bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP‐stimulated cGMP phosphodiesterase from Dictyostelium discoideum
Authors: KESBEKE Fanja , BARANIAK Janina , BULGAKOV Roman , JASTORFF Bernd , MORR Michael , PETRIDIS Georg , STEC Wojciech J. , SEELA Frank , VAN HAASTERT Peter J. M.
Published: March 2005

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant‐mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80‐fold) if cGMP is no longer able to form a hydrogen bond at N2H2 or O2H. Modifications at N7, C8, O3′ and O5′ induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90‐fold.

Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at N1H, C6O, C8 and O3′, while modifications at N2H2, N3, N7, O2′H, and O5′ have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein.

cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3–6.6‐fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.
Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls
Authors: CRASNIER Martine , MOUSTACAS Anne‐Marie , RICARD Jacques
Published: March 2005

At ‘low’ ionic strength, acid phosphatase bound to plant cell walls exhibits an apparent negative co‐operativity, whereas it displays classic Michaelis‐Menten kinetics in free solution. Conversely, at ‘high’ ionic strength, the bound enzyme and the soluble enzyme behave identically. This apparent negative co‐operativity is explained by the existence of an electrostatic partition of the charged substrate by the fixed negative charges of the cell wall. Raising the ionic strength suppresses these electrostatic repulsion effects.

Calcium may be removed from the cell walls by acid treatment and the acid phosphatase is apparently strongly inhibited. This inhibition occurs together with an increased apparent negative co‐operativity of the enzyme. Incubating cell wall fragments previously depleted of calcium with CaCl2 restores the initial behaviour of the enzyme. Calcium, which tightly binds to cell wall pectic compounds, has by itself no effect on the enzyme in free solution. It affects the net charge of the cell wall and therefore the amplitude of electrostatic repulsion effects. Non‐linear least‐square fitting methods make it possible to estimate the density of fixed negative charges as well as the electrostatic partition coefficient, for both the ‘native’ and ‘calcium‐deprived’ cell wall fragments.

It may be shown directly that calcium loading and unloading in the cell wall controls the electrostatic effects, by monitoring proton extrusion from cell wall fragments upon raising the ionic strength. Proton outflux in the bulk phase is considerably enhanced upon removal of calcium from the cell walls.

The main conclusion is that loading and unloading of calcium during cell elongation and division may regulate the activity of cell wall enzymes.
Studies of the GTPase domain of archaebacterial ribosomes
Authors: BEAUCLERK Alan A. D. , HUMMEL Heidi , HOLMES David J. , BÖCK August , CUNDLIFFE Eric
Published: March 2005

Ribosomes from the methanogens Methanococcus vannielii and Methanobacterium formicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF‐2 from rat liver (but not factor EF‐G from Escherichia coli). In this assay, and in poly(U)‐dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolobus solfataricus did not respond to factor EF‐2 (or factor EF‐G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L 11 from E. coli bound well to 23S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.
Systematic application of high‐resolution, phase‐sensitive two‐dimensional 1H‐NMR techniques for the identification of the amino‐acid‐proton spin systems in proteins
Authors: NEUHAUS David , WAGNER Gerhard , VAŠÁK Milan , KÄGI Jeremias H. R. , WÜTHRICH Kurt
Published: March 2005

Novel strategies for elucidation and classification of amino acid 1H‐NMR spin systems in proteins were developed exploiting recently introduced two‐dimensional NMR techniques such as phase‐sensitive double‐quantum‐filtered correlated spectroscopy, relayed coherence transfer spectroscopy, double quantum spectroscopy and nuclear Overhauser spectroscopy. Due to the improved resolution in phase‐sensitive spectra, the fine structure of cross peaks could be exploited as a powerful source of information for establishing 1H‐1H connectivities. Principles for the interpretation of multiplet structures of absorption mode cross peaks are discussed. With these methods the 1H spin systems of rabbit liver metallothionein‐2 were elucidated and classified according to amino acid types. Despite the intrinsically difficult situation arising from the unusual amino acid composition of this protein, a more complete characterization of the 1H spin systems prior to the step of sequential resonance assignments was achieved with the presently introduced methodology than was possible in earlier studies of proteins of similar size.
Effects of retinol on chromatin structure
Authors: FERRARI Nicoletta , VIDALI Giorgio
Published: March 2005

The effect of retinol is studied in 3T3 cultured cells. The vitamin induces a decreased rate of cell proliferation and an augmented sensitivity of chromatin to DNase I digestion.

