bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane fluidization by ether, other anesthetics, and certain agents abolishes P‐glycoprotein ATPase activity and modulates efflux from multidrug‐resistant cells
Authors: Regev Ronit , Assaraf , Yehuda G. , Eytan Gera D.
Published: March 2003

The anesthetics benzyl alcohol and the nonaromatic chloroform and diethyl ether, abolish P‐glycoprotein (Pgp) ATPase activity in a mode that does not fit classical competitive, noncompetitive, or uncompetitive inhibition. At concentrations similar to those required for inhibition of ATPase activity, these anesthetics fluidize membranes leading to twofold acceleration of doxorubicin flip‐flop across lipid membranes and prevent photoaffinity labeling of Pgp with [125I]‐iodoarylazidoprazosin. Similar concentrations of ether proved nontoxic and modulated efflux from Pgp‐overexpressing cells. A similar twofold acceleration of doxorubicin flip‐flop rate across membranes was observed with neutral mild detergents, including Tween 20, Nonidet P‐40 and Triton X‐100, and certain Pgp modulators, such as verapamil and progesterone. Concentrations of these agents, similar to those required for membrane fluidization, inhibited Pgp ATPase activity in a mode similar to that observed with the anesthetics. The mode of inhibition, i.e. lack of evidence for classical enzyme inhibition and the correlation of Pgp ATPase inhibition with membrane fluidization over a wide range of concentrations and structures of drugs favors the direct inhibition of Pgp ATPase activity by membrane fluidization. The unusual sensitivity of Pgp to membrane fluidization, as opposed to acceleration of ATPase activity of ion transporters, could fit the proposed function of Pgp as a ‘flippase’, which is in close contact with the membrane core.
Preferential activation of iron regulatory protein‐2 in cell lines as a result of higher sensitivity to iron
Authors: Recalcati Stefania , Conte , Dario , Cairo Gaetano
Published: March 2003

Iron regulatory proteins (IRP)‐1 and 2 are cytoplasmic mRNA‐binding proteins that control intracellular iron homeostasis by regulating the translation of ferritin mRNA and stability of transferrin receptor mRNA in an iron‐dependent fashion. Although structurally and functionally similar, the two IRP are different in their mode of regulation, pattern of tissue expression and modulation by multiple factors, such as bioradicals. In the present study RNA bandshift assays demonstrated that IRP‐2, but not IRP‐1, activity was higher in cultured cells than in tissues. Increased expression of IRP‐2 in cell lines was not related to immortalization and differentiation but seemed associated to cell proliferation, although not closely dependent on cell growth rate. As a growing cell consumes more iron than its quiescent counterpart, we assessed the iron status of cell lines and found that ferritin content was lower than in tissues. Analysis of IRP activity in cell lines supplemented with heme or non‐heme iron and in livers of iron‐loaded and iron‐deficient rats indicated that IRP‐2 responds more promptly than IRP‐1 to modulations of iron content. We propose that enhanced IRP‐2 activity in cultured cells could be due to a proliferation‐dependent, relative iron deficiency that is sensed first by IRP‐2.
Investigation of the structure of spinach photosystem II reaction center complex
Authors: Tsiotis Georgios , Psylinakis Manolis , Woplensinger Bettina , Lustig Ariel , Engel Andreas , Ghanotakis Demetrios
Published: March 2003

The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single‐particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi‐twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.
Characterization of thermostable RecA protein and analysis of its interaction with single‐stranded DNA
Authors: Kato Ryuichi , Kuramitsu Seiki
Published: March 2003

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 °C but not at 37 °C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single‐stranded DNA (ssDNA)‐dependent ATPase activity, and its activity was maximal at 65 °C. The kinetic parameters, Km and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 m
m and 0.60 s–1 at 65 °C, and 0.34 m
m and 0.28 s–1 at 37 °C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 °C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double‐stranded DNAs (dsDNA), poly(dT)·poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 °C. At 37 °C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 °C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 µm at 25 °C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.
Formation of fatty acid ethyl esters in rat liver microsomes
Authors: Diczfalusy Margareta A. , Björkhem Ingemar , Einarsson Curt , Alexson Stefan E. H.
Published: March 2003

Fatty acid ethyl esters have been detected in high concentrations in organs commonly damaged by alcohol abuse and are regarded as being important non‐oxidative metabolites of ethanol. The formation of fatty acid ethyl esters (FAEEs) has been ascribed to two enzymic activities, acyl‐CoA : ethanol O‐acyltransferase (AEAT) and FAEE synthase.

