bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulfhydryl groups of the uncoupling protein of brown adipose tissue mitochondria
Authors: JEŽEK Petr , DRAHOTA Zdeněk
Published: March 2005

Mersalyl, 5,5′‐dithio‐bis(2‐nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl− transport by the uncoupling protein was unaffected. Using these modifiers and N‐ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p‐diazobenzenesulfonate (N2PhSO2) suppresses only 20–25% of fluorescence of Thiolyte‐DB‐labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80–90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2‐reactive residue is not identical with the SHB.
Evidence for at least four different inositol bisphosphatases in bovine brain
Authors: HOWELL Steven , BARNABY Robert J. , ROWE Tony , RAGAN C. Ian , GEE Nicolas S.
Published: March 2005

Bovine brain supernatant contains at least four enzymes capable of hydrolysing inositol bisphosphates. These activities may be distinguished on the basis of their metal, salt and pH dependence, sensitivity to Li+ ions and thiol‐modification reagents, and on their molecular sizes. In addition to Li+‐sensitive Ins(1,4)P2/Ins(1,3,4)P3 1‐phosphatase [Gee et al. (1988) Biochem. J. 253, 777–782] which has an absolute requirement for Mg2+, two (Li+‐insensitive and Mg2+‐independent) phosphatases, capable of hydrolysing Ins(3,4)P2 and Ins(1,3)P2, respectively, have been identified. Both enzymes were inhibited by only moderate concentrations of salt, although for the former there was no obvious correlation between inhibitory potency and either the nature of the anion/cation or the ionic strength of the buffer. Ins(3,4)P2 phosphatase had a pH optimum of 7.6 and this activity could be resolved on gel‐filtration columns into a two overlapping peaks of molecular mass 170 kDa and 450 kDa.

Mg2+‐independent Ins(1,3)P2 phosphatase had a pH optimum of 7.1 and displayed a single broad activity peak on gel‐filtration columns. However, if assays were performed in the presence of Mg2+, a second Ins(1,3)P2 phosphatase was revealed (35 kDa), which had a pH optimum of 8.8. Ins(1,4)P2/Ins(1,3,4)P3 1‐phosphatase, Ins(3,4)P2 phosphatase, Mg2+‐independent Ins(1,3)P2 phosphatase and inositol monophosphatase were all inhibited by 5,5′‐dithiobis(2‐nitrobenzoic acid) with IC50 values of 34 μM, 65 μM and 560 μM and 1100 μM, respectively. The metabolism of Ins(1,3,4)P3 by brain supernatant was also examined. Product specificity was shown to be entirely dependent on the buffer conditions employed. In Mg2+‐containing buffers, Ins(1,3,4)P3 was hydrolysed predominantly to Ins(3,4)P2, consistent with hydrolysis by Ins(1,4)P2/Ins(1,3,4)P3 1‐phosphatase. In the presence of EDTA, Ins(1,3,4)P3 was degraded exclusively by a 4‐phosphatase enzyme generating Ins(1,3)P2. Under these conditions, high concentrations of Ins(3,4)P2 blocked the hydrolysis of Ins(1,3,4)P3.
Shift to the Na+ from of Na+/K+‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH3)4ATP
Authors: SCHEINER‐BOBIS Georgios , ESMANN Mikael , SCHONER Wilhelm
Published: March 2005

Inactivation of purified Na+/K+‐transporting ATPase by the MgATP complex analogue Co(NH3)4ATP, which binds to the low‐affinity ATP‐binding site, results in the concomitant inhibition of the K+‐activated p‐nitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state.

Complete inactivation of Na+/K+‐transporting ATPase by Co(NH3)4ATP does not alter the ADP/ATP exchange reaction which is considered to be part of the catalytic activity in the E1 conformation.

The enzyme binds cosin at the high‐affinity ATP‐binding site as measured by the change in eosin fluorescence. Eosin binding to the Co(NH3)4ATP‐inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na+. Inactivation by Co(NH3)4ATP increased the half‐maximal opposing effect of K+ on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No eosin fluorescence changes were observed when the Co(NH3)4ATP‐inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable complex at the high‐affinity ATP‐binding site. CrATP thus abolishes eosin binding.

It is concluded, that Co(NH3)4ATP interacts with Na+/K+‐transporting ATPase in the E2 conformation and arrests it there. This affects eosin binding to the high‐affinity ATP‐binding site, since the K+ sensitivity is lost. A possible interpretation of these differing effects of Co(NH3)4ATP on partial reactions of Na+ K+‐transporting ATPase is that the sodium pump works as an (α,β)2 diprotomer. It is likely that the arrest of one α,β protomer in the E2 conformational state by occupancy of the low‐affinity ATP‐binding site with Co(NH3)4ATP induces the Na+ form (E1 form) in the corresponding α,β protomer, as is indicated by the unaffected ADP ATP exchange and the response of the eosin fluorescence on Na+ and K+.
Interaction of non‐classical detergents with the mitochondrial porin
Authors: de PINTO Vito , BENZ Roland , PALMIERI Ferdinando
Published: March 2005

