , Brand Martin D.
Published: July 2002

We investigated the cation permeability of liver mitochondria isolated from hypothyroid or euthyroid rats by measuring the rate of swelling of respiring mitochondria in acetate salts as a function of membrane potential. Mitochondria from hypothyroid rats have a decreased permeability of roughly 3‐fold in the presence of monovalent cations K and tetramethylammonium at any (measured) membrane potential. Since the monovalent cation leak and the proton leak are known to respond similarly to membrane potential our results support the theory that the difference in non‐phosphorylating respiration rate between mitochondria from hypothyroid and euthyroid rats is due to a difference in proton leak.
Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus
Authors: Mollier Pascale , Chwetzoff Serge , Frachon Paule , Ménez André
Published: July 2002

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A2 (PLA2) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti‐notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA2 or PLA2‐containing venoms from other origins. Substitutions or chemical modifications occurring in the C‐terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.
NMR studies of lantibiotics Assignment of the 1H‐NMR spectrum of nisin and identification of interresidual contacts
Authors: Slijper M. , Hilbers C.W. , Konings R.N.H. , van de Ven F.J.M.
Published: July 2002

Nisin is a 34 residue long antimicrobial polypeptide, which contains unusual α,β‐unsaturated amino acids as well as lanthionines. By making use of 2D‐NMR methods, the complete 1H‐NMR spectrum of the polypeptide could be assigned. The NMR data indicate that the molecule adopts a well‐defined three‐dimensional structure.
An infrared spectroscopic study of β‐galactosidase structure in aqueous solutions
Authors: Arrondo J.L.R. , Muga A. , Castresana J. , Bernabeu C. , Goñi F.M.
Published: July 2002

Fourier‐transform infrared spectroscopy has been used to elucidate the secondary structure of E. coli β‐galactosidase in aqueous solution. The structure of this enzyme was previously unknown above the level of the amino acid sequence. Spectra have been recorded in both H2O and D2O media; mutually complementing data are obtained, that provide unambiguous structural information. The results show that β‐galactosidase contains 40% β‐sheet and 35% α‐helical structure, with smaller proportions of random coil (12%) and β‐turns (13%).
Formylmethanofuran dehydrogenase from methanogenic bacteria, a molybdoenzyme
Authors: Karrasch M. , Börner G. , Enßle M. , Thauer R.K.
Published: July 2002

Formylmethanofuran dehydrogenase, a key enzyme of methanogenesis, was purified 100‐fold from methanol grown Methanosarcina barkeri to apparent homogeneity and a specific activity of 34 μmol·min−1·mg protein−1. Molybdenum was found to co‐migrate with the enzyme activity. The molybdenum content of purified preparations was 3–4 nmol per mg protein equal to 0.6–0.8 mol molybdenum per mol enzyme of apparent molecular mass 200 kDa. Evidence is presented that also formylmethanofuran dehydrogenase from H2/CO2 grown Methanobacterium thermoautotrophicum (strain Marburg) is a molybdoenzyme.
Altered patterns of protein phosphorylation in articular chondrocytes treated with interleukin‐1 or synovial cytokines
Authors: Hulkower K.I. , Georgescu H.I. , Evans C.H.
Published: July 2002

Cultures of lapine articular chondrocytes were exposed to purified, human, recombinant interleukin‐1 α or partially purified preparations of lapine, synovial, cytokines in the presence of [32P]orthophosphate. After 30 min incubation, phosphoproteins were extracted from the cells, separated by two‐dimensional gel electrophoresis and visualized autoradiographically. Analysis of the autoradiograms revealed that interleukin‐1 and the synovial factors produced marked changes in the pattern of protein phosphorylation. The synovial cytokines induced many of the same changes as interleukin‐1, as well as a number of unique changes. This finding is consistent with the notion that, in addition to interleukin‐1, synoviocytes secrete other cytokines which modulate the metabolism of chondrocytes. These data support the idea that signal transduction in chondrocytes responding to interleukin‐1 involves the activation of one or more protein kinases.