bluuebunny/crossref_metadata_2025 huggingface dataset:
Carboxyl‐terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin
Authors: SENO Masaharu , SASADA Reiko , KUROKAWA Tsutomu , IGARASHI Koichi
Published: March 2005

The carboxyl‐terminal sequence of basic fibroblast growth factor (bFGF) is rich in basic amino acid residues, a common characteristic amongst fibroblast growth factors, and is considered to contribute greatly to the binding to negatively charged extracellular matrixes such as heparin. To study the relationship between the affinity for heparin and the carboxyl‐terminal structure of bFGF, amino‐ or carboxyl‐terminal truncated molecules were produced in Escherichia coli using recombinant DNA techniques. These terminally truncated bFGFs were applied to a heparin‐affinity HPLC column. Truncation of more than six amino acid residues from the carboxyl‐terminal made the bFGF produced in E. coli markedly difficult to solubilize and weakened its affinity for heparin, though bFGF having up to 46 amino acids removed showed significant stimulation of the DNA synthesis of BALB/c3T3 cells. This stimulation of the DNA synthesis was also recognized by the bFGF having 40 amino acids removed from its amino‐terminal, while the affinity of this peptide for heparin has been shown to be equal to that of the mature bFGF (146 amino acids). These results show that the affinity of bFGF for heparin depends significantly on its carboxyl‐terminal structure and that the essential part for receptor binding is present between Asp41 and Ser100. Moreover, it suggests that the Phe139Leu140Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule.
The contacts of yeast tRNASer with seryl‐tRNA synthetase studied by footprinting experiments
Authors: DOCK‐BREGEON Anne‐Catherine , GARCIA Angela , GIEGÉ Richard , MORAS Dino
Published: March 2005

Yeast tRNASer is a member of the class II tRNAs, whose characteristic is the presence of an extended variable loop. This additional structural feature raises questions about the recognition of these class II tRNAs by their cognate synthetase and the possibility of the involvement of the extra arm in the recognition process. A footprinting study of yeast tRNASer complexed with its cognate synthetase, yeast seryl‐tRNA synthetase (an α2 dimer), was undertaken. Chemical (ethynitrosourea) and enzymatic (nucleases S1 and V1) probes were used in the experiments. A map of the contact points between the tRNA and the synthetase was established and results were analyzed with respect to a three‐dimensional model of yeast tRNASer. Regions in close vicinity with the synthetase are clustered on one face of tRNA. The extra arm, which is strongly protected from chemical modifications, appears as an essential part of the contact area. The anticodon triplet and a large part of the anticodon arm are, in contrast, still accessible to the probes when the complex is formed. These results are discussed in the context of the recognition of tRNAs in the aminoacylation reaction.
Characterization of a soluble form of human CD4
Authors: HARRIS Reed J. , CHAMOW Steven M. , GREGORY Timothy J. , SPELLMAN Michael W.
Published: March 2005

CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen‐presenting cells bearing the class II major histocompatability (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells.

This 368‐amino‐acid glycoprotein contains two potential sites of N‐linked glycosylation (Asn‐271 and Asn‐300) and six cysteine residues. Amino‐terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P. J. et al. (1985) Cell 42, 93–104]. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed‐phase HPLC after digestion of S‐carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin‐digested rCD4 confirmed that the carboxy‐terminus of the protein was Pro‐368. Enzymatic digestion of non‐reduced rCD4 generated disulfide‐bonded fragments that demonstrated the presence of disulfide bonds between Cys‐16 and Cys‐84, Cys‐130 and Cys‐159, and between Cys‐303 and Cys‐345.