The effect of different families of detergents on the solubilization and purification of the pore‐forming protein (porin) of the mitochondrial outer membrane of bovine heart was investigated in detail. With Tritons, dimethylamine oxides and zwittergents, porin solubilization with respect to total mitochondrial membrane protein was more efficient with the more hydrophobic members of each series. With most detergents the protein eluted as protein‐detergent micelles in the void volume of hydroxyapatite/celite columns. In contrast, the protein was bound to the column material and was eluted after the addition of salt to the elution buffer when the detergents octylglucoside, zwittergent Z‐314 and laury(dimethyl)‐amine oxide were used. The protein purified in the presence of the latter detergent had a higher pore‐forming activity in lipid bilayer membranes compared to porin isolated in the presence of Triton X‐100. The binding of porin to the hydroxyapatite/celite column was used to study the lipid content of the active pore‐forming complex. The analysis revealed that the complex contained no phospholipid but rather five molecules of cholesterol/polypeptide chain.
Equilibrium, kinetic and photoaffinity labeling studies of daunomycin binding to P‐glycoprotein‐containing membranes of multidrug‐resistant Chinese hamster ovary cells
Authors: BUSCHE Roger , TÜMMLER Burkhard , CANO‐GAUCI Danielle F. , RIORDAN John R.
Published: March 2005

The binding of daunomycin and its Bolton‐Hunter derivative iodomycin to plasma membranes isolated from multidrug‐resistant Chinese hamster ovary cells (CHO B30) and their drug‐sensitive parents (B1) was investigated. The thermodynamics and kinetics of equilibrium binding monitored by fluorescence titrations and temperature‐jump relaxation spectrometry were compared with the specificity of covalent photolabeling with [3H]daunomycin and [125I]iodomycin. The facts that the uptake of anthracycline from a queous solution into the CHO membranes was not accompanied by any substantial increase of fluorescence anisotropy nor by any spectral shift of the fluorescence emission spectrum and that the partition ratio into the membrane was 20–30‐fold higher when compared to a lecithin bilayer, provided evidence that the non‐covalent drug binding sites are constituted by polar protein domains without any substantial contribution from the surrounding lipids. Photoaffinity labeling with nanomolar concentrations of anthracycline and equilibrium binding curves independently showed that a 150–170‐kDa plasma membrane glycoprotein (P‐glycoprotein), whose overexpression is the major difference between B1 and B30 membranes, provides the binding sites of highest affinity for daunomycin and iodomycin (K∼ 4 × 107 M−1). Comparison of photolabeling and equilibrium data suggested that the same binding sites on P‐glycoprotein were most probably being monitored. The photolabeling of P‐glycoprotein by iodomycin was inhibited in a dose‐dependent manner by other compounds to which multidrug‐resistant cells are either resistant or collaterally sensitive with the following orders of effectiveness: vinblastine > verapamil > nitrendipine > daunomycin ≧ colchicine. Temperature‐jump experiments covering the time range of 1 μs to 1 s revealed a single concentration‐dependent relaxation time of 10–30 μs. The association of daunomycin with its binding sites in the membranes was found to be a diffusion‐controlled process with kon rates of 2–4 × 109 M−1 s−1. Therefore, the selectivity of drug binding was entirely refected in the dissociation rates.
The pathway of the quinol/quinone transhydrogenation reaction in ubiquinol:cytochrome‐c reductase of Neurospora mitochondria
Authors: ZWECK Axel , BECHMANN Georg , WEISS Hanns
Published: March 2005

Ubiquinol:cytochrome‐c reductase, isolated from Neurospora mitochondria as a protein/Triton X‐100 preparation, and reconstituted into phospholipid membranes, catalyses the electron transfer from duroquinol to 2,3‐dimethoxy‐5‐decyl‐6‐methyl‐benzoquinone (decQ) on a myxothiazol‐insensitive, but antimycin‐sensitive, pingpong pathway. Duroquinol reacts first to form the altered, reduced enzyme E′. This reaction is followed by dissociation of duroquinone making way for E′ to bind decQ and convert it into decQH2.
Two genes encode the adenine nucleotide translocator of maize mitochondria
Authors: BATHGATE Brian , BAKER Alison , LEAVER Christopher J.
Published: March 2005

Southern blot hybridisation of maize (Zea mays) genomic DNA with a specific cDNA probe and sequence analysis of several cDNAs have revealed that the mitochondrial adenine nucleotide translocator (ADP/ATP carrier) is encoded by two genes, both of which are expressed in dark‐grown coleoptile tissue. The structural genes (designated ANT‐G1 and ANT‐G2), encoding the two translocator polypeptides, have been isolated from a maize genomic library and characterised by DNA sequence analysis, mung bean nuclease mapping and primer extension. The two genes each contain two introns and encode very similar proteins. Transcripts from both genes have long (approximately 200 nucleotides) 5′ untranslated leaders which are unusual in that they contain several ATG codons upstream of the initiation codon. Transcript analysis of RNA extracted from serial sections of developing maize leaves reveals that the translocator genes are most actively transcribed in the basal meristem, while transcripts were undetectable in green leaves.
Inhibitory effect of nonviable preparations from human immunodeficiency virus 1 on inositol phospholipid metabolism
Authors: MÜLLER Werner E. G. , REUTER Petra , KUCHINO Yoshiyuki , van ROOYEN Jan , SCHRÖDER Heinz C.
Published: March 2005