Biochemical analyses of chromosomal components establish that the rates of radioactive acetate uptake and turnover on histones are increased leaving unaltered the steady‐state level of histone acetylation. The presence of retinol in the culture medium also causes the disappearance of a protein of Mr 20000, which is co‐extracted with the high‐mobility‐group proteins.

The observed changes in chromatin structure and composition are reversible when retinol is removed from the culture medium.
Antiviral activity in L1210 cells of liposome‐encapsulated (2′‐5′)oligo(adenylate) analogues
Authors: BAYARD Bernard , LESERMAN Lee D. , BISBAL Catherine , LEBLEU Bernard
Published: March 2005

Evidence is available for a role of a (2′‐5′)(A)n‐activated endoribonuclease (RNase L) in the antiviral activity of interferon for several RNA viruses. (2′‐5′)(A)n and their analogues might thus provide an interesting alternative to exogenous interferons or their inducers in antiviral chemotherapy.

In addition, the evaluation of the activity of (2′‐5′)(A)n as mediators of interferon's biological activities or as cell growth regulators requires biochemical studies using agonists or antagonists of the system. Non‐disruptive techniques for the introduction of (2′‐5′)(A)n and their analogues into cell lines or tissues are required for these studies since these highly charged compounds are cell impermeable.

(2′‐5′)(A)n oligomers and analogues of increased stability towards phosphodiesterases were derived by chemical modification of their 2′ end and encapsulated in protein‐A‐bearing liposomes. The specific delivery of liposome contents into L1210 mouse leukemic cells was achieved with the help of monoclonal antibodies directed against the appropriate class I major histocompatibility complex‐encoded proteins expressed by these cells. This intracellular delivery led to transient inhibition of protein synthesis and an antiviral activity, both compatible with activation of RNase L. This activity was enhanced for the analogues designed to resist degradation, with respect to the natural product.
Human serum α1‐antichymotrypsin is an inhibitor of pancreatic elastases
Authors: LAINE Anne , DAVRIL Monique , RABAUD Michel , VERCAIGNE‐MARKO Dominique , HAYEM Annette
Published: March 2005

Incubation of human serum α1‐antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. α1‐Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor:enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when α1‐antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1).

Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of α1‐antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when α1‐antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of α1‐antichymotrypsin, so far limited to the inhibition of chymotrypsin‐like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work.

A contribution of α1‐antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.
Temperature‐induced conformational transition in rabbit muscle aldolase studied by temperature dependence of sulfhydryl reactivity
Authors: HEYDUK Tomasz , KOCHMAN Marian
Published: March 2005

A temperature‐induced non‐denaturing conformational transition in rabbit muscle aldolase has been as subject of discussion and controversy for some period of time. In this study the temperature dependence of the reactivity of aldolase SH groups is investigated in order to detect subtle changes in the enzyme conformation.

For model thiol‐containing systems such as cysteine, glutathione and bovine serum albumin, linear Arrhenius plots have been obtained for the reaction with 5,5′‐dithiobis(2‐nitrobenzoic acid). On the other hand, for rabbit muscle pyruvate kinase, a protein which undergoes temperature‐induced conformational transition, the plot obtained is nonlinear with a break at the temperature (18 °C) close to that reported earlier. In the case of aldolase the Arrhenius plots for three slowly reacting SH groups (Cys‐72, 289, 338) and a fast reacting group (Cys‐239) are nonlinear with a break at about 26–27 °C. The fluorescence measurements show that a plot of the fluorescence intensity of tryptophan residues versus temperature exhibits a break at the same temperature. It is shown that the observed conformational change is fully reversible. In the presence of the competitive inhibitor hexitol 1,6‐bisphosphate, which is known to protect Cys‐72 and Cys‐338 from chemical modification, the Arrhenius plot exhibits a break for the fast reacting Cys‐239 residue and is linear for the slowly reacting Cys‐289. It is found that 0.6 M urea increases the transition temperature for all exposed SH groups of aldolase. The above results show that at several points in the aldolase molecule, including the active‐site region, an abrupt change of microenvironments takes place with temperature. The competitive inhibitor protects a portion of aldolase molecule against the thermal transition.
Immunoquantification of epoxide hydrolase and cytochrome P‐450 isozymes in fetal and adult human liver microsomes
Authors: CRESTEIL Thierry , BEAUNE Philippe , KREMERS Pierre , CELIER Chantal , GUENGERICH F. Peter , LEROUX Jean‐Paul
Published: March 2005