The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N‐acetylglucosamine and N‐acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N‐glycosidase F demonstrated that both potential N‐glycosylation sites are utilized. The tryptic map of rCD4 treated with endo‐β‐N‐acetylglucosamine H demonstrated that only complex‐type oligosaccharides are attached to Asn‐271, while Asn‐300 has high‐mannose or hybrid structures attached in addition to complex‐type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn‐300 or Asn‐271 while no galactosamine was observed. This suggests that rCD4 contains no O‐linked oligosaccharides.
Detection of RNA‐dependent DNA polymerase activity in the Xiphophorus melanoma system
Authors: LÜKE Wolfgang , PETRY Harald , HOEFER Klaus , ANDERS Annerose , ANDERS Fritz
Published: March 2005

DNA polymerases have been isolated from muscle and melanoma tissues of Xiphophorus, which are similar to retroviral RNA‐dependent DNA polymerases as they prefer RNA to DNA templates. They appear to associate with submicroscopic structures which exhibit a density of about 1.13 g/ml after sucrose‐density‐gradient centrifugation. The RNA‐dependent‐DNA‐polymerase‐like enzymes could be separated from the DNA‐dependent DNA polymerases by DEAE‐cellulose chromatography. Further purification on phosphocellulose revealed that the muscle enzyme eluted at the void volume and at about 0.6 M KCl, whereas most of the melanoma enzyme eluted at 0.1 M KCl.

Comparison of the template primer specificities of the muscle and melanoma enzymes with those of known DNA polymerases showed obvious similarities to the RNA‐dependent DNA polymerase isolated from Rous sarcoma virus.
Polychlorinated biphenyls increase fatty acid desaturation in the proliferating endoplasmic reticulum of pigeon and rat livers
Authors: BORLAKOGLU Jürgen T. , EDWARDS‐WEBB John D. , DILS Raymond R.
Published: March 2005

Polychlorinated biphenyls (PCB) are abundant and persistent pollutants in the ecosystem. Commercial mixtures (e.g. Aroclor 1254) can contain up to 80 different isomers and congeners, many of which accumulate in biological systems by the ingestion of PCB‐contaminated lipid components of food chains.

Commercial mixtures of PCB induce, in hepatic microsomal membranes in vivo, a variety of different forms of the cytochrome P‐450 components of enzyme systems involved in the metabolism of drugs and other xenobiotics, and can also induce the proliferation of this membrane. Since these microsomal enzyme systems share a number of the requirements of microsomal fatty acid desaturases, we have investigated whether the induction by PCB in vivo of cytochrome‐P‐450‐linked enzymes in the proliferating hepatic microsomal membrane of the pigeon and the rat is accompanied by increased proportions of polyunsaturated fatty acids in this membrane.

The most striking changes observed 120 h after treating pigeons and rats with 1.5 mmol Aroclor 1254/kg body mass were 2.2‐fold and 1.6‐fold increases, respectively, in the proportion of arachidonic acid in the hepatic microsomal membrane. When the effects of this treatment on the proliferation of this membrane and increase in liver mass are taken into account, the amount of arachidonic acid in the total microsomal membrane of pigeon and rat livers increased 6.7‐fold and 1.9‐fold, respectively.

These changes were accompanied by very significant increases in pigeons and rats of the concentration of hepatic microsomal cytochrome P‐450, and in the activity in microsomal protein of a wide range of cytochrome P‐450‐dependent enzyme involved in the metabolism of drugs and other xenobiotics.

This effect of PCB, of increasing in vivo the degree of unsaturation of fatty acids of hepatic microsomal membrane, appears to be a novel finding, and does not seem to have been investigated for other drugs and xenobiotics. Preliminary results have shown that the effect is accompanied by substantial increases in the total activity of Δ6 and Δ5 microsomal fatty acid desaturases converting 18:2 (9, 12) (linoleic acid) to 20:4 (5, 8, 11, 14) (arachidonic acid) [Borlakoglu, J. T., Dils, R. R., Edwards‐Webb, J. D. & Walker, C. H. (1988) Biochem. Soc. Trans. 16, 1072].

