bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane‐bound F420H2‐dependent heterodisulfide reductase in methanogenic bacterium strain Göl and Methanolobus tindarius
Authors: Deppenmeier U. , Blaut M. , Mahlmann A. , Gottschalk G.
Published: July 2002

Washed membrane or cytoplasmic fractions of the methanogenic bacterium strain Göl catalyzed the oxidation of coenzyme F420H2 with a variety of electron acceptors. The F420H2‐oxidizing activity of the cytoplasmic fraction could be assigned to a NADP+:F420 oxidoreductase. The membrane fraction but not the cytoplasmic fraction catalyzed the oxidation of F420H2 with the concomitant reduction of the heterodisulfide of 2‐mercapto‐ethanesulfonate and 7‐mercaptoheptanoylthreonine phosphate (CoM‐S‐S‐HTP) at a rate of 100 protein according to the following equation: F420H2+CoM‐S‐S‐HTP → F420 + CoM + HTP‐SH. The activity depended linearly on the membrane protein up to a concentration of 60 The physiological electron acceptor CoM‐S‐S‐HTP could not be replaced by the corresponding homodisulfides CoM‐S‐S‐CoM and HTP‐S‐S‐HTP or by NADP+. A membrane‐bound F420H2‐dependent CoM‐S‐S‐HTP reductase was also detected in Methanolobus tindarius exhibiting a specific activity of 75 protein. The absence of a F420‐dependent hydrogenase in this organism excludes the involvement of this enzyme in electron transfer from H420H2 to CoM‐S‐S‐HTP.
Transcription and replication silencer element is present within conserved region of human Alu repeats interacting with nuclear protein
Authors: Tomilin Nikolai V. , Iguchi-Ariga Sanae M.M. , Ariga Hiroyoshi
Published: July 2002

Human cells contain a nuclear protein interacting with Alu repeats, and this protein seems to recognize a conserved sequence motif, GGAGGC, present within the RNA polymerase III promoter and within the SV40 T‐antigen‐dependent ARS‐like element. To study the potential functional role of this element, we have inserted the sequence into a chloramphenicolacetyltransferase (CAT) expression vector with a SV40 promoter and enhancer element from the up‐stream region of the human c‐myc gene, and transfected HeLa cells with the resulting plasmid. Analysis of expression by the CAT assay indicates that the Alu‐derived sequence supresses transcription of the CAT gene driven by the c‐myc enhancer/SV40 promoter. The Alu‐derived sequence also inhibits ARS activity of the c‐myc enhancer. The data allow the explaination of the transcriptional inactivity of Alu repeats in HeLa cells, and suggest the existence of a negative control of Alu transcription.
Reversible inhibition of electron transfer in the ubiquinol
Authors: Brustovetsky N.N. , Amerkhanov Z.G. , Popova E.Yu. , Konstantinov A.A.
Published: July 2002

Electron transfer through the ubiquinol:cytochrome c
1‐segment of liver mitochondria isolated from hibernating ground squirrels Citellus undulatus is repressed by 70–80% as compared to mitochondria from the active animals. The inhibition site is likely to be localized between ubiquinone and the cytochrome bc
1, complex. Partial release of the inhibition can be observed upon swelling of the isolated mitochondria in a hypoosmotie medium, the effect being prevented by phospholipase A2 inhibitors. Possible role of phospholipase A2 in regulation of ubiquinol oxidation by complex bc
1, is discussed.
Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei
Authors: Claeyssens Marc , Tomme Peter , Brewer Curtis F. , Hehre Edward J.
Published: July 2002

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl β‐D‐cellotrioside (K
m = 48μM, k
cat = 0.7 min−1) with release of the β‐cellobiose (retention of configuration). The same enzyme catalyzes the (trans‐hydration of cellobial (K
m = 116 μM, K
cat = 1.16 min−1) and lactal (K
m = 135 μM, k
cat = 1.35 min−1), presumably with glycosyi oxo‐carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl β‐cellotrioside, but products formed from these prochiral substrates are again of β configuration. Cellobiohydrolase II from the same microrganism hydrolyzes methyl β‐D‐cellotetraoside (K
m = 4 μM, k
cat = 112 min−1) with inversion of configuration to produce α‐cellobiose. The other reaction product, methyl β‐cellobioside, is in turn partly hydrolysed by Cellobiohydrolase II to form methyl β‐D‐glucoside and D‐glucose, presumably the α‐anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si‐direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.
Periplasmic and membrane‐bound respiratory nitrate reductases in Thiosphaera pantotropha
Authors: Bell Louise C. , Richardson David J. , Ferguson Stuart J.
Published: July 2002

The unusual ability of Thiosphaera pantotropha to catalyze respiratory nitrate reduction under aerobic conditions is shown to correlate with the activity of a periplasmic nitrate reductase that is expressed under both aerobic and anaerobic growth conditions. The organism also synthesizes, but only under anaerobic conditions, a membrane‐bound nitrate reductase which resembles the corresponding enzyme in Paracoccus denitrificans in respect of both catalytic properties and inhibition of activity in intact cells in the presence of oxygen.
Fructose 2,6‐bisphosphate and glycolytic flux in skeletal muscle of swimming frog
Authors: Wegener Gerhard , Krause Ulrike , Thuy Michael
Published: July 2002