Epoxide hydrolase and three cytochrome P‐450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P‐450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P‐450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P‐450 level. As shown previouly, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity. Aldrin epoxidase and benzphetamine‐N‐demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16β‐hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P‐450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16α increased with total P‐450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity toward benzopyrene 4,5‐oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug‐metabolizing enzymes in the human fetus.
Lipopolysaccharides from Yersinia pestis
Authors: DALLA VENEZIA Nicole , MINKA Samuel , BRUNETEAU Maud , MAYER Hubert , MICHEL Georges
Published: March 2005

The chemical structure of the lipid A of lipopolysaccharide I and II from Yersinia pestis, strain EV 40, was studied. It consists of a (1 → 6), β‐linked d‐glucosamine disaccharide which carries two phosphate groups; one phosphate is linked glycosidically with a glucosamine unit, the other one is linked to the non‐reducing glucosamine. Various degradation methods combined with 31P nuclear magnetic resonance spectroscopy showed that the esterbound phosphate group is linked to a 4‐aminoarabinosyl residue and the glycosidically linked phosphate group is linked to a d‐arabinofuranosyl residue in lipopolysaccharide II and to the phosphorylethanolamine in lipopolysaccharide I.

The hydroxyl groups of the disaccharide are acylated by dodecanoic, hexadecenoic, 3‐hydroxytetradecanoic and 3‐dodecanoyloxytetradecanoic acids.

The amino groups of the disaccharide carry 3‐hydroxytetradecanoic and 3‐dodecanoyloxytetradecanoic acids. In addition smaller amounts of 3‐tetradecanoyloxyltetradecanoic and 3‐hexadecanoyloxytetradecanoic acids are present in ester linkage.
Covalent cross‐linking of vasoactive intestinal peptide (VIP) to its receptor in intact colonic adenocarcinoma cells in culture (HT 29)
Authors: MULLER Jean‐Marc , LUIS José , FANTINI Jacques , ABADIE Brigitte , GIANNELLINI Fernand , MARVALDI Jacques , PICHON Jacques
Published: March 2005

[1 2 5I]Monoiodinated vasoactive intestinal peptide (125I‐VIP) was cross‐linked with human colonic adenocarcinoma cells (HT 29 cells) grown as a monolayer using dithiobis(succinimidylpropionate) as cross‐linking reagent. The cross‐linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

A major polypeptide of Mr= 67000 was characterized and it behaved like a high‐affinity binding site for VIP according to the following data.

The concentration of native VIP (0.5 nM) giving half‐maximum inhibition of 125I‐VIP covalent cross‐linking with this polypeptide was very similar to that giving half‐maximum displacement of 125I‐VIP on HT 29 cells (0.6 nM).

Glucagon or insulin was unable to inhibit the labelling of the Mr‐67000 component.

In our experimental conditions neither specific 125I‐VIP binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the Mr‐67000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor.

Preincubation of HT 29 cells with native VIP at 37°C, before 125I‐VIP binding and subsequent crosslinking reaction, decreased the labelling of the Mr‐67000 polypeptide up to 80%.

Assuming one molecule of 125I‐VIP cross‐linked per polypeptide, we have characterized, for the first time, a major polypeptide of Mr= 64000, which belongs to the high‐affinity VIP binding site of an intestinal human cell line.
Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor
Authors: MARMENOUT Anne , FRANSEN Lucie , TAVERNIER Jan , VAN DER HEYDEN José , TIZARD Richard , KAWASHIMA Eric , SHAW Alan , JOHNSON Merrie‐Jo , SEMON Dominique , MÜLLER Rita , RUYSSCHAERT Marie‐Rose , VAN VLIET Adri , FIERS Walter
Published: March 2005

U‐937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size‐fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417–4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724–729]. The 157‐amino‐acid‐long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N‐glycosylation site. When compared to other cytokines, like IFN‐β, IFN‐γ, or IL‐2, there is a remarkably high preference for G · C pairs in the third‐letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803–806], except for some differences in the introns and 5′‐untranslated region.
Structures of N‐terminally acetylated proteins
Authors: PERSSON Bengt , FLINTA Christofer , von HEIJNE Gunnar , JÖRNVALL Hans
Published: March 2005