Previously it was established [Pahwa, S., Pahwa, R., Saxinger, C., Gallo, R. C. & Good, R. A. (1985) Proc. Natl Acad. Sci. USA 82, 8198] that nonviable preparations of human immunodeficiency virus 1 (HIV‐1) abolish the proliferative response of human lymphocytes to phytohemagglutinin A. Now we describe that this effect might be, at least partially, due to an impairment of the function of phospholipase C. It was found that addition of HIV‐1 preparation to lymphocytes diminished the stimulation of phosphatidylinositol phosphorylation caused by phytohemagglutinin A. Moreover, this preparation completely abolished the phytohemagglutinin‐A‐stimulated release of inositol trisphosphate and prevented a translocation of protein kinase C from cytosol to membranes. From this data we conclude that nonviable HIV‐1 preparations inhibit the intracellular signalling pathway, leading to a reduced mitogenic response to phytohemagglutinin A, at the level of protein kinase C.
Myosin heavy‐chain isoforms in adult and developing rabbit vascular smooth muscle
Authors: BORRIONE Anna C. , ZANELLATO Anna M. C. , SCANNAPIECO Gianluigi , PAULETTO Paolo , SARTORE Saverio
Published: March 2005

Two monoclonal antibodies specific for smooth muscle myosin (designated SM‐E7 and SM‐A9) and one monoclonal anti‐(human platelet myosin) antibody (designated NM‐G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC‐1 (205 kDa), and MHC‐2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC‐1 but not MHC‐2. Non‐muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC‐2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non‐muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis.
Purification of the F420‐reducing hydrogenase from Methanosarcina barkeri (strain Fusaro)
Authors: FIEBIG Klaus , FRIEDRICH Bärbel
Published: March 2005

The 8‐hydroxy‐5‐deazaflavin (coenzyme F420)‐reducing and methyl‐viologen‐reducing hydrogenase of the anaerobic methanogenic archaebacterium Methanosarcina barkeri strain Fusaro has been purified 64‐fold to apparent electrophoretic homogeneity. The purified enzyme had a final specific activity of 11.5 μmol coenzyme F420 reduced · min−1· mg protein−1 and the yield was 4.8% of the initial deazaflavin‐reducing activity. The hydrogenase exists in two forms with molecular masses of approximately 845 kDa and 198 kDa. Both forms reduce coenzyme F420 and methyl viologen and are apparently composed of the same three subunits with molecular masses of 48 kDa (α), 33 kDa (β) and 30 kDa (γ). The aerobically purified enzyme was catalytically inactive. Conditions for anaerobic reductive activation in the presence of hydrogen, 2‐mercaptoethanol and KCl or methyl viologen were found to yield maximal hydrogenase activity. Determination of the apparent Km of coenzyme F420 and methyl viologen gave values of 25 μM and 3.3 mM, respectively. The respective turnover numbers of the high molecular mass form of the hydrogenase are 353 s−1 and 9226 s−1.
On the mechanism of inhibition of methanol dehydrogenase by cyclopropane‐derived inhibitors
Authors: FRANK Johannes , van KRIMPEN Simon H. , VERWIEL P. Eugène J. , JONGEJAN Jacob A. , MULDER Antonia C. , DUINE Johannis A.
Published: March 2005

Extraction of cyclopropanol‐inactivated methanol dehydrogenase (MDH) gave a mixture of two interconverting compounds. The same compounds could be prepared from 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐f]quinoline‐4,5‐dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag2O) as a catalyst. Structure elucidation revealed that a C5 3‐propanal adduct of PQQ is formed which is present in the extract as a diastereoisomeric mixture of the ring‐closed form. Cyclopropanone gave an analogous product, while cyclopropylmethanol behaved as a substrate and was oxidized by the enzyme without ring‐opening.

From the work described, several arguments can be derived to reject the idea that inactivation proceeds via formation of a pair of free radicals. The mechanism probably consists of a concerted proton abstraction, rearrangement of the cyclopropoxy anion to a ring‐opened carbanion and attack of the latter on the electrophilic C5 of PQQ. The measured rate of inactivation (3.7 s−1) is in agreement with such a mechanism. The role of the metal oxide and the enzyme in this process is the catalysis of the addition step and possibly a positioning of the reactants. As only a sole type of quinoprotein alcohol dehydrogenase becomes inhibited, the cyclopropane derivatives studied here can be regarded as mechanism‐based inhibitors.

The modified PQQ in cyclopropanone‐inactivated MDH is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of MDH with methanol as a substrate. Its rate of formation and decay and the strongly decreased level of fluorescence in the presence of activator are in accordance with the view that the fluorescing species is the previously found oxidized‐MDH · substrate (MDHox· S) complex. Since the decomposition of this complex requires activator and model studies have failed so far to mimic the enzyme, it seems that the combination of enzyme and activator is essential for the oxidation of the alcohol substrate.
Synthesis, processing and degradation in yeast of precursor human lysozyme with newly designed signal sequences
Authors: YAMAMOTO Yoshio , KIKUCHI Masakazu
Published: March 2005

Recently, we reported that the synthesis of human lysozyme in yeast is inhibited when artificially designed non‐native signal sequences are used [Yamamoto et al. (1987) Biochem. Biophys. Res. Commun. 149, 431–436; (1989) Biochemistry 28, 2728–2732]. Pulse/chase experiments described in the present paper have now revealed that precursor lysozymes are actually synthesized with these signal sequences, but that they are spontaneously degraded, suggesting that the enzyme is destabilized by the signal sequences. The data further show a good correlation between the secretory capability of the artificial signal sequences and the efficiency of the processing of precursor lysozyme.
Binding of nidogen and the laminin‐nidogen complex to basement membrane collagen type IV
Authors: AUMAILLEY Monique , WIEDEMANN Hanna , MANN Karlheinz , TIMPL Rupert
Published: March 2005