 In the present study we determined AEAT and FAEE synthase activities in isolated rat liver microsomes and further characterized the microsomal AEAT activity in more detail. The determined AEAT and FAEE synthase activities were found to be similar (about 1.7 nmol·min–1·mg–1). However, the AEAT activity was increased about sixfold by the addition of 250 µm bis‐(4‐nitrophenyl) phosphate (a serine esterase inhibitor) to the incubation whereas FAEE synthase activity was completely inhibited. p‐Hydroxymercuribenzoic acid (a cysteine‐reacting compound) also stimulated AEAT activity (about fourfold) but had no effect on FAEE synthase activity. The effects of the inhibitors suggest that the formation of FAEEs by AEAT was severely counteracted by enzymic hydrolysis of the substrate (acyl‐CoA) and to a lesser extent the product by serine esterases. dl‐Melinamide, a hypocholesterolaemic drug, was found to be a very potent inhibitor of AEAT activity with an IC50 value of about 2.5 µm.

 Furthermore, we compared the activities of two purified microsomal carboxylesterases, ES‐4 and ES‐10, and identified ES‐4 as the enzyme responsible for hydrolysis of FAEEs. The two carboxyesterases were also tested for FAEE synthase activity, but neither had any detectable activity. Esterase ES‐4 was found to have some AEAT activity, but it was low.

 When measured under optimal conditions without competing hydrolysis the capacity of AEAT is thus considerably higher than FAEE synthase and the results are consistent with an important role for AEAT in the formation of ethyl esters. As the ratio acyl‐CoA/non‐esterified fatty acids is high under normal conditions, AEAT is probably the most important enzyme in fatty acid ethyl ester formation.
Binding of heptapeptides or unfolded proteins to the chimeric C‐terminal domains of 70‐kDa heat shock cognate protein
Authors: Wu Shin‐Jen , Wang Chung
Published: March 2003

Seventy‐kDa heat shock cognate protein (hsc70) and its homologs in bacteria, yeast and vertebrates are known to form complexes with S‐carboxymethyl‐α‐lactalbumin (CMLA), an unfolded protein; and, this activity has been attributed to its C‐terminal 30‐kDa domain. Herein, we show that hsc70s isolated from the seeds of mung bean and peas, however, are not effective in complexing with CMLA, and that the 30‐kDa domain of Arabidopsis hsc70 (At30) cannot form stable complexes with CMLA either. Moreover, chimeric 30‐kDa domains, either composed of rat 18‐kDa and Arabidopsis 10‐kDa subdomains (R18At10) or with Arabidopsis 18‐kDa and rat 10‐kDa subdomains (At18R10), were prepared and tested for their ability to complex with CMLA or a heptapeptide FYQLALT. At18R10 cannot complex with both CMLA and FYQLALT. On the other hand, R18At10 is capable of forming complexes with FYQLALT at a level similar to that of the rat 30‐kDa domain (R30). R18At10 also forms complexes with CMLA, but the amount of the R18At10/CMLA complexes is much less than that of R30/CMLA. The results imply that the 18‐kDa subdomain dictates the binding specificity for heptapeptide, and that the C‐terminal 10‐kDa subdomain may also provide some selection or restriction for unfolded proteins to form complexes with hsc70.
Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1
Authors: Darnis Sophie , Juge Nathalie , Marino Cristina , Avilés Francesc Xavier , Puigserver Antoine , Chaix Jean‐Claude , Guo Xiao‐Jun
Published: March 2003

A full‐length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa‐long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three‐dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg·L−1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35‐kDa active enzyme.
The structure of an entire noncovalent immunoglobulin kappa light‐chain dimer (Bence‐Jones protein) reveals a weak and unusual constant domains association
Authors: Roussel Alain , Spinelli Silvia , Déret Sophie , Navaza Jorge , Aucouturier Pierre , Cambillau Christian
Published: March 2003