Primary structures of 250 characterized proteins with N‐terminally acetylated residues were correlated with residue distributions and other data. Excluding multiple forms derived from characterized species variants, the structures represent 105 different types of acetylated proteins. Results of comparisons extend previous suggestions based on fewer structures and define relationships further. The N‐terminal residue that is acetylated is of a limited type and is frequently a small residue, with a heavy over‐representation of serine and alanine. However, the occurrence of methionine at the acetylated position is also high, whereas that of glycine is less frequent than previously estimated. Lysine is over‐represented in the N‐terminal region, as is aspartic and glutamic acids at a few positions close to the acetylated N‐terminus (especially the adjacent position). Finally. distributions of branched‐chain residues in the N‐terminal region of acetylated proteins are altered in relation to those of proteins in general, isoleucine is over‐represented, and leucine and valine are under‐represented. The results suggest that α‐amino‐acetylated proteins have special residues in N‐terminally non‐hydrophobic structures. Data are compatible with a protective function for acetylation but do not exclude further role(s) in processing or other special functions.
Kinetic co‐operativity of monomeric mnemonical enzymes
Authors: RICARD Jacques , NOAT Georges
Published: March 2005

The expression of the kinetic Hill coefficient for a two‐substrate, two‐product mnemonical enzyme has been derived. Its relation with the Λ coefficient, that is the slope of the reciprocal plots for 1[A] →0, has been established. The variation of this Hill coefficient, as a function of the second substrate and product concentrations, has been studied theoretically. Whereas the Λ coefficient does not vary as a function of the substrate and first product concentrations, the kinetic Hill coefficient does. If the enzyme is positively co‐operative, the Hill coefficient increases upon increasing the second substrate concentration and decreases if the first product concentration is increased. The converse is expected to occur if the enzyme displays a negative co‐operativity. The last product may either reverse a positive co‐operativity into a negative one or, alternatively, strengthen an already negative co‐operativity. The co‐operativity generated by the mnemonical model has been compared to the kinetic behaviour of a random model. These two models have been shown to be discriminated on the basis of the departure they show with respect to the Michaelis‐Menten behaviour.

These theoretical considerations have been applied to previously published data, obtained with wheat germ hexokinase LI. This monomeric enzyme has a negative co‐operativity with respect to the preferred substrate, glucose. The Hill coefficient decreases with MgADP concentration and decreases with glucose‐6‐phosphate concentration. This is exactly what is to be exactly what is to be expected on the basis of the above theory of kinetic co‐operativity.
Structure of the capsular antigen of Neisseria meningitidis serogrup K
Authors: VAN DER KAADEN Arie , GERWIG Gerrit J. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , TIESJEMA Rudy H.
Published: March 2005

The capsular antigen isolated from the culture liquid of a Neisseria meningitidis serogroup K(1811) fermentation consists of the 2‐acetamido‐2‐deoxymannuronic acid disaccharide repeating unit as follows:
→3)‐β‐d‐ManpNAcA‐(1→4)‐β‐d‐ManpNAcA‐(1→

The polysaccharide is O‐acetylated at the non‐glycosylated C‐4. Structural evidence has been obtained from sugar analysis, methylation analysis, as well as 1H and 13C NMR spectroscopy.
Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes
Authors: AGIUS Loranne , ALBERTI K. George M. M.
Published: March 2005

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions.

The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1‐14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1‐14C]pyruvate correted for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by substracting the flux through PDH from the total pyruvate metabolized.

With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h‐starved rats.

In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59–76%) and PC (80–83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats.

In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC.

2[5(4‐Chlorophenyl)pentyl]oxirane‐2‐carboxylate (100 μM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate‐stimulated but not the octanoate‐stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the β‐oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation.

It is concluded that the effect of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.
Interaction of α‐crystallin with lens plasma membranes
Authors: MULDERS John W. M. , STOKKERMANS Jack , LEUNISSEN Jack A. M. , BENEDETTI Ennio L. , BLOEMENDAL Hans , DE JONG Wilfried W.
Published: March 2005