The laminin‐nidogen complex and purified nidogen both bind collagen IV but not other collagens, as shown by solid‐state ligand‐binding and inhibition assays. Laminin purified from the dissociated complex and a variety of laminin proteolytic fragments failed to bind collagen IV. Complexes formed in solution between nidogen or laminin‐nidogen and collagen IV were visualized by rotary shadowing which identified one major binding site about 80 nm away from the C‐terminus of the collagen triple helix. A second, weaker binding site may exist closer to its N‐terminus. Binding sites of nidogen were assigned to its C‐terminal globular domain which also possesses laminin‐binding structures. A more diverse collagen‐IV‐binding pattern was observed for the laminin‐nidogen complex, whereby interactions may involve both nidogen and short‐arm structures of laminin.
Interaction of elongation factor‐Tu from Escherichia coli with aminoacyl‐tRNA carrying a fluorescent reporter group on the 3′ terminus
Authors: OTT Günther , FAULHAMMER Heinz G. , SPRINZL Mathias
Published: March 2005

Transfer ribonucleic acids containing 2‐thiocytidine in position 75 ([s2C]tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3′‐shortened tRNA using ATP(CTP):tRNA nucleotidyl‐transferase. [s2C]tRNA was selectively alkylated with fluorescent N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)ethyl‐enediamine (1,5‐I‐AEDANS) on the 2‐thiocytidine residue. The product [AEDANS‐s2C]aminoacyl‐tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore. From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast [AEDANS‐s2C]Tyr‐tRNATyr, yeast Tyr‐tRNATyr, and the homologous E. coli Phe‐tRNAPhe, respectively, interacting with E. coli elongation factor Tu · GTP. The measurement of the association and dissociation rates of the interaction of [AEDANS‐s2C]Tyr‐tRNATyr with EF‐Tu · GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol−1 K−1 for ΔS°′ and –34.7 kJ mol−1 for ΔH°′ of this reaction.
Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli
Authors: FARROW Stuart N. , KAMIYA Hiroyuki , MIURA Kazunobu , OHTSUKA Eiko , NISHIMURA Susumu
Published: March 2005

A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli. The 942‐base‐pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E. coli trp promoter. Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E. coli.
Immunological detection of phytoene desaturase in algae and higher plants using an antiserum raised against a bacterial fusion‐gene construct
Authors: SCHMIDT Arno , SANDMANN Gerhard , ARMSTRONG Gregory A. , HEARST John E. , BÖGER Peter
Published: March 2005

Immunological characterization of phytoene desaturase, a key enzyme of carotenoid biosynthesis, is reported. For this purpose, a phytoene‐desaturase fusion protein has been employed. For its construction 921 base pairs of the crtI gene were fused to the N‐terminal region of the Escherichia coli lacZ gene. Plasmid pGABX2 resulted from insertion of a BglI–XhoI fragment from the Rhodobacter capsulatus carotenoid biosynthesis gene cluster, carrying the crtI, crtA and crtB genes, into pBR322. A 968‐base‐pair SalI‐fragment from pGABX2 was cloned into pUR288 at the 3′ end of the lacZ gene. Isopropyl‐β‐D‐thio‐galactopyranoside‐dependent activation of the lacZ fusion gene resulted in expression of a stable 150‐kDa protein. After electroelution from SDS/polyacrylamide slab gels, the protein was used for antibody production. The heterogenic antiserum obtained was tested by Western blotting against proteins from Rhodobacter capsulatus and several different photoautotrophic organisms including higher plants. Apparent molecular masses of immunoreactive proteins from Rhodobacter, Aphanocapsa, rape and spinach were around 64 kDa. In Bumilleriopsis a 55‐kDa protein was found instead. The antibody also inhibited in vitro desaturation of phytoene when detergent‐solubilized membranes were employed.
Guanylate kinase from Saccharomyces cerevisiae
Authors: BERGER Albin , SCHILTZ Emile , SCHULZ Georg E.
Published: March 2005

This paper describes a large‐scale purification of guanylate kinase (ATP + GMP · ADP + GDP) from Saccharomyces cerevisiae, the crystallization of the enzyme and preliminary X‐ray investigations. Furthermore the complete amino acid sequence of the enzyme has been determined and was compared to adenylate kinase sequences.

Guanylate kinase was purified in five steps to homogeneity: crude extract, ion‐exchange chromatography, affinity chromatography and gel filtration twice.

The enzyme was crystallized to single octahedral bipyramids with sizes up to 500 × 200 × 150 μm3. Preliminary X‐ray results are given.

The final sequence shows 186 amino acids (Mr= 20 548), containing one cysteine and one tryptophan. It was determined from peptides of five cleavages of the whole protein. Three cleavages were used for determination of the whole polypeptide chain. From the other two, only some peptides were used to secure overlaps and the cysteine position. The N‐terminal blocking group was identified by 1H‐NMR spectroscopy.