Monoclonal free light chains secreted in immunoproliferative disorders are frequently involved in renal complications, including a specific proximal tubule impairment, Fanconi’s syndrome. The latter is characterized in most cases by intracellular crystallization including a light‐chain variable‐domain fragment which resists lysosomal proteases. Bence‐Jones protein (BJP) DEL was isolated from a patient with myeloma‐associated Fanconi’s syndrome. The crystal structure of this human κ immunoglobulin light‐chain noncovalent dimer was determined using molecular replacement with the structure of molecule REI, as the variable domain, and that of BJP LOC as the constant domain. To our knowledge, DEL is the first complete κ BJP structure described to date. The R‐factor is 20.7% at 2.8 Å resolution. The BJP DEL dimer was compared with other light‐chain dimers and with Fab fragments with a κ light chain. Although the domain‐folding pattern was similar, the relative positions of the constant domains differed. BJP DEL showed a noncanonical quaternary structural arrangement which may be attributable to the poor CL–CL affinity and lack of an interchain disulfide bridge, combined with the conformational editing effect of the crystal‐packing forces. Our results suggest that, in the absence of a disulfide bridge, most BJP CLs are probably mobile in solution. This may explain their high susceptibility to proteases and the absence of naturally occurring crystals for these dimers. Furthermore, these findings of an unusual quaternary structure of an immunoglobulin light‐chain association extend our knowledge about the large and highly diverse structures of the immunoglobulin superfamily.
Mapping of structural determinants for the oligomerization of p58, a lectin‐like protein of the intermediate compartment and cis‐Golgi
Authors: Lahtinen Ulla , Svensson Kerstin , Pettersson Ralf F.
Published: March 2003

Shortly after synthesis, p58, the rat homologue of the mannose‐binding lectin ERGIC‐53/MR60, which localizes to pre‐Golgi and cis‐Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane‐proximal half of the lumenal domain consists of four predicted α‐helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled‐coil interactions. Using single‐stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the α‐helical domains was probed by internal deletions. The N‐terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild‐type protein. The two membrane‐proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual α‐helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to d‐mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild‐type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58.
Hydroxyl‐radical production in physiological reactions
Authors: Chen Si‐xue , Schopfer Peter
Published: March 2003

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2·−) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (˙OH) from H2O2 in the presence of O2·−. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2·− into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of ˙OH from O2·− and H2O2 (iron‐catalyzed Haber–Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2·− and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The ˙OH‐producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2·− and ˙OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1–2 orders of magnitude more active than Fe‐EDTA, an inorganic catalyst of the Haber–Weiss reaction. Particularly high ˙OH‐producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase‐type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the ˙OH‐producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.
A 75‐kDa Na+,K+‐ATPase competitive inhibitor protein isolated from rat brain cytosol binds to a site different from the ouabain‐binding site
Authors: Roy Koushik , Mandal Atin K. , Sen Parimal C.
Published: March 2003

A Na+,K+‐ATPase inhibitor protein has been purified to homogeneity from rat brain cytosol by ammonium sulphate precipitation, DEAE anion‐exchange chromatography and hydroxyapatite adsorption column chromatography. The purified protein migrates as a single polypeptide band of 75 kDa on 7.5% SDS/PAGE. Amino acid composition data shows the presence of a high number of acidic amino acids in the molecule in relation to the pI value of 4.6. The inhibitor binds Na+,K+‐ATPase reversibly and blocks ATP binding sites at micromolar concentrations with an I50 of ≈700 nm. As a result, formation of the phosphorylated intermediate of Na+,K+‐ATPase is hindered in the presence of the inhibitor. It does not affect p‐nitrophenylphosphatase activity. Tryptophan fluorescence studies and CD analysis suggest conformational changes of Na+,K+‐ATPase on binding to the inhibitor.
Effect of cations on purine·purine·pyrimidine triple helix formation in mixed‐valence salt solutions
Authors: Floris Romina , Scaggiante Bruna , Manzini Giorgio , Quadrifoglio Franco , Xodo Luigi E.
Published: March 2003

The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex‐forming oligonucleotides, designed to bind to biologically relevant polypurine–polypyrimidine sequences occurring in the promoters of the murine Ki‐ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent‐ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self‐association equilibria involving the G‐rich triplex‐forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K+. However, footprinting experiments also showed that the inhibitory effect of K+ on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2+, which upon coordinating to N7 of guanine are expected to enhance hydrogen‐bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G‐rich triplex‐forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex‐forming oligonucleotides.
Initiation of galactosaminoglycan biosynthesis
Authors: Moses Jonatan , Oldberg Åke , Fransson Lars‐Åke
Published: March 2003