It is postulated that there is a significant link between increased fatty acid desaturation and the induction of cytochrome‐P‐450‐linked enzymes, and this is discussed in terms of the mechanisms involved in the metabolism of foreign compounds.
Antisuppression by mutations in elongation factor Tu
Authors: TAPIO Soile , ISAKSSON Leif A.
Published: March 2005

Two slow‐growing kirromycin‐resistant Escherichia coli mutants with altered EF‐Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene. Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here. Ap, the tufA gene product of strain D2216 (from A. Parmeggiani), has previously been shown to give an increased GTPase activity. The slow cellular growth rates of both EF‐Tu mutants are correlated with decreased translational elongation rates. Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs [tyrT(Su3), trpT(Su9), glyT(SuAGA/G)], but have only little or no effect on misreading by wild‐type tRNAs. A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF‐Tu mutations. The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double‐mutant strain has an elevated temperature optimum (42°C) for growth rate, translation rate and nonsense suppression. Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.
Impaired in vitro kinetics of EF‐Tu mutant Aa
Authors: TAPIO Soile , BILGIN Nes'e , EHRENBERG Måns
Published: March 2005

The kirromycin‐resistant EF‐Tu mutant Aa, previously shown to be an antisuppressor for nonsense and missense suppressor tRNAs, has been characterised in a poly(U)‐primed translation system in vitro. Two major defects were found in the function of the mutant. First, the dissociation constant for Aa binding to Phe‐tRNAPhe was increased tenfold compared to wild‐type EF‐Tu. Second, kcat/Km for the interaction between the EF‐Tu · GTP · aa‐tRNA complex and the ribosome was decreased by the mutation to one third of its wild‐type value. No differences were observed between mutant and wild‐type factor in the regeneration of EF‐Tu · GTP from EF‐Tu · GDP via EF‐Ts or in the mistranslation frequency by Leu‐tRNALeu4. The relation between the in vitro results and the mutant phenotype in vivo is discussed.
The complex formed between plastocyanin and cytochrome c
Authors: BAGBY Stefan , DRISCOLL Paul C. , GOODALL Kevin G. , REDFIELD Christina , HILL H. Allen O.
Published: March 2005

Spinach plastocyanin and horse heart cytochrome c have been shown, by monitoring the behaviour of the hyperfine‐shifted heme resonances of Fe(III) cytochrome c on titration with Cu(II) plastocyanin, to form a 1:1 complex with a dissociation constant of 67 mM (D2O, pH* 7.0, 300 K). The interaction sites on the plastocyanin surface have been investigated in one‐ and two‐dimensional NMR experiments involving competition for plastocyanin between cytochrome c and the paramagnetic cation Cr(NH3)3+6. The plastocyanin resonances which are paramagnetically broadened in the spectrum of plastocyanin alone are also broadened in the spectrum of the mixture of the two proteins. This shows that, on the NMR time scale, no plastocyanin residues are hidden from Cr(NH3)3+6 by complexation with cytochrome c. [It has been shown that Cr(NH3)3+6 does not disrupt formation of the complex between the two proteins.] It appears that initial complexation of cytochrome c takes place at the acidic east site of plastocyanin, and that the extensive negative electrostatic surface of plastocyanin accommodates the paramagnetic probe and cytochrome c simultaneously in a dynamic ternary complex. The location of the electron transfer site on plastocyanin is discussed.
Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria
Authors: LAW Ruby H. P. , DEVENISH Rodney J. , NAGLEY Phillip
Published: March 2005

This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y8‐1, which includes an N‐terminal‐cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti‐F1‐β monoclonal antibody. Preliminary experiments showed that N9L/Y8‐1 imported into normal rho+ mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit− mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit− mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8‐1 was brought under the control of a inducible promoter GALI. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL‐1. Galactose control of the expression of N9L/Y8‐1 was demonstrated by the ability of strain YGL‐1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL‐1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective Fo sectors. These mitochondria demonstrated very efficient import of N9L/Y8‐1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes.
Platelet protein phosphorylation and protein kinase C activation by phorbol esters with different biological activity and a novel synergistic response with Ca2+ ionophore
Authors: BROOKS Susan F. , GORDGE Philip C. , TOKER Alex , EVANS A. Tudor , EVANS Fred J. , AITKEN Alastair
Published: March 2005