Glycolytic flux in skeletal muscle is controlled by 6‐phosphofructokinase but how this is achieved is controversial. Brief exercise (swimming) in frogs caused a dramatic increase in the phosphofructokinase activator, fructose 2,6‐bisphosphate, in working muscle. The kinetics of phosphofructokinase suggest that in resting muscle, the enzyme is inhibited by ATP plus citrate and that the increase in fructose 2,6‐bisphosphate is part of the mechanism to activate phosphofructokinase when exercise begins. When exercise was sustained, fructose 2,6‐bisphosphate in muscle was decreased as was the rate of lactate accumulation. Glycolytic flux and the content of fructose 2,6‐bisphosphate appear to be closely correlated in working frog muscle in vivo.
The epidermolytic toxins are serine proteases
Authors: Dancer Stephanie J. , Garratt Richard , Saldanha Jose , Jhoti Harren , Evans Robert
Published: July 2002

Certain strains of Staphylococcus aureus usually belonging to phage group II produce epidermolytic toxins (ETA and ETB) which cause intraepidermal splitting in mice, neonates and occasionally adults. Amino acid sequences of ETA and ETB have been reported but the mechanism of epidermolysis remains unknown. A search of the NBRF‐PIR computer database showed the toxins to have significant sequence similarity with staphylococcal V8 protease and that the catalytic triad of V8 protease is present in ETA and ETB. Comparison of ETA, ETB and V8 protease with other members of the trypsin‐like serine protease family revealed little homology save for the immediate vicinity of the residues constituting the catalytic triad. The toxins, therefore, exhibit a distant relationship to mammalian serine proteases. A potential Ca2+‐binding loop was identified in ETA (but not ETB) on the basis of sequence similarity with the second calcium‐binding loop of rat intestinal calcium‐binding protein. Epidermolysis produced by ETA in the mouse bioassay was shown to be inhibited by the presence of EDTA consistent with a Ca2+‐dependent mechanism.
Arachidonic acid epoxygenase
Authors: Karara Armando , Dishman Elizabeth , Jacobson Harry , Falck J.R. , Capdevila Jorge H.
Published: July 2002

Mass spectral and Chromatographic analysis demonstrates the presence of 14,15‐, 11,12‐ and 8,9‐epoxyeicosatrienoic acids (44%, 33% and 23% of the total, respectively) in human kidney cortex. Chiral analysis of the human renal expoxyeicosatrienoic acids shows the formation of 8,9‐, 11,12‐ and 14,15‐epoxyeicosatrienoic acids in a 1:1, 4:1 and 2:1 ratio of antipodes, respectively. These results demonstrate the biosynthetic origin of the human kidney 11,12‐ and 14,15‐epoxyeicosatrienoic acids and suggest a role for renal cytochrome P‐450 in the bioactivation of endogenous pools of arachidonic acid.
Identification of a 66 KDa protein associated with yeast mitochondrial ATP synthase as heat shock protein hsp60
Authors: Gray Robyn E. , Grasso David G. , Maxwell Ronald J. , Finnegan Patrick M. , Nagley Phillip , Devenish Rodney J.
Published: July 2002

A 66 kDa protein, denoted P66, not hitherto classified as an integral component of yeast mitochondrial ATPase, is often observed in preparations of this enzyme complex. A physical association exists between P66 and the assembled ATPase complex since both components are coimmunoprecipitated by anti‐F1β monoclonal antibody. Two recombinant clones expressing proteins immunologically similar to P66 were isolated from a yeast genomic library in λgt11 by screening with a polyclonal anti‐holo‐ATPase antibody. Based on restriction site mapping and partial nucleotide sequence analysis, both clones encompass the gene encoding the yeast heat shock protein hsp60. The identification of P66 with hsp60, taken together with its demonstrated association with the mitochondrial ATPase complex, is consistent with recent suggestions that hsp60 is involved in assembly of the ATP synthase complex.
Purification and internal amino acid sequence of the 80 kDa protein kinase C substrate from Swiss 3T3 fibroblasts
Authors: Brooks S.F. , Erusalimsky J.D. , Totty N.F. , Rozengurt E.
Published: July 2002

The acidic 80 kDa protein kinase C (PKC) substrate was purified from 2.3 × 1010 Swiss 3T3 fibroblasts. Partial amino add sequence data were obtained from five peptides generated by S. aureus V8 cleavage of the protein, enabling a total of 91 amino acid residues to be assigned. The sequences of these five peptides were compared to the deduced amino acid sequences of acidic 80–87 kDa PKC substrates from both actively proliferating A431 epidermal carcinoma cells, and fully differentiated neural tissue. Despite their similar physical properties, there was no homology between the peptides derived from the fibroblast 80 kDa protein and the PKC substrate from A431 cells. However, there was 66% homology with the 87 kDa bovine brain protein within the regions covered by the peptides (about 30% of the total protein). Furthermore, comparison of the peptides from the fibroblast 80 kDa protein with proteolytic peptides derived from the acidic 80 kDa rat brain protein revealed an overall homology of 89%. These data provide the first direct evidence that the 80 kDa PKC substrate from Swiss 3T3 fibroblasts is closely related to the 80–87 kDa PKC substrates detected in fully differentiated neural tissue.
Changes of fluorescence anisotropy in plasma membrane of human polymorphonuclear leukocytes during the respiratory burst phenomenon
Authors: Fiorini Rosamaria , Curatola Giovanna , Bertoli Enrico , Giorgi Pier Luigi , Kantar Ahmad
Published: July 2002