Since guanylate kinase shows the mononucleotide binding pattern GXXGXGK, it was compared to other proteins containing this pattern. But no further homology signal could be detected. A comparison with adenylate kinases revealed significant similarity in another chain segment. This led to the conclusion that guanylate kinase is at least partially homologous to the adenylate kinases.
Nematocysts (stinging capsules of Cnidaria) as Donnan‐potential‐dominated osmotic systems
Authors: WEBER Jakob
Published: March 2005

Isolated nematocysts (capsules of stinging cells in Cnidaria) from the freshwater polyp Hydra vulgaris [Weber, J., Klug, M. & Tardent, P. (1987) Comp. Biochem. Physiol. 88B, 855–862] are under a constant internal osmotic pressure of 12.5 MPa. The capsular wall which withstands this considerable pressure has an average elasticity modulus of approximately 1 GPa, enabling the cyst to swell from its relaxed state to more than double its volume. If the large concentrations of Mg2+ and Ca2+ within isolated nematocysts are substituted by alkali ions, the capsular volume increases by 15% and the final osmotic pressure rises to 15.3 MPa whereas after substitution by Ca2+ a decrease to 90% of the volume and 6.5 MPa is observed.

Evidence obtained from the osmotic behavior of nematocysts, as well as data from in vitro exchange of their cations, are consistent with a physicochemical model in which the internal osmotic pressure of nematocysts and thus their ability to discharge is governed by the cationic composition of the content and the salt concentrations in the environment.
Characterization of a mitogen‐activated, Ca2+‐sensitive microtubule‐associated protein‐2 kinase
Authors: HOSHI Minako , NISHIDA Eisuke , SAKAI Hikoichi
Published: March 2005

We have previously found that treatment of quiescent mammalian fibroblast cells with several mitogenic factors activates in common a Ca2+‐sensitive serine/threonine‐specific protein kinase activity toward microtubule‐associated protein 2 (MAP2) [Hoshi, M., Nishida, E. and Sakai, H. (1988) J. Biol. Chem. 263, 5396–5401]. Here, we characterized the mitogen‐activated MAP2 kinase activity in rat 3Y1 cells. The activated kinase activity was detected in the cytosolic fraction but not in the membrane fraction. The inhibitory effect of Ca2+ on the kinase activity was reversible. Kinetic analyses revealed that the apparent Km values of the kinase activity for MAP2 and ATP were 1.6 μM and 30 μM, respectively. Free Ca2+ at 4 μM decreased apparent Vmax values for MAP2 and ATP without changing the apparent Km values. The MAP2 kinase had an apparent molecular mass of about 40 kDa as determined by gel filtration and by sucrose density gradient centrifugation. Myelin basic protein as well as MAP2 could serve as good substrates for this kinase, but 40S ribosomal protein S6, casein, histone, phosphorylase b, protamine, tubulin, actin and tau could not. These properties of the enzyme indicate that the Ca2+‐sensitive MAP2 kinase may be a previously unidentified enzyme. Down‐regulation of protein kinase C by prolonged phorbol ester treatment abolished the MAP2 kinase activation by phorbol ester, but did not prevent the MAP2 kinase activation by epidermal growth factor (EGF) or fresh serum. This suggests that the Ca2+‐sensitive MAP2 kinase could be activated through protein‐kinase‐C‐dependent and ‐independent pathways. Activation of the MAP2 kinase occurred shortly after the addition of EGF or phorbol ester even in the presence of protein synthesis inhibitors (cycloheximide, puromycin and emetin). Moreover, treatment of the EGF‐ or phorbol‐ester‐activated MAP2 kinase with acid phosphatase inactivated the kinase activity. Thus, the MAP2 kinase may be activated through phosphorylation.
2‐Aminopurine selectively blocks the transcriptional activation of cellular genes by virus, double‐stranded RNA and interferons in human cells
Authors: WATHELET Marc G. , CLAUSS Isabelle M. , PAILLARD Florence C. , HUEZ Georges A.
Published: March 2005

Interferons, double‐stranded RNA and viruses induce the transcription of partly overlapping sets of cellular genes. We have studied the regulation of 11 interferon‐inducible genes by these agents and found that four of them were also directly inducible by virus and double‐standed RNA, and two by virus only. We have investigated whether an inhibitor of interferon‐ß gene activation, 2‐aminopourine, would block this induction process. Induction of these genes by virus and double‐stranded RNA was indeed blocked by 2‐aminopurine. Since a single cis‐acting element can confer inducibility both to interferons, and to virus and double‐stranded RNA, we tested the effect of 2‐aminopurine on gene activation by interferon‐α and interferon‐γ. Remarkably, in all the cell lines tested, these induction processes and the establishment of an antiviral state were blocked by the drug. These observations contrast with previous reports. The inhibitory effect of this drug on gene induction was exerted in a selective fashion and at the transcriptional level. This indicate that for the virus‐, double‐stranded‐RNA‐, and interferons‐mediated gene induction, an early and similar step in signal transduction was affected by 2‐aminopurine.
Two distinct nonmuscle myosin‐heavy‐chain mRNAs are differentially expressed in various chicken tissues
Authors: KATSURAGAWA Yoshinari , YANAGISAWA Masashi , INOUE Akihiro , MASAKI Tomoh
Published: March 2005