By using various radiolabelled precursors, glycosylation and phosphorylation of decorin in a rat fibroblast cell line was investigated in the presence of increasing concentrations of p‐nitrophenyl‐O‐β‐d‐xylopyranoside. Decorin core protein glycanation was suppressed to ≈25% of the normal level in the presence of 2 mm and 3 mm xyloside. Glycans/saccharides were released from the core protein and size‐separated by gel chromatography. The intracellular decorin obtained from cells treated with 2 mm xyloside was substituted with Xyl and also with Gal‐Xyl and Gal‐Gal‐Xyl, but not with longer saccharides. Only the trisaccharide contained an almost fully phosphorylated Xyl. We conclude that galactosylation of endogenous, xylosylated decorin and exogenous xyloside probably follow separate pathways or that xylosides and early decorin glycoforms are kept separated. At the addition of the first glucuronic acid the two pathways seem to merge and dephosphorylation of decorin takes place. Xyloside‐primed and secreted galactosaminoglycan chains produced simultanously retained phosphorylated Xyl. Inadequate dephosphorylation could be due to excess substrate or to a short transit.time. As shown previously [Moses, J., Oldberg, Å., Eklund, E. & Fransson, L.‐Å. (1997) Eur. J. Biochem.248, 767–774], brefeldin A‐arrested decorin is substituted with the linkage‐region extended with an undersulphated and incomplete galactosaminoglycan chain. In cells treated with this drug, xylosides were unable to prime galactosaminoglycan synthesis and unable to inhibit glycosylation and phosporylation of decorin.
Structural and serological studies on the O‐antigen of Proteus mirabilis O14, a new polysaccharide containing 2‐[(R)‐1‐carboxyethylamino]ethyl phosphate
Authors: Perepelov Andrei V. , Ujazda Elzbieta , Senchenkova Sof’ya N. , Shashkov Alexander S. , Kaca Wieslaw , Knirel Yuriy A.
Published: March 2003

An O‐specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain d‐galactose, 2‐acetamido‐2‐deoxy‐d‐glalactose, phosphate, N‐(2‐hydroxyethyl)‐d‐alanine (d‐AlaEtn), and O‐acetyl groups. Studies of the initial and O‐deacetylated polysaccharides using one‐ and two‐dimensional 1H‐ and 13C‐NMR spectroscopy, including COSY, TOCSY, NOESY, H‐detected 1H,13C heteronuclear multiple‐quantum coherence, and heteronuclear multiple‐bond correlation experiments, demonstrated the following structure of the repeating unit:

This is the second bacterial polysaccharide reported to contain α‐d‐Galp6PAlaEtn, whereas the first one was the O‐antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska‐Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem.,188, 645–651]. Anti‐(P. mirabilis O14) serum cross‐reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O‐antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O‐antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.
Alkyl‐dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes
Authors: Biermann Jan , Just Wilhelm W. , Wanders Ronald J. A. , van den Bosch Henk
Published: March 2003

Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl‐GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross‐linking. After cross‐linking and immunoblot analysis alkyl‐GrnP synthase could be detected in a 210‐kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two‐dimensional SDS/PAGE demonstrated that GrnP‐acyltransferase is also cross‐linked in a 210‐kDa complex. Co‐immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl‐GrnP synthase can form a homotrimeric complex in the absence of GrnP‐acyltransferase as was demonstrated by immunoblot analysis after cross‐linking experiments with either GrnP‐acyltransferase deficient human fibroblast homogenates or recombinant (His)6‐tagged alkyl‐GrnP synthase. We conclude that alkyl‐GrnP synthase interacts selectively with GrnP‐acyltransferase in a heterotrimeric complex and in the absence of GrnP‐acyltransferase can also form a homotrimeric complex.
Interaction of the GM2‐activator protein with phospholipid–ganglioside bilayer membranes and with monolayers at the air–water interface
Authors: Giehl Andrea , Lemm Thorsten , Bartelsen Oliver , Sandhoff Konrad , Blume Alfred
Published: March 2003