Steady state fluorescence anisotropy (r
s) of TMA‐DPH was measured to study the effect of respiratory burst activation with PMA, FMLP, and PAF on the physico‐chemical structure of PMNs plasma membrane. Our results show a significant increase in r
s during the respiratory burst activation. In the presence of NADPH‐oxidase inhibitor DPI, only PAF induces changes in r
s values. This suggests a non‐specific effect of PAF on plasma membrane. Azide, which induces a supranormal release of H2O2, fails to increase the basal r
s value after activation. Moreover, the catalase does not abolish the increase in rr
s induced upon activation. This rules out the possibility that changes of r
s during the respiratory burst activation are attributed mainly to H2O2 release. We conclude that multiple processes accompanying the respiratory burst activation are responsible for the changes in the physico‐chemical properties of PMNs plasma membrane.
Interleukin 1 and tumor necrosis factor stimulate cGMP formation in rat renal mesangial cells
Authors: Pfeilschifter Josef , Schwarzenbach Heidi
Published: July 2002

Treatment of mesangial cells with recombinant human interleukin 1β (IL‐1β) or recombinant human tumor necrosis factor α (TNFα) dose‐dependently increased cGMP formation. Both IL‐1β and TNFα‐stimulated formation of cGMP occurred after a initial lag period of 4 to 8 hours. Treatment of cells with actinomycin D, cycloheximide or dexamethason completely abolished cytokine‐induced cGMP formation. Furthermore, the guanylate cyclase inhibitor Methylene blue completely blocked IL‐1β‐ and TNFα‐stimulated cGMP generation. NG‐mono‐methyl‐L‐arginine attenuated IL‐1β‐ and TNFα‐induced cGMP production, an effect that was reversed by L‐arginine.
Extracellular matrix
Authors: Labat-Robert J. , Bihari-Varga M. , Robert L.
Published: July 2002

A succinct overview of recent results on the biochemistry of extracellular matrix (ECM) is presented. The rapid expansion of this discipline over the best decades renders impossible to give an even approximately complete coverage of matrix biology. Some selected results concerning the four major families of macromolecules composing the ECM, that is, collagene (14 types described), elastin(s). proteoglycans and structural glycoproteins (especially fibronectin) are described. Special attention is directed to a crucial aspect of matrix biology: cell‐matrix interactions. A number of cell membrane receptors were recently described mediating the two way information flow from the cells to the matrix via the ‘programme’ of ECM synthesis coded in the genome and unfolding during differentiation and from the ECM to the cells through the membrane receptors which contact the cytoskeleton. One of them at least, the elastin receptor was shown to be linked through a G‐protein‐phospholipase C‐IP3 mediated relay to the regulation of intracellular calcium. Modifications of the ECM will therefore influence cell behaviour. Derangements of this informational feed back mechanisms appear to be involved in most age‐related connective tissue diseases.
The quantum yield of bacteriorhodopsin
Authors: Tittor Jörg , Oesterhelt Dieter
Published: July 2002

The conflicting reports on the quantum yield of the primary reaction in the photocycle of bacteriorhodopsin led us to reinvestigate the quantum yield under various experimental conditions. Quantitation of the molecules in the photocycle was done by measuring the number of molecules being in the M intermediate state 1 ms after laser flash excitation via determination of the absorption change at 602 nm. It was shown that this procedure provides to the lower limit of the quantum yield. A value of 0.64 ∓ 0.04 at pH 7.0 and room temperature (19°C) was obtained, independent from the wavelength of excitation in the range 500–600 nm and the ionic strength between 1 mM and 2 M salt.
Eclosion hormone of the silkworm Bombyx mori Expression in Escherichia coli and location of disulfide bonds
Authors: Kono Takaharu , Nagasawa Hiromichi , Kataoka Hiroshi , Isogai Akira , Fugo Hajime , Suzuki Akinori
Published: July 2002

A gene encoding eclosion hormone (EH) from the silkworm, Bombyx mori was chemically synthesized, inserted into a secretion vector and expressed in Escherichia coli, leading to the production of biologically active EH. Sequence analysis of cystine‐containing peptides in a thermolysin digest of this EH established the locations of 3 disulfide bonds in the molecule. Evidence was also obtained that the 6 residues at the NH2‐terminal are dispensable but 4 residues at the COOH‐terminal play an important role in EH activity.
Polypeptides traverse the mitochondrial envelope in an extended state
Authors: Rassow Joachim , Hartl Franz-Ulrich , Guiard Bernard , Pfanner Nikolaus , Neupert Walter
Published: July 2002

Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through the contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b
2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 30 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes.
Cloning and expression of cDNA encoding 25‐hydroxyvitamin D3 24‐hydroxylase
Authors: Ohyama Yoshihiko , Noshiro Mitsuhide , Okuda Kyuichiro
Published: July 2002

A cDNA encoding 25‐hydroxyvitamin D3 24‐hydroxylase (P450cc24) was isolated from a rat kidney cDNA library using specific antibodies to the enzyme. The isolated cDNA wax 3.2 kbp long and contained a 1542‐bp open reading frame encoding 514 amino acids. The deduced amino acid sequence contained a presequence typical or mitochondrial enzymes in the N‐terminal region. The amino acid sequence shows less than 30% similarity to those of any other cytochrome P450s so far reported and, therefore, P450cc24 constitutes a novel family or P450, COS‐7 cells transfected with the cDNA produced a protein that was reactive with the antibodies and catalyzed NADPH‐dependent 24‐hydroxylation of 25‐hydroxy‐vitamin D3 in the presence or adrenodoxin and NADPH‐adrenodoxin reductase. Using the cDNA as a probe we demonstrated that the increase of 24‐hydroxylation activity caused by administration or vitamin D3 into rats was accompanied by an increase of the mRNA.
Non‐viral cellular substrates for human immunodeficiency virus type 1 protease
Authors: Shoeman Robert L. , Kesselmeier Cornelia , Mothes Elfriede , Höner Bernd , Traub Peter
Published: July 2002