Two distinct cDNA clones for nonmuscle myosin heavy chain (MHC) were isolated from a chicken fibroblast cDNA library by cross‐hydridization under a moderate stringency with chicken gizzard smooth muscle MHC cDNA. These two fibroblast MHC and the gizzard MHC are each encoded in different genes in the chicken genome. Northern blot analysis showed that both of the nonmuscle MHC mRNAs were expressed not only in fibroblasts but also in a variety of tissues including brain, lung, kidney, spleen, and skeletal, cardiac and smooth muscles. However, the relative contents of the two nonmuscle MHC mRNAs varied greatly among tissues. The encoded amino acid sequences of the nonmuscle MHCs were highly similar to each other (81% identity) and to the smooth muscle MHC (81–84%), but much less similar to vertebrate skeletal muscle MHCs (38–41%) or to protista nonmuscle MHCs (35–36%). A phylogenic tree of MHC isoforms was constructed by calculating the similarity scores between these MHC sequences. An examination of the tree showed that the vertebrate sarcomeric (skeletal and cardiac) MHC isoforms are encoded in a very closely related multigene family, and that the vertebrate non‐sarcomeric (smooth muscle and nonmuscle) MHC isoforms define a distinct, less conserved MHC gene family.
One‐ and two‐dimensional NMR studies of yeast phosphoglycerate kinase
Authors: FAIRBROTHER Wayne J. , BOWEN Derrick , HALL Len , WILLIAMS Robert J. P.
Published: March 2005

One‐ and two‐dimensional proton NMR studies have been carried out on yeast phosphoglycerate kinase (Mr∼ 45000) in order to identify amino‐acid spin systems and obtain sequence‐specific assignments. A number of sequence‐specific assignments have been made using a combination of structural information contained in nuclear Overhauser effect spectra and X‐ray crystallographic data. The results of substrate binding studies (both 3‐phosphoglycerate and Mg · ATP), which indicate mutual reorientation of certain assigned aromatic residues in the inter‐domain region of the protein, are discussed.
Arginase of Agrobacterium Ti plasmid C58
Authors: SCHRELL Andreas , ALT‐MOERBE Juliane , LANZ Thomas , SCHROEDER Joachim
Published: March 2005

Agrobacterium nopaline Ti plasmids code for three enzymes of nopaline [N2‐(1,3‐dicarboxypropyl)‐l‐arginine] degradation: nopaline oxidase, arginase, and ornithine cyclodeaminase. We describe the DNA sequence of the arginase gene, a comparison of the deduced protein sequence with eucaryotic arginases, and properties of the procaryotic enzyme. The results show that the agrobacterial arginase is related with arginases from yeast, rat liver, and human liver (28–33% identity). The Ti plasmid enzyme revealed several properties which appear common to all arginases, but it does not utilize l‐canavanine as substrate, and its Mn2+ requirement is not satisfied by Fe2+, Co2+, or Ni2+. The properties of arginase and ornithine cyclodeaminase are discussed as part of the mechanisms which avoid depletion of l‐arginine and l‐ornithine pools for biosynthetic reactions during catabolic utilization of nopaline.
Enantiotopic differentiation in horse‐liver alcohol‐dehydrogenase‐catalyzed oxidoreduction studied with novel substrates having organometallic moieties
Authors: YAMAZAKI Yoshimitsu , UEBAYASI Masami , HOSONO Kuniaki
Published: March 2005

Horse‐liver alcohol‐dehydrogenase‐catalyzed oxidation of 1,2‐bis(hydroxymethyl)ferrocene (1) gave (1R)‐(+)‐1‐formyl‐2‐hydroxymethylferrocene (3) (86 ± 2% enantiomeric excess, e.e.), while the reduction of the corresponding dialdehyde 1,2‐diformylferrocene (2) gave the antipode (1S)‐(–)‐3 (94 ± 2% e.e.). This fact indicates that the pro‐R group in both 1 and 2 was preferentially converted by the enzyme.

When one of two substituents on the substrate was replaced by a methyl group or moved to the β‐site, the stereoselectivity in the reaction decreased as evidenced by the enantiomeric purity of the products (5–64% e.e.). Treatment of racemic 1‐hydroxyethylferrocene (14) with horse‐liver alcohol dehydrogenase (HLADH) gave optically pure (R)‐(–)‐14together with acetylferrocene. The reduction of 2 with HLADH, NAD and (2H6)ethanol gave (–)‐(1S,2R)‐1‐formyl‐2‐[(R)‐hydroxy(2H1)methyl]ferrocene and that of 1,2‐di[(2H)formyl]ferrocene with HLADH, NAD and ethanol gave (–)‐(1S,2R)‐1‐(2H)formyl‐2‐[(S)‐hydroxy(2H1)methyl]ferrocene. These configurations indicate that the enzymic reduction occurred on the re‐face of pro‐R formyl group. The re‐face selectivity was also found in the enzymic reduction of (6‐benzaldehyde)tricarbonylchromium and its (2H)formyl analogue.