Phorbol esters with different biological activities have been tested for their ability to induce the phosphorylation of human platelet proteins. We have shown that only the potent platelet aggregatory phorbol esters were able to stimulate the phosphorylation of proteins of 76, 68, 47, 30 and 20 kDa in intact platelets. The ability of these esters to stimulate phosphorylation of the 47‐kDa protein (‘p47’) correlated with their ability to cause platelet aggregation. When a non‐platelet aggregatory deoxyphorbol (12‐deoxyphorbol 13‐phenylacetate 20‐acetate) was combined with a subthreshold dose of the Ca2+ ionophore, A23187, a large increase in phosphorylation of p47 and a fourfold decrease in Ka was observed. This was in contrast to a barely detectable stimulation of phosphorylation at micromolar levels of this phorbol ester in the absence of the ionophore. This synergism was not evident for the potent platelet aggregatory derivatives. The Ka for DOPPA with a mixture of total platelet protein kinase C was 530 nM in the absence of calcium decreasing to 120 nM in the presence of calcium. In the presence of calcium, 12‐deoxyphorbol 13‐phenylacetate 20‐acetate was shown to stimulate preferentially one of the isoforms of protein kinase C.
Structure and biosynthesis of the signal‐sequence receptor
Authors: PREHN Siegfried , HERZ Joachim , HARTMANN Enno , KURZCHALIA Teymuras V. , FRANK Rainer , ROEMISCH Karin , DOBBERSTEIN Bernhard , RAPOPORT Tom A.
Published: March 2005

The signal‐sequence receptor (SSR) has previously been shown to be a component of the environment which nascent polypeptides meet on passage through the endoplasmic reticulum (ER) membrane. We report here on the primary structure of the SSR as deduced from cDNA clones and from direct protein sequencing. The glycoprotein is synthesized with a cleavable amino‐terminal signal sequence and contains only one classical membrane‐spanning segment. Its insertion into the ER membrane during biosynthesis depends on the function of the signal‐recognition particle. SSR shows a remarkable charge distribution with the amino terminus being highly negatively charged, and the cytoplasmic carboxyl terminus positively charged. The SSR can be phosphorylated in its cytoplasmic tail both in intact cells and in a cell‐free system, suggesting a regulation of its function. The localization of the protein in the ER membrane was confirmed by immunofluorescence microscopy.
Purification, biological properties and partial sequence analysis of 67‐kDa calcimedin and its 34‐kDa fragment from chicken gizzard
Authors: KOBAYASHI Ryoji , TASHIMA Yohtalou
Published: March 2005

Calcimedin is a group of proteins, originally isolated from chicken gizzard, which are able to bind to several hydrophobic matrices in the presence of Ca2+. Although the molecular properties have been partially discovered, the physiological functions of calcimedins have not yet been clearly defined. In this study, we describe the isolation and characterization of 67‐kDa calcimedin and its 34‐kDa fragment from chicken gizzard. Both structural and functional studies establish that 67‐kDa calcimedin is a member of the calpactin/lipocortin family: it displays phospholipase A2 inhibitory activity, Ca2+‐dependent F‐actin binding and phospholipid binding activity similar to those of calpactins (lipocortins). By comparing the sequence of 67‐kDa calcimedin with the predicted sequence of 67‐kDa calelectrin, we concluded that the primary structure of these 67‐kDa proteins is highly conserved. In particular, the sequences GLGTDEGAIIXVLTQR and EGAGTDESTLIEIMATR conform with the annexin consensus sequence which is characteristic of the calpactin/lipocortin family.