A Computer search revealed 10 proteins with homology to the sequence we originally identified in vimentin as the site of cleavage by human immunodeficiency virus type 1 (HIV‐1) protease. Of these 10 proteins (actin, α‐actinin, spectrin, tropomyosins, vinculin, dystrophin, MAP‐2, villin, TRK‐1 and lg μ‐chain), we show that 4 of the first 5 were cleaved in vitro by this protease, as arc MAP‐1 and ‐2 [(1990) J. Gen. Virol. 71, 1985–1991]. In these proteins, cleavage is not restricted to a single motif, but occurs at many sites. However, cleavage is not random, since 9 other proteins including the cytoskeletal proteins filamin and band 4.1 are not cleaved in the in vitro assay. Thus, the ability of HIV‐1 protease to cleave specific components or the cytoskeleton may be an important, although as yet unevaluated aspect of the life cycle of this retrovirus and/or may directly contribute to the pathogenesis observed during infection.
Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C
Authors: Taniyama Kohtaro , Takeda Koichiro , Ando Hiroshi , Kuno Takayosi , Tanaka Chikako
Published: July 2002

The functional GABAB receptor was expressed in Xenopus oocytes by injecting mRNA obtained from the cerebellum of the rat. Application of GABA in the presence of bicuculline induced a hyperpolarization under current‐clamp conditions and an outward current under voltage‐clamp conditions. Baclofen mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagonized by phaclofen. The GABA‐induced outward current was slightly inhibited by treatment with GDP‐β‐S and was completely inhibited by treatment with GTP‐γ‐S. The activation of protein kinase C by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), but not 4α‐phorbol‐12,13‐didecanoate, suppressed the GABAB receptor‐mediated hyperpolarization, and the effect of TPA was antagonized by sphingosine. Thus, activation of protein kinase C inhibits the expressed GABAB receptor‐mediated response.
Modulation of a Shaker potassium A‐channel by protein kinase C activation
Authors: Moran Ofira , Dascal Nathan , Lotan Ilana
Published: July 2002

Brain fast transient K??? channel (A channel) is known to be modulated by PKC activation. We studied, by two‐electrode voltage clamp, the molecular mechanism of modulation by PKC activation of A‐channels expressed in Xenopus oocytes from the Shaker H4 clone. The modulation is inhibitory affecting primarily the maximal conductance of the channels. A secondary effects is a small change in the voltage‐dependence of activation and inactivation of the channel.
Specific activation of Gs by synthetic peptides corresponding to an intracellular loop of the β‐adrenergic receptor
Authors: Cheung Anne H. , Huang Ruey-Ruey C. , Graziano Michael P. , Strader Catherine D.
Published: July 2002

Peptides corresponding to the amino acid sequence of the hamster β2‐adrenergic receptor (β2AR) were synthesized and their ability to activate purified G‐proteins determined. Two peptides, comprising the N‐ and C‐terminal 15 amino acids of the putative third intracellular loop region of the β2AR were found to activate the G‐protein Gs but not to activate a preparation of Gi/Go. Other peptides corresponding to the internal portions of this loop and the C‐terminal tail region failed to activate either G‐protein. The presence of phospholipid vesicles was required for this activation. The observation that peptides with sequences corresponding to the ends of the third intracellular loop of the βAR can specifically activate Gs confirms the results of previous mutagenesis studies on the receptor and demonstrates that the secondary structure conferred by the amino acid sequences in these regions is sufficient for the activation of G‐proteins.
Heat shock proteins and cell proliferation
Authors: Pechan Paul Michael
Published: July 2002

Heat shock proteins (Hsps) carry out a number of essetial functions in the cell. These functions could be utilized, in a developmentally regulated manner, to affect proteins necessary for cell growth and proliferation. Data showing Hsp involvement in cell proliferation under non‐stress conditions are perhaps not altogether surprising in view of the important roles of Hsps in cell metabolism. In a number of cases stress can facilitate cell proliferation in cells which could otherwise not be amenable to this developmental pathway. It is proposed that Hsps could play an essential helper role in initiating this process.
Effects of hypothalamic peptides on electrical activity and membrane currents of ‘patch perforated’ clamped GH3 anterior pituitary cells
Authors: Barros Francisco , Delgado Luis M. , Maciá Carmen , de la Peña Pilar
Published: July 2002

‘Perforated‐patch’ recordings of rat anterior pituitary GH, cells allow long and stable monitoring of electrical activity and membrane currents. Under current clamp conditions, the biphasic effect of thryotropin releasing hormone (TRH) consisting of a transient hyperpolarization followed by a longer phase of increased action potential frequency is fully preserved. Somatostatin suppresses action potential activity and antagonizes the second phase of enhanced spiking caused by TRH. Voltage clamp records of isolated currents indicate that TRH affects calcim‐dependent potassium currents, but does not alter either voltage‐dependent potassium or calcium currents at times and concentrations at which the electrical activity is increased.
Does aminotriazole inhibit import of catalase into peroxisomes by retarding unfolding?
Authors: Middelkoop Esther , Strijland Anneke , Tager Joseph M.
Published: July 2002

Fusion of complementary cell lines from patients with diseases of peroxisome biogenesis leads to peroxisome assembly in the heterokaryons and to uptake of cytosolic catalase by the newly peroxisomes. Here we show that catalase import is inhibited by prior binding to catalase of the inhibitor 3‐amino‐1,2,4‐triazole, which appears to retard unfolding of the protein.
Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo
Authors: Towatari Takae , Nikawa Takeshi , Murata Mitsuo , Yokoo Chihiro , Tamai Masaharu , Hanada Kazunori , Katunuma Nobuhiko
Published: July 2002