Docking of 2 into the active site of HLADH was examined using computer graphics. It has been suggested that the enantioselectivity to the pro‐R side in the oxidoreduction of 1 and 2 by HLADH is a natural consequence of the re‐face selectivity, which is caused by a steric interaction between the ligand and the side chain of Phe‐93 or the Zn complex and strengthened by an interaction between the unreactive polar α‐substituent and the protein, probably by hydrogen bond formation.
Octanoate metabolism in isolated hepatocytes and mitochondria from fetal, newborn and adult rabbit
Authors: PÉGORIER Jean‐Paul , DUÉE Pierre‐Henri , CLOUET Pierre , KOHL Claude , HERBIN Catherine , GIRARD Jean
Published: March 2005

Ketogenesis from endogenous fatty acids or from exogenous octanoate has been studied in isolated hepatocytes from fetal, 24‐h‐old newborn and adult rabbit. In fed adult rabbits, endogenous ketogenesis is low and increases sixfold in the presence of 2 mM octanoate. At birth, endogenous ketogenesis is low and markedly increases 24 h after birth but, in both cases, the addition of 2 mM octanoate does not increase the rates of ketone body production. Hepatocytes isolated from 24‐h‐old newborn or fed adult rabbits and incubated with [1‐14C]octanoate show a preferential channeling of fatty acid into oxidation (84–92% of octanoate metabolized). In contrast, esterification represents 43% of the amount of octanoate metabolized at birth. Chromatographic analysis of labelled triacylglycerols shows that 76 ± 2% of labelled fatty acids are identified as octanoate and all of the radioactivity in the octanoate peak is due to the carboxyl carbon. In hepatocytes from term fetus, the low capacity for octanoate oxidation is associated with a high capacity for esterification, whatever the octanoate concentration in the medium. Octanoate activated to octanoyl‐CoA in the cytosol of fetal hepatocyte is not oxidized in the mitochondria since carnitine acyltransferase I has a low activity at birth in the rabbit. This suggests that only a part of the octanoate pool is activated outside the mitochondria. Factors involved in the direct esterification of octanoate into triacylglycerols in term fetal hepatocytes are discussed.
Analysis of the α‐amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera
Authors: STRASSER Alexander W. M. , SELK Regine , DOHMEN R. Jürgen , NIERMANN Thomas , BIELEFELD Martina , SEEBOTH Peter , TU Guihong , HOLLENBERG Cornelis P.
Published: March 2005

We have cloned and characterized the α‐amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for α‐amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512‐amino‐acid polypeptide with a calculated Mr of 56 500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzaeα‐amylases. The AMY1 gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.
Vascular anticoagulant β: a novel human Ca2+/phospholipid binding protein that inhibits coagulation and phospholipase A2 activity
Authors: HAUPTMANN Rudolf , MAURER‐FOGY Ingrid , KRYSTEK Edeltraud , BODO Gerhard , ANDREE Harrie , REUTELINGSPERGER Chris P. M.
Published: March 2005

A cDNA was cloned coding for a new member of the human Ca2+‐modulated phospholipid‐binding protein family termed annexins. Due to its 56% identity to the human vascular anticoagulant (VAC) the new protein is named VAC‐β, renaming the previous VAC as VAC‐α. Northern analysis detects one hybridizing mRNA species of 2.2 kb in human placenta. Genomic Southern blot analysis shows a VAC‐β gene of comparable complexity to the VAC‐α gene. The cDNA was expressed in Escherichia coli and the recombinant protein purified to homogeneity. Antiserum raised against VAC‐β weakly cross‐reacts with VAC‐α. The properties of VAC‐β as an anticoagulant and as an inhibitor of phospholipase A2 activity were analyzed and compared to those of VAC‐α.
Thermodynamic nonideality in enzyme catalysis
Authors: BERGMAN Douglas A. , WINZOR Donald J.
Published: March 2005

The enhanced catalytic reduction of pyruvate by rabbit muscle lactate dehydrogenase that results from the addition of serum albumin [Nichol, L. W., Sculley, M. J., Ward, L. D. & Winzor, D. J. (1983) Arch. Biochem. Biophys. 222, 574–581] is shown to emanate solely from an increase in maximal velocity, there being no discernible effect of this inert space‐filling macromolecular solute on the Michaelis constant for either pyruvate or NADH. As part of the search for a mechanistic explanation of this kinetic phenomenon, the space‐filling effects of albumin have been used to eliminate the possibility that the increase in sedimentation coefficient of lactate dehydrogenase effected by inclusion of oxamate with enzyme‐NADH complex reflects preferential binding of this pyruvate analog to a more compact isomeric state of the binary complex. The enzyme kinetic results are therefore considered in terms of a reaction scheme entailing gross conformational changes during the formation of ternary enzyme‐NADH‐pyruvate complex and its isomerization to an activated transition state. The experimentally observed insensitivity of the Michaelis constant for pyruvate to albumin concentration is in keeping with theoretical prediction, but incorporation of the measured extent of maximal velocity enhancement into the kinetic model leads to a predicted volume for the fully saturated transition‐state complex that is far too small to be experimentally feasible. A more complex mechanistic model involving additional isomerizations of enzyme‐substrate species is thus required to achieve quantitative description of the albumin effect solely in terms of thermodynamic nonideality.
Interaction of muscle and non‐muscle tropomyosins with deoxyribonuclease I
Authors: CLARK Ian D. , BURTNICK Leslie D.
Published: March 2005