A 34‐kDa fragment of 67‐kDa calcimedin was also purified and their relatedness has been confirmed by antibody cross‐reactivity. The sequence data further support that the 34‐kDa fragment is derived from the C‐terminal portion of 67‐kDa calcimedin by limited proteolysis. The 34‐kDa fragment, which contains the annexin consensus sequence, preserves the phospholipase A2 inhibitory activity, and binds F‐actin and phospholipids.
Glucose requirement for postischemic recovery of perfused working heart
Authors: MALLET Robert T. , HARTMAN David A. , BÜNGER Rolf
Published: March 2005

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload‐controlled, isolated working guinea pig hearts. A 25‐min global but low‐flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15‐min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential ([ATP]/([ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia‐reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde‐3‐phosphate dehydrogenase/3‐phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde‐3‐phosphate dehydrogenase/3‐phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/([ADP][Pi]) during creatine kinase dysfunction in the ischemia‐reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2‐deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de‐energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low‐flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate‐perfused hearts is due in part to intensification of glyceraldehyde‐3‐phosphate dehydrogenase/3‐phosphoglycerate kinase activity. (3) 2‐Deoxyglucose depletes the glyceraldehyde‐3‐phosphate pool and effects intracellular phosphate fixation in the form of 2‐deoxyglucose 6‐phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia‐reperfusion transitions in presence of excess oxidative substrate.
A low‐molecular‐mass Kazal‐type protease inhibitor isolated from rat hepatocytes is identical to rat pancreatic secretory trypsin inhibitor II
Authors: KIDO Hiroshi , YOKOGOSHI Yutaka , KATUNUMA Nobuhiko
Published: March 2005

A low‐molecular‐mass serine protease inhibitor was purified from hepatocytes and liver of rats. It was found to be a single polypeptide of 56 amino acid residues corresponding to Mr= 6224, a value that is in agreement with the molecular mass determined by gel chromatography. The inhibitor formed a complex in a molar ratio of 1:1 with trypsin. Its complete amino acid sequence was identical with that of pancreatic secretory trypsin inhibitor II (PSTI‐II) in pancreatic juice, but not with that of PSTI‐I [Uda, K., Ogawa, M., Shibata, T., Murata, A., Mori, T., Kikuchi, N., Yoshida, N., Tsunasawa, S. & Sakiyama, F. (1988) Biol. Chem. Hoppe‐Seyler 369, 55–61]. PSTIs have been reported to be primarily pancreatic secretory products, but in patients immunoreactive PSTI was found in the plasma and urine during acute inflammatory disease and shown to be produced ectopically in cancer tissues. Here we report for the first time that PSTI‐II is present in other normal tissues besides the pancreas.
Studies on prophenoloxidase activation in the mosquito Aedes aegypti L.
Authors: ASHIDA Masaaki , KINOSHITA Kuninori , BREY Paul T.
Published: March 2005

This study, the first of its kind in a mosquito vector species, demonstrates the feasibility of studying prophenoloxidase activation in an insect containing not more than a few microliters of hemolymph. Mosquito phenoloxidase was found to be in an inactive proenzyme form, prophenoloxidase. Mosquito prophenoloxidase required bivalent cation for its activation; Ca2+ was found to be the most efficient for activation. Concomitant amidase activity was also observed prior to phenoloxidase activity. Through Western blotting, using a cross‐reactive silkworm anti‐prophenoloxidase antibody, our results strongly suggest that mosquito prophenoloxidase activation resulted from limited proteolysis. Protease inhibitor studies reinforced this contention showing the involvement of (a) serine protease(s) with trypsin‐like activity in the activation of mosquito prophenoloxidase.
Purification and properties of L‐aspartate aminotransferase of Chlamydomonas reinhardtii
Authors: LAIN‐GUELBENZU Blanca , MUÑOZ‐BLANCO Juan , CÁRDENAS Jacobo
Published: March 2005