New derivatives of E‐64 (compound CA‐030 and CA‐074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K
1 values for cathepsin B were about 2–5 nM, like that of E‐64‐c, whereas their initial K
1 values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us intraperitoneal injection of compound CA‐030 or CA‐074 into rats, compound CA‐074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA‐030 does not show selectivity for cathepsin B, although both compounds CA‐030 and CA‐074 show complete selectivity for cathepsin B in vitro.
DNA damage by oxygen‐derived species Its mechanism and measurement in mammalian systems
Authors: Halliwell Barry , Aruoma Okezie I.
Published: July 2002

When cells are exposed to oxidative stress, DNA damage frequently occurs. The molecular mechanisms causing this damage may include activation of nucleases and direct reaction of hydroxyl radicals with the DNA. Several oxygen‐derived species can attack DNA, producing distinctive patterns of chemical modification. Observation of these patterns and measurement of some of the products formed has been used to determine the role of different oxygen‐derived species in DNA cleavage reactions, to assess the extent of oxidative damage to DNA in vivo and to investigate the mechanism of DNA damage by ionizing radiation and chemical carcinogens.
Structural and functional modifications induced by diamide on the F0 sector of the mammalian ATP synthase
Authors: Dabbeni-Sala Federica , Lippe Giovanna , Sorgato M.Catia
Published: July 2002

In this report data are presented which firmly establish that by treating isolated F0 with the thiol reagent diamide, two 25 kDa F0 subunits react to form a dimer of 45 kDa apparent molecular mass. This dimerising effect is correlated to the impairment of the binding of F1 to F0, both at μM and mM diamide concentrations. Under the latter condition, modification of other F0 subunits also occurs. Passive proton conductance through F0, as well as its sensitivity to N,N′‐dicyclohexylcarbodiimide, are affected at low diamide concentration. Thus perturbation of the cysteine residue of the 25 kDd F0 subunit is sufficient for altering the ATP synthase proton channel.
Pyruvate‐formate‐lyase‐deactivase and acetyl‐CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE
Authors: Kessler Dorothea , Leibrecht Iris , Knappe Joachim
Published: July 2002

A 4.8 kb DNA‐fragment was cloned and sequenced encompassing the structural gene of PFL‐deactivase (2.7 kb) and 2 kb of the 5 flanking region that contains the elements for anaerobic induction. A mutant lacking deactivase was shown to require exogenous electron acceptors for anaecrobic growth with glucose. This revealed the identity of PFL‐deactivase with the alcohol and acetaldehyde dehydrogenases of E. coli. The multienzyme represents a homopolymeric protein (∼ 40 × 96 kDa) requiring Fe2+ for all functions.
Interaction of mitochondrial creatine kinase with model membranes A monolayer study
Authors: Rojo Manuel , Hovius Ruud , Demel Rudy , Wallimann Theo , Eppenberger Hans M. , Nicolay Klaas
Published: July 2002

The interaction of mitochondrial creatine kinase (Mi‐CK; EC 2.7.3.2) with phospholipid monolayers and spread mitochondrial membranes at the air/water interface has been investigated. It appeared that Mi‐CK penetrated into these monolayers as evidenced by an increase in surface pressure upon incorporation of Mi‐CK. The increase in surface pressure was dependent on (1) the amount and (2) the oligometric form of Mi‐CK in the subphase, as well as on (3) the initial surface pressure and (4) the phospholipid composition of the monolayer. In this experimental system Mi‐CK was able to interact equally well with both inner and outer mitochondrial membranes.
Spectroscopic characterization of the copper(I)‐thiolate cluster in the DNA‐binding domain of yeast ACE1 transcription factor
Authors: Casas-Finet Jose R. , Hu Stella , Hamer Dean , Karpel Richard L.
Published: July 2002

A polypeptide containing the amino‐terminal region of ACEI (residues 1–122; 122*), the activator of yeast Cu‐methallothionein gene transcription, shows charge‐transfer and metal‐centered UV absorption bands, and orange luminescence which are characteristic of Cu‐cysteinyl thiolate cluster structures. These spectral features are abolished by the Cu(1) complexing agents CN* and diethyldithiocarbamate or exposure to acid, but not by the Cu(II)chelator. EDTA. Binding of the polypeptide to its specific DNA recognition site, but not to calf‐thymus double‐stranded DNA, induces quenching of its Tyr and Cu‐S cluster luminescence emission. The CD spectrum is characteristic of a tightly folded structure that may be organized around the Cu cluster.
Purification and characterization of a carbohydrate‐binding peptide from Bauhinia purpurea lectin
Authors: Yamamoto Kazuo , Konami Yukiko , Kusui Kaoru , Osawa Toshiaki
Published: July 2002

In order to examine the correlation between the amino acid sequence and sugar binding specificity of Bauhinia purpurea lectin (BPA), a galactose and lactose binding lectin, a peptide which interacts with lactose was purified from an Asp‐N endoproteinase digest of BPA by means of affinity chromatography on a column of lactose‐Sepharose. The amino acid sequence of this peptide is Asp‐Thr‐Trp‐Pro‐Asn‐Thr‐Glu‐Trp‐Ser. A tryptic, fragment having the ability to interact with lactose was also purified and found to contain the above sequence, consisting of 9 amino acids. The chemical synthesis of this peptide was carried out by the solid‐phase method and the synthetic peptide was found to exhibit lactose binding activity in the presence of calcium.
A radioiodinated linear vasopressin antagonist: A ligand with high affinity and specificity for V1a receptors
Authors: Schmidt A. , Audigier S. , Barberis C. , Jard S. , Manning M. , Kolodziejczyk A.S. , Sawyer W.H.
Published: July 2002