The binding of the major water‐soluble lens protein α‐crystallin to the lens plasma membrane has been investigated by reassociating purified α‐crystallin with α‐crystallin‐depleted membranes and with phospholipid vesicles in which the lens membrane protein MP26 had been reconstituted. α‐Crystallin reassociates at high affinity (Kd= 13 × 10−8 M) with alkali‐washed lens plasma membranes but not with lens plasma membranes treated with guanidine/HCl, nor with phospholipid vesicles or erythrocyte membranes. Binding to lens plasma membranes is dependent on salt, temperature and pH and occurs in a saturable manner. Reconstitution of MP26 into phospholipid vesicles and subsequent analysis of α‐crystallin binding suggests the involvement of this transmembrane protein. Binding ist not influenced by pretreatment of membranes with proteases, suggesting that the 4‐kDa cytoplasmic fragment of MP26 is not necessary for α‐crystallin binding. Labeling experiments using (trifluoromethyl)‐3‐(m‐[125I]iodophenyl)diazirine as a probe for intrinsic membrane proteins further showed that α‐crystallin contains hydrophobic regions on its surface which might enable this protein to make contact with the lipid bilayer. Newly synthesized α‐crystallin, in lens culture, is not associated with the plasma membrane, suggesting that the assembly of α‐crystallin aggregates does not take place in a membrane‐bound mode.
Action of serine carboxypeptidases on endopeptidase substrates, peptide‐4‐methyl‐coumaryl‐7‐amides
Authors: KUNUGI Shigeru , FUKUDA Mitsuhiro , HAYASHI Rikimaru
Published: March 2005

Carboxypeptidase Y hydrolyzed N‐substituted peptide‐4‐methylcoumarin‐7‐amides (peptide‐NH‐Mec) at pH 7 by releasing 7‐amino‐4‐methylcoumarin (NH2‐Mec) which was then followed by carboxypeptidase action. In particular, a chymotrypsin‐directed substrate, Suc‐Leu‐Leu‐Val‐Tyr‐NH‐Mec, was hydrolyzed by the enzyme with a second‐order rate constant of 7200 M−1 S−1, which is compatible with the rate for an anilide substrate and some N‐substituted dipeptides. The activity was completely inhibited by phenylmethylsulfonyl fluoride and competitively depressed by the presence of an N‐substituted dipeptide. Dependences of kinetic parameters on pH were different from those of carboxypeptidase, esterase, amidase, and anilidase activities.

Carboxypeptidases P from Penicillium janthinellum and W from wheat also hydrolyzed some of these peptide‐NH‐Mec derivatives in a similar manner but at a rather low rate. Thus, the NH2‐Mec‐releasing activity may be considered to be intrinsic to serine carboxypeptidases in general.

Taking into consideration this endopeptidase‐like activity of serine carboxypeptidases, fluorogenic substrates should be used carefully to specify endopeptidases in crude extracts.
Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments
Authors: CARETTA Antonio , CAVAGGIONI Andrea , SORBI Robert Tibor
Published: March 2005

The high‐affinity binding of the cGMP analogue 8‐(5‐thioacetamidofluorescein)‐cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean ± SD): (a) membranes contain about one cGMP binding site per 130 rhodopsin molecules; (b) the concentration of free ligand for half saturation is 2.0 ± 0.6 μM; (c) the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 ± 0.5; (d) half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed.
Specificity of the uridine‐diphosphate‐N‐acetylmuramyl‐l‐alanyl‐d‐glutamate: meso‐2,6‐diaminopimelate synthetase from Escherichia coli
Authors: ABO‐GHALIA Mohamed , MICHAUD Catherine , BLANOT Didier , van HEIJENOORT Jean
Published: March 2005

To investigate the specificity of the uridine‐diphosphate‐N‐acetylmuramyl‐l‐alanyl‐d‐glutamate: meso‐2,6‐diaminopimelate synthetase, various compounds mimicking more or less different parts of the UDP‐MurNAc‐l‐Ala‐d‐Glu substrate were prepared. Their size ranged from that of uridine or l‐Ala‐d‐Glu to that of the whole nucleotide substrate. Chemical synthesis led to Nα‐acyl‐dipeptides, in which the acyl group mimicked the MurNAc moiety, and to glycopeptides MurNAc(α or β‐Me)‐l‐Ala‐d‐Glu, in which the anomeric function is blocked. Partial degradation or chemical modification of the substrate UDP‐MurNAc‐l‐Ala‐d‐Glu afforded: MurOHNAc‐l‐Ala‐d‐Glu, p1‐MurNAc‐l‐Ala‐d‐Glu, and DDP‐MurNAc‐l‐Ala‐d‐Glu (DDP = dihydrouridine‐diphosphate). All these compounds were tested as substrates or (and) inhibitors of the reaction catalyzed by the A2pm‐adding enzyme, which, after partial purification, was obtained in two active forms. Among the compounds tested as substrates, only DDP‐MurNAc‐l‐Ala‐d‐Glu was a good one. The Km for this compound was 97 μM versus 55 μM for the natural substrate. Among the various compounds tested as inhibitors, only P1‐MurNAc‐l‐Ala‐d‐Glu, and MurNAc(α or β‐Me)‐l‐Ala‐d‐Glu had a significant inhibitory effect at 1 mM. Apparently, no particular portion of the molecule is predominantly responsible for its recognition by the enzyme. In other words, multiple sites located over the whole molecule are required for a proper recognition and determine the high specificity of this activity. Therefore, to obtain efficient competitive inhibitors it is necessary to synthesize molecules very similar in size and structure to the natural substrate.
Studies on the formation of intrachain disulphide bonds in newly biosynthesised bovine prolactin
Authors: KADERBHAI Mustak Ali , AUSTEN Brian M.
Published: March 2005