Differential scanning calorimetry (DSC) and film balance measurements were performed to study the interactions of the GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glc1→1′Cer (GM2)‐activator protein with phospholipid/ganglioside vesicles and monolayers. The nonglycosylated form of the GM2‐activator protein, added to unilamellar lipid vesicles of different composition, causes differential effects on the gel to liquid–crystalline phase transition peaks. The phase transition temperature (Tm) of pure dimyristoylglycerophosphocholine (DMPC) bilayer is slightly decreased. When lipids which specifically bind the GM2‐activator protein are incorporated into the vesicles (e.g. a sulfatide or gangliosides) a shoulder in the thermograms at higher temperatures is observed, indicating an increase of the stability of the gel phase in relation to the liquid‐crystalline phase. We also studied the surface activity of a glycosylated and a nonglycosylated GM2‐activator protein at the air–water interface. The glycosylated form showed a slightly lower surface activity than the GM2‐activator protein without oligosaccharide moiety. When the GM2‐activator protein is added to the sub‐phase of a surface covered with a lipid monolayer, it can only insert into the monolayer and reach the air–water interface below a monolayer pressure of 25 mN·m−1, depending on the lipid composition, and not when the monolayers are at the bilayer equivalence pressure of 30–35 mN·m−1. Particularly for Galβ1→3GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glc1→1′Cer (GM1) and GM2 containing films, the critical pressures (πcrit) when no additional increase in surface pressure is observed after addition of the protein into the subphase, are much lower. This leads to the conclusion that binding of the GM2 activator protein to the ganglioside headgroups prevents the protein from reaching the air–water interface. The protein is then located preferentially at the lipid–water interface and cannot penetrate into the chain region.
Hydrophobic sequences can substitute for the wild‐type N‐terminal sequence of cystatin A (stefin A) in tight binding to cysteine proteinases
Authors: Ylinenjärvi Karin , Widersten Mikael , Björk Ingemar
Published: March 2003

A phage‐display library of the cysteine‐proteinase inhibitor, cystatin A, was constructed in which variants with the four N‐terminal amino acids randomly mutated were expressed on the surface of filamenteous phage. Screening of this library for binding to papain gave predominantly variants with a glycine residue in position 4. This finding is in agreement with previous conclusions that glycine in this position is essential for tight binding of cystatin A to cysteine proteinases by allowing optimal interaction of the N‐terminal region of the inhibitor with the enzyme. In contrast, the first three residues of the variants obtained by the screening were more variable. Two variants were identified with similar affinities for papain as the wild‐type inhibitor, but with these residues, Val‐Phe‐Thr‐ or Ile‐Leu‐Leu, differing appreciably from those of the wild‐type, Met‐Ile‐Pro. Other sequences of the N‐terminal region, presumably mainly hydrophobic, can thus substitute for the wild‐type sequence and contribute similar energy to the inhibitor–proteinase interaction. The two variants binding tightly to papain differed in their affinity for cathepsin B, demonstrating that cystatin variants with increased selectivity for a particular target cysteine proteinase can be obtained by phage‐display technology.
Human high‐Km 5′‐nucleotidase
Authors: Rampazzo Chiara , Gazziola Cinzia , Ferraro Paola , Gallinaro Lisa , Johansson Magnus , Reichard Peter , Bianchi Vera
Published: March 2003

5′‐Nucleotidases participate, together with nucleoside kinases, in substrate cycles involved in the regulation of deoxyribonucleotide metabolism. Three major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol. The two cytosolic classes have been named high‐Km nucleotidases and 5′(3′)‐nucleotidases. Starting from two plasmids with partial sequences (Oka, J., Matsumoto, A., Hosokawa, Y. & Inoue, S. (1994) Biochem. Biophys. Res. Commun.205, 917–922) we cloned the complete cDNA of the human high‐Km nucleotidase into vectors suitable for transfection of Escherichia coli or mammalian cells. After transfection, E. coli overproduced large amounts of the enzyme. Most of the enzyme was present in inclusion bodies that also contained many partially degraded products of the protein. Part of the enzyme, corresponding to ≈2% of the soluble proteins, was in a soluble active form. Stably transfected human 293 cells were obtained with a vector where the 3′‐end of the nucleotidase coding sequence is linked to the 5′‐end of the green fluorescent protein coding sequence. Several green clones overproduced both mRNA and fusion protein. Two clones with 10‐fold higher enzyme activity were analyzed further. The nucleotidase activity of cell extracts showed the same substrate specificity and allosteric regulation as the high‐Km enzyme. The growth rate of the two clones did not differ from the controls. The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate dideoxyinosine. Both ribonucleoside and deoxyribonucleoside triphosphate pools were decreased slightly, suggesting participation of the enzyme in their regulation.
The reduction state of the Q‐pool regulates the electron flux through the branched respiratory network of Paracoccus denitrificans
Authors: Otten Marijke F. , Reijnders Willem N. M. , Bedaux Jacques J. M. , Westerhoff Hans V. , Krab Klaas , Van Spanning Rob J. M.
Published: March 2003