A linear vasopressin antagonist, Phaa‐D‐Tyr(Me)‐Phe‐Gin‐Asn‐Arg‐Pro‐Arg‐Tyr‐NH2 (Linear AVP Antag) (Phaa = Phenylacetyl), was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. This antagonist appeared to be a highly potent anti‐vasopressor peptide with a pA2 value in vivo of 8.94. It was demonstrated to bind to rat liver membrane preparations with a very high affinity (K
d = 0.06 nM). The affinity for the rat uterus oxytocin receptor was lower (K
i = 2.1 nM), and affinities for the rat kidney‐ and adenohypophysis‐vasopressin receptors were much lower (K
i 47 nM and 92 nM, respectively), resulting in a highly specific vasopressin V18 receptor ligand. Autoradiographical studies using rat brain slices showed that this ligand is a good tool for studies on vasopressin receptor localization and characterization.
Analysis of 13C and 15N CPMAS NMR‐spectra of soil organic matter and composts
Authors: Almendros G. , Fründ R. , Gonzalez-Vila F.J. , Haider K.M. , Knicker H. , Lüdemann H.-D.
Published: July 2002

15N and 13C CPMAS spectra of composted plants are presented. The plants (L. rigidium and Zea mays) were grown in 15N enriched medium and fennented for several months until an approx. 80% of the dry matter was lost. In all 15N spectra the secondary amide/peptide peaks a 87 ppm contributes more than 80% of the total intensity. No new 15N peaks are formed during the fermentation process. Older attempts to assign a significant fraction of humic acid nitrogen to heteroaromatic structures formed in the fermentation process are thus most probably wrong.
Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritima
Authors: Schumann Judith , Wrba Alexander , Jaenicke Rainer , Stetter Karl Otto
Published: July 2002

The hyperthermophilic cubacterium Thermotoga maritima uses starch as a substrate, without releasing amylase activity into the culture medium. The enzyme is associated with the ‘toga’. Its expression level is too low to allow the isolation of the pure enzyme. Using cycloheptaamylose and acarbose affinity chromatography and common chromatographic procedures, two enzyme fractions are obtained. They differ in specificity, pH‐optimum, temperature dependence and stability. Substrate specificity and Ca2* dependence indicate α‐, β‐ and gluco‐amylase activity. Compared with α‐amylase from Bacillus licheniformis (T
max = 75°C), the amylasex from Thermotoga maritima show exceedingly high thermal stability with an upper temperature limit at 95°C. Significant turnover occurs only 70 and 100°C, i.e. in the range of viability of the microorganism.
Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity
Authors: Witzemann Veit , Sakmann Bert
Published: July 2002

The levels of mRNAs coding for the myogenic factors MyoD and myogenin were measured during synapse formulation in developing muscle and in adult muscle, after denervation and reinnervation and after muscle paralysis induced by blocking of neuromuscular transmission by neurotoxins known to alter the density and localization of synaptic proteins such as the acetylcholine receptor (AChR). The mRNA levels of both factors depend on usage of the neuromuscular synapses, but they change to different extents. Myogenin mRNA levels decrease drastically with innervation and increase strongly following blocking of transmission whereas the level of MyoD mRNA showed only a small decrease in response to innervation, denervation of muscle paralysis by neurotoxins. Neither mRNA showed a synapse‐related cellular distribution. The results suggest that nerve‐induced electrical muscle activity determines the cellular ratio of MyoD and myogenin mRNAs in adult muscle.
Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency
Authors: Galanis Maria , Devenish Rodney J. , Nagley Phillip
Published: July 2002

We describe a novel method for enhancing protein import into mitochondria, by tandemly duplicating the N‐terminal cleavable leader peptide using a gene manipulation strategy. The import into isolated yeast mitochondria of passenger proteins (yeast mitochondrial ATP synthase subunits 8 and 9 and some mutagenised derivatives) that show little or no import when endowed with one such leader (that of Neurospora crassa mitochondrial ATP synthase subunit 9) is remarkably improved when the leader is tandemly duplicated. The import of these chimacric proteins bearing a double leader is so rapid that a series of partially processed precursor intermediates accumulates inside the mitochondria before the final proteolytic release of leader sequences from the passenger proteins. It is considered that the duplicated leader greatly accelerates delivery of the import precursors to outer membrane receptor elements and the associated translocation systems, thereby enhancing precursor uptake into mitochondria.
The proteinase yscA‐inhibitor, IA3, gene Studies of cytoplasmic proteinase inhibitor deficiency on yeast physiology
Authors: Schu Peter , Wolf Dieter H.
Published: July 2002

The gene of the proteinase yscA inhibitor IA
3, , of the yeast Saccharamyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library and sequenced. The gene codes for a single protein of 68 amino acids. The structural gene was deleted in vitro by oligonucleotide‐site‐directed mutagenesis. The mutated allele was introduced via homologous recombination into the genome of wild‐type yeast and into the genome of a yeast mutant, which lacks the second cytoplasmic proteinase‐inhibitor, IB
2. The deficiency of either or of both inhibitors has no effect on the cell viability under various physiological conditions. The inhibitor mutants, however, show an increase in the general in vivo protein degradation rate. The IA
3 mutant has a 2–3‐fold increased protein degradation rate in the first 6 h after a shift from rich medium onto starvation‐medium, whereas the IB
2 mutant shows a constantly increased degradation rate of 20–50% under the same conditions. The inhibitor double null mutant has the same protein degradation rate as the IA
3 null mutant. These results suggest an in vivo interaction between the vacuolor endopeptidases and their cytoplasmic inhibitors.
A synthetic β‐casein phosphopeptide and analogues as model substrates for casein kinase‐1, a ubiquitous, phosphate directed protein kinase
Authors: Meggio Flavio , Perich John W. , Reynolds Eric C. , Pinna Lorenzo A.
Published: July 2002