Native disulphide‐bonded prolactin (band III) was distinguished from reduced prolactin (band II) and intermediate unstable disulphide‐linked conformations by: (a) faster mobility of the former in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and (b) high‐pressure liquid chromatography analyses of tryptic‐digested peptides derived from prolactin in various conformations during its refolding pathway from reduced, unfolded to native conformation. The electrophoretic separation has been used to examine the state of disulphide bonding in newly synthesised prolactin translated from bovine pituitary mRNA in a rabbit reticulocyte translation system supplemented with nuclease‐treated dog pancreatic microsomal membranes. The formation of correct disulphide pairing in prolactin (band III), synthesised in the in vitro translation system in the presence of pancreatic microsomes, required the presence of a thiol oxidant such as oxidised glutathione during the translation. The action of thiol oxidants on the in vitro biosynthesised and microsomally processed prolactin were both dose‐dependent and catalytic; non‐thiol oxidants such as NAD+ and NADP+ were ineffective. Examination of the time course of addition of oxidised glutathione to translating lysates showed that efficient and correct disulphide pairing in newly biosynthesised prolactin occurred when the oxidant was present co‐translationally, but much lower yields of correctly disulphide‐bonded prolactin were obtained when the oxidant was added after translation and processing were complete. The presence of protein‐disulphide isomerase in dog pancreatic microsomes. employed in the in vitro translation system to process preprolactin, was demonstrated by (a) two‐dimensional polyacrylamide gel electrophoresis of the membrane proteins, and (b) enzymic activity to accelerate reactivation of scrambled ribonuclease. Protein‐disulphide isomerase activity was latent in intact microsomal vesicles, full activity being expressed upon sonication. A procedure has been devised to prepare pancreatic microsomal vesicles depleted of protein‐disulphide isomerase which are active in processing and segregating in vitro biosynthesised prolactin. These membranes in the presence of low concentrations of oxidised glutathione are less active but in the presence of saturating levels of oxidised glutathione are fully competent in forming correct disulphide bridges in newly synthesised prolactin.
Metabolism of polyunsaturated fatty acids by rabbit reticulocytes
Authors: SALZMANN‐REINHARDT Ulrike , KÜHN Hartmut , WIESNER Rainer , RAPOPORT Samuel
Published: March 2005

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1‐14C]linoleic acid and [1‐14C]arachidonic acid by three different pathways.

Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono‐ di‐ and triacylglycerols, but not cholesterol esters).

Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14‐eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15‐Hydroxy‐5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13‐hydroxy‐9,11(Z,E)‐octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via β‐oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids.

β‐Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via β‐oxidation to 14CO2. Addition of 5,8,11,14‐eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished β‐oxidation.

Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.
Different conformations of tRNA in the ribosomal P‐site and A‐site
Authors: JØRGENSEN Tony , SIBOSKA Gunhild E. , WIKMAN Friedrik P. , CLARK Brian F. C.
Published: March 2005

Footprinting studies involving radioactively end‐labelled tRNA species bound at either the ribosomal P‐or A‐site have yielded information that the tRNA's conformation is different in the two sites. Appropriate controls showed the relevance of using poly(U)‐directed tRNAPhe binding in the P‐site and Phe‐tRNAPhe in the A‐site. Digestion of the tRNA species was effected by RNases T1, T2 and cobra venom RNase. Experiments were performed with tRNAs 32P‐labelled at either end to establish positions of primary cuts more confidently. In addition to the common protection of the aminoacyl‐stem and anticodon‐arm, footprinting experiments revealed striking differences in the accessibility of the T‐and d‐loops of tRNAs bound in the P‐and A‐sites. We observed a more open structure for the tRNA in the A‐site. These results are consistent with a dynamic structure of tRNA during the translocation step of protein biosynthesis.
Purification and characterization of esterases D1 and D2 from human erythrocytes
Authors: MATSUO Kiyosato , KOBAYASHI Kunihiko , HAGIWARA Keiji , KAJII Tadashi
Published: March 2005

Esterase D1 and esteras D2, two common esterase D (EC 3.1.1.1) isozymes, were isolated and purified from human erythrocytes. Their substrate specificity, pH profile and Km values were essentially identical. Their molecular mass was the same at 34 kDa on sodium dodecyl sulfate/polyacrylamide electrophoresis and at 27 kDa on Sephadex G‐100 gel filtration. Antisera to each of the esterase D1 and esterase D2 isozymes were successfully raised in chickens; each antiserum reacted identically with both isozymes. These findings indicate that the isozymes are close to each other in structure.