In this work we demonstrate how the reduction state of the Q‐pool determines the distribution of electron flow over the two quinol‐oxidising branches in Paracoccus denitrificans: one to quinol oxidase, the other via the cytochrome bc1 complex to the cytochrome c oxidases. The dependence of the electron‐flow rate to oxygen on the fraction of quinol in the Q‐pool was determined in membrane fractions and in intact cells of the wild‐type strain, a bc1‐negative mutant and a quinol oxidase‐negative mutant. Membrane fractions of the bc1‐negative mutant consumed oxygen at significant rates only at much higher extents of Q reduction than did the wild‐type strain or the quinol oxidase‐negative mutant. In the membrane fractions, dependence on the Q redox state was exceptionally strong corresponding to elasticity coefficients close to 2 or higher. In intact cells, the dependence was weaker. In uncoupled cells the dependence of the oxygen‐consumption rates on the fractions of quinol in the Q‐pool in the wild‐type strain and in the two mutants came closer to that found for the membrane fractions. We also determined the dependence for membrane fractions of the wild‐type in the absence and presence of antimycin A, an inhibitor of the bc1 complex. The dependence in the presence of antimycin A resembled that of the bc1‐negative mutant. These results indicate that electron‐flow distribution between the two quinol‐oxidising branches in P. denitrificans is not only determined by regulated gene expression but also, and to a larger extent, by the reduction state of the Q‐pool.
Motor and cargo interactions
Authors: Sheetz Michael P.
Published: March 2003

The movements of intracellular cargo along microtubules within cells are often saltatory or of short duration. Further, calculations of the fraction of membrane vesicles that are moving at any period, indicate that active motor complexes are rare. From observations of normal vesicle traffic in cells, there appears to be position‐dependent activation of motors and a balance of traffic in the inward and outward directions. In‐vitro binding of motors to cargo is observed under many conditions but motility is not. Multi‐component complexes appear to be involved in producing active organelle movements by a graded activation system that is highly localized in the cell. The basis of the activation of motility of the organelle motor complexes is still unknown but phosphorylation has been implicated in many systems. In the case of the motor‐binding protein, kinectin, it has been linked to active organelle movements powered by conventional kinesin. From the coiled‐coil structure of kinectin and the coiled‐coil tail of kinesin, it is postulated that a coiled‐coil assembly is responsible for the binding interaction. Many other cargoes are transported but the control of transport will be customized for each function, such as axonemal rafts or cytoskeletal complexes. Each function will have to be analyzed separately and motor activity will need to be integrated into the specific aspects of the function.
Investigation on the detergent role in the function of secondary quinone in bacterial reaction centers
Authors: Agostiano Angela , Milano Francesco , Trotta Massimo
Published: March 2003

In this paper are reported studies on the detergent role in isolated reaction centers (RC) from Rhodobacter sphaeroides, over a large range of lauryldimethylamino‐N‐oxide (LDAO) concentrations, in influencing the thermodynamics of the quinone exchange reaction as well as the protein aggregation. The occurrence of the quinone exchange reaction between the QB‐binding site (where QB is the second quinone molecule of two in the RC) and the ubiquinone 0 dissolved in the different environments (water, LDAO micelles and detergent phase of the protein–detergent complex) has also been analyzed. Measurements carried out in QB‐depleted RC to which exogenous quinone has been added show that the relative amplitudes of the slow and fast phase of the recombination reaction depend on this parameter. The overall amount of the restored QB‐functionality is affected by the concentration of the LDAO in solution. Interpolation of the titration curves with a quadratic function obtained by simple considerations allowed the binding constant of UQ0 to the QB‐binding site to be calculated. From the fitting procedure, the distribution of the quinone in the different environments present in solution was evaluated, indicating that the exchange reaction can take place only between the QB‐site and the detergent phase. The dependence of the quinone pool size upon the volume of the phase in which the interacting quinone is solubilized is also discussed. The increasing difficulty in saturating the QB‐pocket above the LDAO critical micellar concentration is finally related to the association of protein–detergent complexes to form large protein clusters.
Phosphorylation control of cardiac acetyl‐CoA carboxylase by cAMP‐dependent protein kinase and 5′‐AMP activated protein kinase
Authors: Dyck Jason R. B. , Kudo Naomi , Barr Amy J. , Davies Stephen P. , Hardie D. Grahame , Lopaschuk Gary D.
Published: March 2003