An enzyme which catalyzes the transamination of L‐aspartate with 2‐oxoglutarate has been purified 400‐fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138 000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5‐phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L‐aspartate. L‐Aspartate or pyridoxal 5‐phosphate, but not 2‐oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L‐phenylalanine and L‐tyrosine with 2‐oxoglutarate, and L‐serine, L‐alanine and L‐glutamine with oxaloacetate. L‐Aspartate aminotransferase exhibited hyperbolic kinetics for 2‐oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L‐aspartate and L‐glutamate. Apparent Km values were 0.55 mM for 2‐oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L‐aspartate and 3.88 mM for L‐glutamate. Transamination of L‐aspartate in C. reinhardtii is a bisubstrate reaction with a bi‐bi ping‐pong mechanism, and is not inhibited by substrates.
Triggering of mannosyltransferase activity in inner mitochondrial membranes by dolichyl‐monophosphate incorporation mediated through phospholipids or fatty acids
Authors: ARDAIL Dominique , GATEAU‐ROESCH Odile , LOUISOT Pierre , MORELIS Renée , PRIVAT Jean Paul , EGRET‐CHARLIER Marguerite , PTAK Marius
Published: March 2005

The activity of GDPmannose: dolichyl monophosphate mannosyltransferase in inner mitochondrial mem‐branes can be triggered by dolichyl‐monophosphate incorporation mediated through phospholipids or fatty acids. The efficiency of this incorporation and the efficiency of the enzyme activity are not equivalent. Among a variety of amphiphiles which were tested, the highest mannosyltransferase activity was obtained with the mixture of lipids extracted from the outer mitochondrial membranes. The results presented here appear consistent only with a mechanism involving collisional contacts of the phospholipid vesicles and fusion with the membranes. ESR spectroscopy confirms that (a) the incorporation process is followed by solubilization of dolichyl monophosphate molecules in the lipid phase and (b) the general organization of the inner mitochondrial membranes is not perturbed by the addition of dolichyl monophosphate.
Occurrence of tyrosine sulfate in proteins – a balance sheet
Authors: HILLE Annette , BRAULKE Thomas , von FIGURA Kurt , HUTTNER Wieland B.
Published: March 2005

The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein‐bound carbohydrate sulfate.

HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse‐chase and/or long‐term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65–92% of the protein‐bound tyrosine sulfate and 44–84% of the protein‐bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8–22 times higher in proteins secreted into the medium than in cellular proteins.

All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine‐sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation.

Lysosomes were found to contain small, but significant, amounts of protein‐bound tyrosine sulfate in addition to protein‐bound carbohydrate sulfate. Protein‐bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein‐bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them.

Our results show that secretory proteins are the most abundant, but not exclusive,in vivosubstrates for tyrosine sulfation and suggest the presence of soluble tyrosine‐sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.
Properties and primary structure of the L‐malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus
Authors: HONKA Erika , FABRY Stefan , NIERMANN Thomas , PALM Peter , HENSEL Reinhard
Published: March 2005

L‐Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deduced from the coding gene.

The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specifity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52°C, as was found for glyceraldehyde‐3‐phosphate dehydrogenase from the same organism.

With respect to the primary structure, the archaebacterial L‐malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L‐malate dehydrogenases and L‐lactate dehydrogenases of eubacteria and eukaryotes.

The phylogenetic meaning of this relationship is discussed.
Purification and further characterization of the second nitrate reductase of Escherichia coli K12
Authors: IOBBI‐NIVOL Chantal , SANTINI Claire‐Lise , BLASCO Francis , GIORDANO Gérard
Published: March 2005

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr‐230000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum‐cofactor‐deficient NADPH‐dependent nitrate reductase activity from a Neurospora crassa nit‐1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme.

Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin.