The fact that the molecular mass of the esterase D1 and esterase D2 isozymes computed on sodium dodecyl sulfate/polyacrylamide electrophoresis was close to that obtained on Sephadex G‐100 gel filtration in nondissociating buffer indicates that the isozymes are not dimers bound by disulfide bonds or a noncovalent force. These facts together indicate that the esterase D isozymes are monomers, contrary to the prevailing view that they are dimers.
Autophagic‐lysosomal and mitochondrial sequestration of [14C]sucrose
Authors: TOLLESHAUG Helge , SEGLEN Per O.
Published: March 2005

[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37°C. The sedimentation characteristics of particle‐associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome.

Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08‐1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large‐size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3‐methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy.
Structures of the major carbohydrates of natural human interleukin‐2
Authors: CONRADT Harald S. , GEYER Rudolf , HOPPE Jurgen , GROTJAHN Lutz , PLESSING Annelore , MOHR Harald
Published: March 2005

Purified human interleukin‐2 secreted by peripheral blood lymphocytes from healthy donors was found to exist in several forms. These forms were (partially) resolved by reversed‐phase high‐performance liquid chromatography and sodium dodecy1 sulfate/polyacrylamide gel electrophoresis. Two major plypeptide species (interleukin‐2 N1 and N2, 16.5 kDa) were shown to be glycosylated on the basis of [3H]galactose/[3H]glucosamine incorporation and determination of amino sugars after acid hydrolysis. A third component (interleukin‐2 M, 14.5 kDa) represents a nonglycosylated form. The amino acid composition and the NH2‐terminal sequence of both forms are consistent with the data deduced from the cDNA coding for interleukin‐2 after removal of a leader peptide of 20 amino acids. Carbohydrates are O‐linked to the IL‐2 protein via threonine‐3 of the polypeptide chain. The ologosaccharides were released by reuctive β‐elimination and were purified by gel filtration and high performance liquid chomatography. Applying methylation analysis, exoglycosidase digestion and fast atom bombardment mass spectrometry the following major carbohydrate structures were identified: N1, NeuAc(α 2‐3) Gal(β 1‐3) GalNAc‐ol; and N2, NeuAc(α 2‐3) Gal(β 1‐3) [NeuAc(α 2‐6)] GalNAc‐ol.
High‐uptake and low‐uptake forms of proteoglycans secreted by arterial smooth muscle cells
Authors: SCHMIDT Annette , BUDDECKE Eckhart
Published: March 2005

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans, designated as proteoglycan A and B, into the culture medium. They are isolated as immunologically distinct monomers with relative molecular masses of 280 × 103 and 180 × 103 and are characterized as chondroitin‐sulfate‐rich (A) and dermatan‐sulfate‐rich (B) proteoglycans. Both proteoglycan A and B were labelled with [35S]sulfate and used for studies of endocytosis.

Uptake of proteoglycan B by arterial smooth muscle cells shows saturable kinetics. At saturation (500 μ) one cell may endocytose up to 1.5 × 106 proteoglycan B molecules/h. Proteoglycan A is internalized at a 10‐fold lower rate. No saturation kinetics were observed at high proteoglycan A concentrations (500 μ).

Endocytosis of proteoglycan B in the presence of an excess of proteoglycan A and vice versa suggest that proteoglycan A and B do not compete for the same receptor site. Free hyaluronate or chondroitin sulfate do not inhibit the uptake of proteoglycan B or A.

The results suggest that proteoglycan B is internalized by arterial smooth muscle cells via a high‐affinity receptor‐mediated process, whereas proteoglycan A is taken up by fluid endocytosis and/or by low‐affinity endocytotic processes.
Steady‐state kinetics of skeletal muscle myosin light chain kinase indicate a strong down regulation by products
Authors: GEUSS Ursula , MAYR Georg W. , HEILMEYER Ludwig M. G.
Published: March 2005

The kinetic behaviour of myosin light chain kinase isolated from skeletal muscle was studied under steadystate conditions using highly purified phosphorylatable light chains 2 (LC2).