The phosphopeptide Ser (P)‐Ser(P)‐Ser‐(P)‐Glu‐Glu‐Ser11‐Ilc‐Thr, reproducing the 17‐24 segment of β‐casein Λ1 including the seryl residue (Ser‐22) which is targeted by casein kinase‐1 was synthesized and used as model substrate for this enzyme. Its phosphorylation efficiency is actually higher than that of intact β‐casein (similar Vmax
and 14 μM vs 50 μM Km
). Conversely the fully dephosphorylated peptide SSSEESIT is not affected by CK‐1 to any detectable extent and its glutamyl derivative EEEEESIT displays a more than 50‐fold higher Km
and a 5‐fold lower Vmax
as compared to the parent phosphopeptide. The relevance of the individual phosphoseryl residues has been assessed by comparing the phosphorylation efficiencies of the phosphopeptides EESpEESIT, ESpEEESIT and SpEEEESIT; while the first is a substrate almost as good as the tris Ser (P)‐peptide (Km
=62 μM), and the third one is almost as poor as EEEEESIT (Km
=1.55mM), ESpEEESIT displays a intermediate efficiency (Km
=277 μM). These data in conjunction with the finding that the phosphopentapeptide Ser(P)‐Ser(P)‐Ser‐(P)‐Ser‐Ser(P), but neither Ser(P)‐Ser(P)‐Ser‐Ser(P) nor Ser‐Ser(P)‐Ser(P)‐Glu‐Glu and Ser‐Ala‐Ala‐Ser(P)‐Ser(P), is readily phosphorylated by CK‐1, support the concept that CK‐1 is a phosphate directed protein kinase recognizing the Ser(P)‐X‐X‐Ser‐X and, less efficiently, the Ser(P)‐X‐X‐X‐Ser‐X motifs.
Multiple receptor‐like protein tyrosine phosphatases in the form of chondroitin sulfate proteoglycan
Authors: Maeda Nobuaki , Hamanaka Hiroki , Shintani Takafumi , Nishiwaki Taeko , Noda Masaharu
Published: July 2002

The possibility that some of the brain proteoglycans are receptor‐like protein tyrosine phosphatases (PTPases) was investigated. Membrane‐bound proteoglycan fractions were prepared from the postnuclear membrane fraction of 8‐day‐old rat brain by DEAE ion‐exchange chromatography and CsCl density gradient centrifugation. The isolated proteoglycan fractions showed high PTPase specific activities together with the typical PTPase characteristics. Renaturation experiments indicated that chondroitin sulfate proteoglycans with 380‐ and 170‐kDa core proteins carried the PTPase activity. The proteoglycan with 380‐kDa core protein was identified as RPTPβ/ζ bearing HNK‐1 carbohydrate.
Selectivity of DNA polymerases toward α and β nucleotide substrates of d and l series
Authors: Semizarov Dmitry G. , Victorova Lyubov S. , Dyatkina Natalia B. , von Janta-Lipinski Martin , Krayevsky Alexander A.
Published: July 2002

The substrate properties of four carbocyclic d and l nucleoside 5′‐triphosphate analogs toward HIV and AMV reverse transcriptases and terminal deoxynucleotidyl transferase were evaluated. The compounds of the d‐β and l‐β series were found to be terminating substrates for these enzymes, while the derivatives of the d‐α and l‐α series were recognized only by terminal deoxynucleotidyl transferase, suggesting that for the template‐independent enzyme the mutual orientation of the two fragments is of no significance. A hypothesis for binding of nucleotides to the DNA polymerase active center was proposed.
Isolation and characterization of cDNA clones specifically expressed in testicular germ cells
Authors: Tanaka Hiromitsu , Yoshimura Yasuhide , Nishina Yukio , Nozaki Masami , Nojima Hiroshi , Nishimune Yoshitake
Published: July 2002

We have cloned cDNAs involved in germ cell‐specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild‐type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.
Interaction between the zinc(II) and the heparin binding site of the Alzheimer's disease βA4 amyloid precursor protein (APP)
Authors: Multhaup Gerd , Bush Ashley I. , Pollwein Peter , Masters Colin L.
Published: July 2002

The Alzheimer's disease βA4 amyloid precursor protein (APP) has been suggested to be involved in regulation of cell growth, neurite outgrowth and adhesiveness through binding to heparin sulfate proteoglycans. In order to unravel the molecular mechanisms underlying those functions in vitro we show that APP binds in a time dependent and saturable manner to the glycosaminoglycan side‐chains of proteoglycans but not to chondroitinsulfate. We also demonstrate an interaction between the high affinity heparin binding site within the carbohydrate domain of APP and the zinc(II) binding site of APP. We show that the affinity for heparin is increased two‐ to four‐fold in the presence of micromolar zinc(II). Thus micromolar concentrations of zinc(II) appear to be able to modulate the binding of APP to heparin side‐chains of proteoglycans and as shown previously [Science 265 (1994) 1464–1467] to induce the aggregation of soluble amyloid βA4 protein.
Novel bioactive lipodepsipeptides from Pseudomonas syringae: The pseudomycins
Authors: Ballio A. , Bossa F. , Di Giorgio D. , Ferranti P. , Paci M. , Pucci P. , Scaloni A. , Segre A. , Strobel G.A.
Published: July 2002