Acetyl‐CoA carboxylase (ACC) is regarded in liver and adipose tissue to be the rate‐limiting enzyme for fatty acid biosynthesis; however, in heart tissue it functions as a regulator of fatty acid oxidation. Because the control of fatty acid oxidation is important to the functioning myocardium, the regulation of ACC is a key issue. Two cardiac isoforms of ACC exist, with molecular masses of 265 kDa and 280 kDa (ACC265 and ACC280). In this study, these proteins were purified from rat heart and used in subsequent phosphorylation and immunoprecipitation experiments. Our results demonstrate that 5′ AMP‐activated protein kinase (AMPK) is able to phosphorylate both ACC265 and ACC280, resulting in an almost complete loss of ACC activity. Although cAMP‐dependent protein kinase phosphorylated only ACC280, a dramatic loss of ACC activity was still observed, suggesting that ACC280 contributes most, if not all, of the total heart ACC activity. ACC280 and ACC265 copurified under all experimental conditions, and purification of heart ACC also resulted in the specific copurification of the α2 isoform of the catalytic subunit of AMPK. Although both catalytic subunits of AMPK were expressed in crude heart homogenates, our results suggest that α2, and not α1, is the dominant isoform of AMPK catalytic subunit regulating ACC in the heart. Immunoprecipitation studies demonstrated that specific antibodies for both ACC265 and ACC280 were able to coimmunoprecipitate the alternate isoform along with the α2 isoform of AMPK. Taken together, the immunoprecipitation and the purification studies suggest that the two isoforms of ACC in the heart exist in a heterodimeric structure, and that this structure is tightly associated with the α2 subunit of AMPK.
Roles of key functional groups in ω‐conotoxin GVIA
Authors: Flinn James P. , Pallaghy Paul K. , Lew Michael J. , Murphy Roger , Angus James A. , Norton Raymond S.
Published: March 2003

The contributions of various functional groups to the pharmacophore of the N‐type calcium‐channel blocker, ω‐conotoxin GVIA (GVIA), have been investigated using structural and in‐vitro functional studies of analogues substituted at one or two positions with non‐native residues. In most cases the structure of the analogue was shown to be native‐like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two‐dimensional NMR. Three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N‐type calcium‐channel activity. The data provide a more detailed picture of the roles in GVIA structure and activity of the crucial Lys2 and Tyr13, as well as all other positively charged residues, Tyr22, the hydroxyproline residues and the C‐terminal amido moiety, many of which were identified as being important for activity in an alanine scan [Lew et al. (1997) J. Biol. Chem.272, 12014–12023]. Substitutions of Lys2 with nonstandard amino acids and arginine quantified the roles of the length and charge of the Lys side chain. The orientation of the Tyr13 side chain and its hydroxyl moiety was shown to be important by substitution with d‐Tyr and the d‐form and l‐form of the constrained analogue 7‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hyp21 hydroxyl groups, investigated by proline substitutions, appear to be more structural (as monitored by NMR) than functional, although small decreases in potency were observed in some assays. The reversibility of the channel blockade was also studied, and several analogues with faster wash‐out characteristics than native GVIA were identified. Rapid reversibility (as in the case of ω‐conotoxin MVIIA) may be beneficial for therapeutic applications. Disubstituted analogues revealed some interesting cooperative effects, which were not predicted from single‐residue substitutions. A disubstituted chimera of GVIA and ω‐conotoxin MVIIA was more potent than either native molecule. The more detailed description of the GVIA pharmacophore obtained here provides a better basis for the future design of truncated peptide and peptidomimetic analogues.
Characterization of MB‐1
Authors: Hefford Mary Alice , Dupont Claude , MacCallum Jillian , Parker Matthew H. , Beauregard Marc
Published: March 2003

MB‐1 is a de‐novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four‐helix bundle protein. MB‐1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases [Beauregard, M., Dupont, C., Teather, R.M. & Hefford, M.A. (1995) Bio/Technology13, 974]. Here we report an analysis of the secondary, tertiary and quaternary structures in MB‐1 using circular dichroism, fluorospectroscopy and size‐exclusion chromatography. Our data indicate that the MB‐1 structure is close to the target structure, an α‐helical bundle, in many respects and is highly helical in solution. The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design.