Forward reaction, product inhibition, and reverse reaction data indicate a sequential mechanism which can be interpreted best by a rapid‐equilibrium random bi‐bi reaction model. The forward reaction parameters are KATP= 150 μM, KLC2= 5.3 μM, and Ki, LC2= 7.6 μM. The enzyme forms a dead‐end complex with ADP and light chain 2; Kd, ADP of this complex is 50 μM. The forward reaction is also strongly inhibited by the phosphorylated light chain 2, Ki, LC2P is 1.5 μM. An equilibrium constant Keq of about 70 can be calculated from the kinetic parameters which agrees with the directly measured value of about 60.

The role of the two inhibitory mechanisms in the regulation of the enzyme and of the high energy of the light chain phosphate bond as deducible from Keq are discussed.
Solubilization and reconstitution of the membrane‐bound carotenogenic enzymes from daffodil chromoplasts
Authors: BEYER Peter , WEISS Günter , KLEINIG Hans
Published: March 2005

The membrane‐bound carotenogenic enzymes of daffodil (Narcissus pseudonarcissus) chromoplast membranes, i.e. dehydrogenation, cis‐trans isomerization and cyclization activities, were solubilized in an enzymatically inactive form using the zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethylamino]‐1‐propanesulfonate (Chaps).

Full enzymatic activities were regained upon reconstitution of the solubilized proteins into liposomes. These preparations converted radiolabelled cis‐phytoene into β‐carotene in a high yield. The reconstituted enzymatic sequence behaved as a tight ‘assembly line’.

In the enzymatic sequence of the reconstituted system the cis‐trans isomerization reaction occurred on the stage of cis‐phytofluene after a dehydrogenation of cis‐phytoene.
Human α1 ‐antitrypsin expression in Xenopus oocytes
Authors: ERRINGTON Deborah M. , BATHURST Ian C. , CARRELL Robin W.
Published: March 2005

Injection of equivalent amounts of normal (PiMM) or abnormal (PiZZ) α1‐antitrypsin mRNA into Xenopus oocytes resulted in secretion of both the normal and abnormal α1‐antitrypsin. A much lower proportion of the abnormal protein was secreted, and the Z α1‐antitrypsin that was not secreted accumulated within the cell in a high‐mannose form. The time taken for secretion of the normal and abnormal proteins was identical. Both the secreted and intracellular α1‐antitrypsin synthesized by oocytes were functionally active.
Differential regulation of interferon‐induced mRNAs and c‐myc mRNA by α‐ and γ‐interferons
Authors: KELLY John M. , GILBERT Christopher S. , STARK George R. , KERR Ian M.
Published: March 2005

The extent and kinetics of induction have been determined for eight mRNAs induced by α‐ and γ‐interferons in HeLa cells. The mRNAs which code for a 2‐5A synthetase, metallothionien II, HLA class I antigen and five unknown proteins were induced 2 ‐ > 100‐fold by α‐interferons. In the continued presence of α‐interferon some mRNAs were maintained at the induced levels until at least 40 h, whereas others were induced only transiently. When the effects of α‐ and γ‐interferons were compared, the induced levels and kinetics were very similar for one mRNA (1‐8) but were significantly different for the others. One mRNA (6‐16) was induced more than 100‐fold by α‐interferon but not significantly by γ‐interferon. Parallel analysis of the level of c‐myc mRNA showed it to decrease twofold in response to α‐interferon, but to increase more than threefold in response to γ‐interferon, despite a more profound inhibition of cell growth by the latter. There must, therefore, be differences in how the levels of different mRNAs are sustained by α‐interferons and how α‐ and γ‐interferons regulate the levels of the same mRNAs.
Modification of nuclear matrix proteins by ADP‐ribosylation
Authors: WĘSIERSKA‐GĄDEK Józefa , SAUERMANN Georg
Published: March 2005

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP‐ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography.

In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP‐ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography.

By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa.

Approximately 2% of the total nuclear ADP‐ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix‐associated enzyme catalyzes the incorporation of [32P]ADP‐ribose into acid‐insoluble products of molecular mass 116 kDa and above, in a 3‐aminobenzamide‐inhibited, time‐dependent reaction.

The possible function of ADP‐ribosylation of nuclear matrix proteins and of the attachment of ADP‐ribosyltransferase to the nuclear matrix in the regulation of matrix‐associated biochemical processes is discussed.