The sulfhydryl‐selective fluorescent reagent acrylodan (6‐acryloyl‐2‐dimethylaminonaphthalene) was used to label tropomyosins from rabbit cardiac muscle and from equine platelets. Addition of bovine pancreatic deoxyribonuclease I to solutions of acrylodan‐modified tropomyosins significantly altered the emission properties of the samples. Muscle and non‐muscle tropomyosin fluorescence were affected in qualitatively similar manners; emission maxima were red‐shifted by about 8 nm to 522–525 nm and maximal intensities were reduced by approximately 15%. Addition of KI to each of the fluorescent samples caused a greater degree of fluorescence quenching in the presence of DNase I than in its absence. The slopes of Stern‐Volmer plots were 15–25% steeper in the presence of DNase I. Fluorescence polarization values for acrylodan‐labelled tropomyosin samples were 25–35% lower in the presence of DNase I. Each of these effects could be saturated by addition of about a two‐fold molar excess of DNase I to tropomyosin. Together they suggest that interaction with DNase I causes localized unfolding of tropomyosin, thereby allowing the fluorescent label to become more exposed to the solvent and less restricted in its local motions. Circular dichroism measurements support this idea. Addition of DNase I to solutions of either labelled or unlabelled tropomyosin results in a net 14–18% loss in ellipticity near 220 nm, indicative of unfolding of α‐helix.
Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor
Authors: BIHOREAU Nicolas , SAUGER Annick , YON Jeannine M. , van de POL Hendrik
Published: March 2005

Factor VIII was purified 1200‐fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti‐(80‐kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor‐VIII forms obtained, consisted of 80‐kDa light chains, each being associated with one more‐or‐less fragmented heavy chain ranging over 90–210 kDa. The specific activity of these different forms was 7000 U/mg.

At differnt stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70‐kDa light chain (generated from the 80‐kDa light chain) with heavy chains ranging over 90–210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80‐kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal factor VIII, the ratio of von Willebrand activity to factor‐VIII activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin.

In the second step, the 90‐kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70‐kDa proteolyzed light chain, generating the final activated complex (45–50–70 kDa).

The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.
The role of subunit 4, a nuclear‐encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex
Authors: PAUL Marie‐Francoise , VELOURS Jean , ARSELIN de CHATEAUBODEAU Geneviéve , AIGLE Michel , GUERIN Bernard
Published: March 2005

The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3. Transformation of the Saccharomyces cerevisiae strain D273‐10B/A/U produced a mutant unable to grow on glycerol medium. The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected. ATPase was rendered oligomycin‐insensitive and the F1 sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of F0revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0. It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex. Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes. Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type. In addition, a high percentage of spontaneous rho−mutants was detected.
Characterization and N‐terminal amino acid sequence of multiple ferredoxins in kidney and adrenal mitochondria
Authors: DRISCOLL William J. , OMDAHL John. L.
Published: March 2005

Two separate ferredoxins that differ in molecular mass by about 1.5 kDa were isolated from both pig kidney and bovine adrenal mitochondria. The proteins had different biochemical and immunological properties and appeared to be distinct gene products. The smaller ferredoxin from pig kidney (renodoxin, Mr∼ 13 500) was very similar to bovine adrenodoxin (Mr= 14048). Both proteins had nearly identical N‐terminal amino acid sequences and electron‐transfer activities. However, renodoxin and adrenodoxin expressed distinct antigenic determinants, although they were immunologically cross‐reactive. The larger kidney (∼ 15‐kDa) and adrenal (∼ 15.3‐kDa) ferredoxins were biochemically similar to each other but they had lower specific activities and their N‐terminal sequences were different when compared to renodoxin and adrenodoxin. Each of the four ferredoxins had a visible absorption spectrum characteristic of a [2Fe‐2S] chromophore. But in addition, the larger ferredoxins displayed a prominent A276 peak due to their higher tyrosine content and the presence of tryptophan, which is absent in adrenodoxin and renodoxin. Consequently, the larger ferredoxins were termed Trp‐ferredoxin. Using antibody to pig kidney Trp‐ferredoxin, the larger adrenal and kidney ferredoxins were found to be very similar immunologically; however, the Trp‐containing and adrenodoxin‐type ferredoxins did not cross‐react in immunoblot analysis. Nevertheless, it was shown from competition ELlSA and activity‐inhibition analysis that the two ferredoxin types had limited common antigenic determinants. Trp‐ferredoxin was the major iron‐sulfur protein in kidney whereas adrenodoxin was the dominant molecular form in adrenal gland.
Reconstitution of a light‐stimulated adenylate cyclase from retina and Neurospora crassa preparations
Authors: MUSCHIETTI Jorge P. , BIANCHINI Graciela M. , MARTINETTO Horacio E. , CARRICARTE Valentina C. , GIUSTO Norma , FARBER Débora B. , TORRES Héctor N. , FLAWIA Mirtha M.
Published: March 2005

Adenylate cyclase catalytic subunits from Neurospora crassa membranes may interact with regulatory factors from membranes of bovine retinal rod outer segments (pretreated with N‐ethylmaleimide), reconstituting a heterologous system which, in the presence of light, is catalytically active in assay mixtures containing MgATP. Maximal activation was observed at 550 nm. Transducin‐depleted retinal membranes were not capable of reconstituting the heterologous light‐stimulated adenylate cyclase system. Addition of a transducin preparation to depleted membranes restored the reconstitution capacity of these membranes.

A similar heterologous adenylate cyclase system was reconstituted with Neurospora and mouse retinal whole membranes (pretreated with N‐ethylmaleimide). Membranes from mice suffering photoreceptor degeneration (rd homozygotes) did not reconstitute an heterologous adenylate cyclase system.