Furthermore, MB‐1 was found to be native‐like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB‐1 has very low affinity for 8‐anilino‐1‐naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins. On the other hand, our analysis revealed two aspects that command further attention. The folding stability of MB‐1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size‐exclusion chromatography experiments and analysis of MB‐1 denaturation indicate that MB‐1 is dimeric, not monomeric as designed. In light of these results, the utility and the current limitations of our design approach are discussed.
New inhibitors of Helicobacter pylori urease holoenzyme selected from phage‐displayed peptide libraries
Authors: Houimel Mehdi , Mach Jean‐Pierre , Corthésy‐Theulaz Irène , Corthésy Blaise , Fisch Igor
Published: March 2003

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25‐mer combinatorial library and two newly constructed random 6‐mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24‐mer TFLPQPRCSALLRYLSEDGVIVPS and 6‐mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 µm for the 24‐mer and 30 µm for the 6‐mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.
Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum
Authors: Struglics André , Håkansson Gunilla
Published: March 2003

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4‐kDa soluble protein was phosphorylated in the presence of EDTA when [γ‐32P]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4‐kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4‐kDa protein was purified from mitochondrial soluble proteins using ATP‐agarose and anion exchange chromatography. Amino‐acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C‐terminal of NDPKs. Acid and alkali treatments of [32P]‐labelled pea mitochondrial NDPK indicated the presence of acid‐stable as well as alkali‐stable phosphogroups. Thin‐layer chromatography experiments revealed serine as the acid‐stable phosphogroup. The alkali‐stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.
O‐linked N‐acetylglucosamine levels in cerebellar neurons respond reciprocally to pertubations of phosphorylation
Authors: Griffith Lee S. , Schmitz Brigitte
Published: March 2003

The novel intracellular carbohydrate O‐linked N‐acetylglucosamine (O‐GlcNAc) is present on proteins ranging from those of viruses to those of humans and include cytosolic, nuclear and plasma‐membrane proteins. In this report we have examined the effect of manipulation of phosphorylation on the levels of O‐GlcNAc in cerebellar neurons from early postnatal mice. Our results indicate a reciprocal response of O‐GlcNAc levels to phosphorylation. Activation of protein kinase A or C, for example, results in reduced levels of O‐GlcNAc specifically in the fraction of cytoskeletal and cytoskeleton‐associated proteins, while inhibition of the same kinases results in increased levels of O‐GlcNAc. These data are in keeping with a reciprocal action of O‐GlcNAc with respect to phosphorylation and suggest that this modification may have a role in signal transduction.
Genomic origin and transcriptional regulation of two variants of cGMP‐binding cGMP‐specific phosphodiesterases
Authors: Kotera Jun , Fujishige Kotomi , Imai Yuji , Kawai Eri , Michibata Hideo , Akatsuka Hiroyuki , Yanaka Noriyuki , Omori Kenji
Published: March 2003

We have reported alternative splice variants of cGMP‐binding cGMP‐specific phosphodiesterases (PDE5A), i.e. rat PDE5A2, human PDE5A1, canine PDE5A1 and PDE5A2, which possess distinct N‐terminal sequences. In this study, the DNA sequences corresponding to the unique N‐terminal portions of PDE5A1 and PDE5A2 were shown to be tandemly located upstream of exons encoding the common region of PDE5A in both human and rat PDE5A genes. The presence of human PDE5A2 and rat PDE5A1 transcripts in lung was confirmed by reverse transcriptase–PCR. These results indicated that two variant forms of PDE5A exist in humans, canines and rats. We examined the tissue distribution of the two variants of human PDE5A in adult and fetal humans. The patterns of expression of the two alternatively spliced transcripts of human PDE5A in human tissues differed. Many putative regulatory elements including cAMP response elements were observed in the 5′‐untranslated region and intron of the PDE5A gene. The levels of the PDE5A transcripts, especially the PDE5A2 transcripts, were increased by a cAMP analogueue in cultured rat vascular smooth muscle cells, indicating that the PDE5A2 is an inducible variant of PDE5A in rats.