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon‐generated mutant of a Pseudomonas syringae wild‐type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25‐A and 25‐B, pseudomycins A, B, C, C′ are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to l‐Ser‐d‐Dab‐l‐Asp‐l‐Lys‐l‐Dab‐l‐aThr‐Z‐Dhb‐l‐Asp(3‐OH) ‐l‐Thr(4‐Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N‐terminal Ser. This is in turn N‐acylated by 3,4‐dihydroxytetradecanoate in pseudomycin A, by 3‐hydroxytetradecanoate in pseudomycin B, by 3,4‐dihydroxyhexadecanoate in pseudomycin C, and by 3‐hydroxyhexadecanoate in pseudomycin C′. Some preliminary data on the biological activity of pseudomycin A are reported.
6‐Ketocholestanol abolishes the effect of the most potent uncouplers of oxidative phosphorylation in mitochondria
Authors: Starkov A.A. , Dedukhova V.I. , Skulachev V.P.
Published: July 2002

The effect of a keto‐derivative of cholesterol, namely, 6‐ketocholestanol (5α‐cholestan‐3β‐ol‐6‐one; kCh) on the uncoupling of oxidation and phosphorylation by various uncouplers was studied in rat heart mitochondria. kCh was found to completely abolish the uncoupling effect (the increase in the respiration rate under the respiratory control conditions and the decrease in the membrane potential) caused of FCCP, CCCP and SF6847 and partially by TTFB at low concentrations of uncouplers. It was without effect on the uncoupling by PCP, DNP and palmitate. Carboxyatractylate, a specific inhibitor of the ADP/ATP‐antiporter, was shown to almost completely abolish the uncoupling induced by palmitate and partially by low concentration of TTFB, PCP and DNP. Effects of high concentrations of all these uncouplers as well as of any concentrations of gramicidin proved to be kCh‐ and carboxyatractilate‐insensitive. The data are discussed in terms of the hypothesis on the protein‐mediated mechanism of the protonophorous uncoupling.
Purification of type 1 protein (serine/threonine) phosphatases by microcystin‐Sepharose affinity chromatography
Authors: Moorhead Greg , MacKintosh Robert W. , Morrice Nick , Gallagher Timothy , MacKintosh Carol
Published: July 2002

A microcystin (MC)‐Sepharose column was prepared by addition of 2‐aminoethanethiol to the α,β‐unsaturated carbonyl of the N‐methyldehydroalanine residue of MC‐LR, followed by reaction of the introduced amino group with N‐hydroxysuccinimide‐activated CH‐Sepharose. The MC‐Sepharose bound protein phosphatase‐1 (PPI) with high capacity and purified human PP1γ in one step from E. coli extracts. It was also used to purify forms of PPI bound to myofibrils from skeletal muscle. Two of these comprised PP1 completed to N‐terminal fragments of the M‐subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N‐terminal fragment of the glycogen‐binding (G)‐subunit.
Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils
Authors: Carreras Maria Cecilia , Pargament Griselda A. , Catz Sergio D. , Poderoso Juan J. , Boveris Alberto
Published: July 2002

Nitric oxide (.NO) release, oxygen uptake and hydrogen peroxide (H2O2) production elicited by increasing phorbol 12‐myristate 13‐acetate (PMA) concentrations were measured in human neutrophils. Half‐maximal activities were sequentially elicited at about 0.0001–0.001 μg (.NO) and 0.001‐0.01 μg (H2O2). At saturated PMA concentrations, .NO production, oxygen uptake and H2O2 release were 0.56 ± 0.04, 3.32 ± 0.52 and 1.19±0.17 nmol · min−1 · 106 cells−1. .NO production accounts for about 30% of the total oxygen uptake. Luminol‐dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half‐maximally activated at about 0.001‐0.01 μg . Preincubation with N
G‐monomethyl‐l‐arginine (l‐NMMA) decreased O2 uptake and .NO release but increased H2O2 production, while superoxide dismutase (SOD) increased .NO detection by 30%. Chemiluminescence was also reduced by preincubation with l‐NMMA and/or SOD. The results indicate that .NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of ″NO and O2
.− release favour the formation of other oxidants like peroxynitrite.
Inhibitor analysis of calf thymus DNA polymerases α, δ and ϵ
Authors: Wright George E. , Hübscher Ulrich , Khan Naseema N. , Focher Federico , Verri Annalisa
Published: July 2002

Quantitative effects of inhibitors of the replicative DNA polymerases (pol) α, δ and ϵ from calf thymus are reported under similar assay conditions. Carbonyldiphosphonate was a competitive inhibitor of pols δ and ϵ, with 4‐ to 6‐fold selectivity compared to pol α. Aphidicolin inhibited pols α and δ with 6‐ to 10‐fold selectivity compared to pol ϵ. The ‘butylphenyl’ nucleotides, BuPdGTP and BuAdATP, inhibited pol α with at least 1000‐fold selectivity compared to pols δ and ϵ. The use of these inhibitors under similar assay conditions permits the discrimination of the three enzymes.
Monomeric and dimeric forms of cholesterol esterase from Candida cylindracea
Authors: Kaiser Rudolf , Erman Mary , Duax William L. , Ghosh Debashis , Jörnvall Hans
Published: July 2002

Cholesterol esterase from Candida cylindracea was separated into two fractions, corresponding to a dimeric and a monomeric form. Fingerprint analysis after lysine cleavages shows identical patterns, suggesting lack of primary differences. Crystals obtained from the two proteins differ and suggest the possibility of an equilibrium between the two forms, influenced by the substrate cholesterol linoleate, which appears to stabilize the more active, dimeric form. All crystals have dimers as the asymmetric unit. The primary structure of the enzyme was determined at the peptide level and shows only one difference, Leu‐350 instead of Ile, from a DNA‐deduced amino acid sequence, and conservation of features typical for cholesterol esterases characterized.