An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.
Protease resistance of the β subunit of the hamster fibronectin receptor
Authors: SCZEKAN Melanie M. , JULIANO R. L.
Published: March 2005

The structural stability of the hamster fibronectin receptor has been studied using limited proteolytic digestion and anti‐(fibronectin receptor) monoclonal antibodies of known specificity. Treatment of the solubilized intact receptor or of the dissociated α and β chains with any one of several proteases generated large protease‐resistant fragments (92–110 kDa). Western blot analysis of tryptic digests using subunit‐specific monoclonal antibodies revealed the large trypsin‐generated fragment to be of β‐subunit origin. No products from the α subunit were detected. The protease‐resistant fragment is lost upon exposure to reducing conditions; thus, the highly disulfide‐bonded region of the β subunit is important in the maintenance of the tertiary structure of the entire subunit. In contrast to solubilized fibronectin receptor, membrane‐bound receptor is much more stable to proteolysis, and tryptic cleavage results in two large immunoreactive fragments of approximately 100 kDa and 95 kDa. This suggests a difference in the conformation and/or oligomeric organization of the membrane‐bound receptor as compared with the solubilized heterodimeric receptor.
Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus
Authors: VENTUROLI Giovanni , MELANDRI B. Andrea , GABELLINI Nadia , OESTERHELT Dieter
Published: March 2005

The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25%l‐α‐phosphatidylglycerol).

At low ionic strength, the kinetics of cytochrome‐c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 μM, the process was strictly bimolecular (second‐order rate constant, k= 1.7 × 109 M−1 s−1), while at higher concentrations a fast oxidation process (half‐time lower than 20 μs) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 μM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction‐center–cytochrome‐c2 complex of about 105M−1 was evaluated. From the fraction of photo‐oxidized reaction centers promptly re‐reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome‐c2 site was exposed to the external compartment.

Both the second‐order oxidation reaction and the formation of the reaction‐center–cytochrome‐c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCI, the value of the second‐order rate constant was decreased to 7.0 × 107 M−1 s−1 and no fast oxidation of cytochrome c2 could be observed at 10 μM cytochrome c2.

The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double‐flash experiments, varying the dark time between photoactivations over the range 30 μs to 5 ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 × 103 s−1 and a lower limit for k2 of approximately 2 × 104 s−1 (corresponding to a second‐order rate constant of approximately 3 × 109 M−1 s−1).
Fate of interleukin‐6 in the rat
Authors: CASTELL José , KLAPPROTH Jan , GROSS Volker , WALTER Eike , ANDUS Tilo , SNYERS Luc , CONTENT Jean , HEINRICH Peter C.
Published: March 2005

Iodinated recombinant human interleukin‐6(125I‐rhIL‐6) was intravenously injected into rats and its fate was studied during 24 h. Between 10–20 min after a single‐dose injection, 125I‐rhIL‐6 accumulated in liver as previously reported [Castell et al. (1988) Eur. J. Biochem. 177, 357–361]. After 1 h. the radioactivity disappeared from the liver and accumulated in skin, reaching 35% of injected 125I‐rhIL‐6 5–8 h after injection. No comparable accumulation of radioactivity was found in skin when [125I]iodide or rat serum 125I‐albumin was administered. Finally the radioactivity was detected as [125I]iodide in urine. Autoradiograhic analysis of skin sections 5 h after 125‐I‐rhIL‐6 injection showed radioactivity in the interstitium.

When the experiments were carried out with [35S]rhIL‐6, essentially the same results were obtained: a decrease in radioactivity in the liver after 20 min, and a substantial increase in skin 7 h after injection.

In vitro experiments showed that 125I‐rhIL‐6 is degraded by rat and human fibroblasts, whereas no degradation was observed with rat hepatoma cells (Fao) or human hepatocytes. These observations suggest the involvement of skin in the catabolism of IL‐6.
Translational regulation of M13 gene II protein by its cognate single‐stranded DNA binding protein
Authors: ZAMAN Guido J. R. , SCHOENMAKERS John G. G. , KONINGS Ruud N. H.
Published: March 2005

To unravel the mechanism by which the single‐stranded DNA binding protein encoded by gene V of the filamentous phage M13 regulates the synthesis of its cognate DNA replication protein encoded by gene II, an in vivo test system has been developed. The system consists of two recombinant plasmids with compatible replication origins. One plasmid contains M13 gene V under the control of the inducible araB promoter of Salmonella typhimurium. The other plasmid contains a fusion gene, whose expression is dependent upon the M13 gene‐II‐promoter and which consists of the 5′ end of M13 gene II and the 5′‐truncated β‐galactosidase gene of Escherichia coli. Induction of the synthesis of wild‐type gene V protein by arabinose resulted in a specific reduction of both the β‐galactosidase activity and the amount of fusion protein produced. These specific inhibitory effects were not observed when the synthesis of the fusion protein was studied in the presence of an amber mutant of gene V. Comparison of the relative concentrations of the fusion protein mRNAs, as present in arabinose‐induced and noninduced cells, provided solid and direct evidence for the conclusions made in earlier publications, that gene V protein exerts its regulatory effect at the level of translation. Since the transcript of the fusion gene only contains the first 74 nucleotides of gene II mRNA, it is furthermore concluded that these nucleotides are already sufficient for gene V protein to exert its regulatory effect.
Protein kinase C and T cell activation
Authors: BERRY Nicola , NISHIZUKA Yasutomi
Published: March 2005

Understanding the intracellular mechanisms by which binding of ligands, such as hormones and growth factors, to their specific receptors elicits the appropriate cellular response has long been a topic of great interest. Considerable excitement was generated when it was recognised that several receptor‐ligand interactions operate via the hydrolysis of inositol phospholipids. This yields, at least, two ‘second messengers’, namely, inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3], which causes the release of Ca2+ from intracellular stores, and 1,2‐diacylglycerol (ac2Gro), which activates the serine/threonine‐specific enzyme, protein kinase C(PKC), reviewed in [1] and [2]. The pertinent question that follows is, how do PKC activation and elevation of the intracellular Ca2+ concentration evoke cell responses? In this review, attention has been focussed on PKC, and the consequences of its activation in resting human T cells. Evidence that PKC activity is, at least partially, responsible for activation of resting human T cells will be examined, and some of the more recent research investigating how PKC activation elicits this cell response will be described.
Structural features of neutral protease from Bacillus subtilis deduced from model‐building and limited proteolysis experiments
Authors: SIGNOR Giovanni , VITA Claudio , FONTANA Angelo , FRIGERIO Francesco , BOLOGNESI Martino , TOMA Salvator , GIANNA Roberto , De GREGORIIS Enrico , GRANDI Guido
Published: March 2005

The overall folding of neutral protease from Bacillus subtilis has been predicted by computer‐aided modelling, taking as a basis the known three‐dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two‐domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop region. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20:1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214‐Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The ‘nicked’B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1–214 and 215–300 are separated under protein‐denaturing conditions. Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.
Spin‐equilibrium and heme‐ligand alteration in a high‐potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism
Authors: SARAIVA Lígia M. , LIU Ming Y. , PAYNE William J. , LEGALL Jean , MOURA José J. G. , MOURA Isabel
Published: March 2005

A c‐type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme‐methionine coordination (low‐spin form) coexisting with a minor high‐spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high‐spin form at all pH values, attaining a minimum at intermediate pH values. The mid‐point redox potential determined by visible spectroscopy at pH 7.2 is + 150 mV.

The pH‐dependent spin equilibrum and other relevant structural features were studied by 300‐MHz 1H‐NMR spectroscopy. In the oxidized form, the 1H‐NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H‐NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low‐field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H‐NMR spectrum is characterized by down‐field hyperfine‐shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at ≃ 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH‐dependent features of the 1H‐NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti‐Curie temperature dependence.

In the ferrous state, the 1H‐NMR spectrum shows a methyl proton resonance at −3.9 ppm characteristic of methionine axial ligation. The electron‐transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 × 104 M−1 s−1 at pH 5.

EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax≃ 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.
Multiple fatty acid binding to albumin in human blood plasma
Authors: BRODERSEN Rolf , ANDERSEN Signe , VORUM Henrik , NIELSEN Søren Ulrik , PEDERSEN Anders Overgaard
Published: March 2005

Binding equilibria of long‐chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observation in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non‐esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I.

The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.