bluuebunny/crossref_metadata_2025 huggingface dataset:
A phosphotyrosyl‐protein phosphatase activity associated with acid phosphatase from human prostate gland
Authors: LI Heng‐Chun , CHERNOFF Jonathan , CHEN Lan Bo , KIRSCHONBAUM Alexander
Published: March 2005

Using [32P]P‐Tyr‐IgG and [32P]P‐Tyr‐casein phosphorylated by pp60v‐src as substrates, studies on the phosphotyrosyl‐protein phosphatase activity in human prostate gland indicate that it is associated with prostatic acid phosphatase. Evidence to support this conclusion include the following: (a) these two enzymatic activities co‐purify to apparent homogeneity; (b) they co‐migrated on polyacrylamide gel electrophoresis, ion‐exchange and gel filtration chromatographies; (c) the exhibit identical thermostability; and (d) the phosphotyrosyl‐protein phosphatase activity is sensitive to inhibition by p‐nitrophenyl phosphate and by several classical inhibitors of prostatic acid phosphatase including l(+)‐tartrate, molybdate, vanadate and NaF. The purified enzyme exhibits high specificity towards phosphotyrosyl‐proteins with little activity towards several phosphoseryl‐proteins and phosphothreonyl‐proteins examined. The present findings indicate that prostatic acid phosphatase may function in vivo as a phosphotyrosyl‐protein phosphatase.
Properties of monoclonal antibodies to nicotinic acetylcholine receptor from chick muscle
Authors: MEHRABAN Fuad , KEMSHEAD John T. , DOLLY J. Oliver
Published: March 2005

1. Four stable, hybrid‐cell lines secreting monoclonal antibodies to distinct determinants on the nicotinic acetylcholine receptor from chick muscle have been established. These were characterised by the following criteria: immunoglobulin isotype, ability to produce experimental autoimmune myasthenia gravis in mice and reactivity towards homologous and hetereologous acetylcholine receptor proteins.

2. Two monoclonal antibodies were found to inhibit the reaction of α‐bungarotoxin with homologous acetylcholine receptor; in addition one of these, on binding to receptor‐toxin, induced a rapid dissociation of the complex (t1/2= 0.5 h at 23 °C).

3. Three of the antibody preparations recognised epitopes on this receptor from muscle of other species and two of these caused experimental autoimmune myasthenia gravis in BALB/c mice following passive transfer.

4. The latter two recognised to significant extents the α‐bungarotoxin binding component purified from chick optic lobe and brain cortex.

5. Sedimentation analysis demonstrated that two of the monoclonal antibodies form a distinct size (s20, w = 12S) of complex with the receptor of chick muscle which most probably corresponds to a 1:1 attachment of antibody and receptor; this may involve cross‐linking of two determinants within the same oligomer. A similar observation was made with the α‐bungarotoxin binding component from optic lobe using one of the cross‐reacting antibodies. Another monoclonal antibody was found to be capable of forming much heavier complexes with the receptor from chick muscle, these are thought to involve inter‐molecular cross‐linking of oligomers.

6. The observed properties of these antibodies are discussed in relation to their myasthenogenicity and with reference to the extent of structural similarities between the peripheral nicotinic acetylcholine receptor and the α‐bungarotoxin binding protein from brain.
Cytosolic glutamine synthetase in higher plants
Authors: HIREL Bertrand , McNALLY Sheila F. , GADAL Pierre , SUMAR Nazira , STEWART George R.
Published: March 2005

Cytosolic glutamine synthetase (GS1) was purified to homogeneity from etiolated barley leaves by DEAE‐Sephacel and hydroxyapatite chromatography, gel filtration and polyacrylamide gel electrophoresis. Specific antibodies against the purified protein were raised by the immunization of rabbits. Immunoprecipitation experiments demonstrated that cytosolic glutamine synthetases isolated from the leaves of different plant species were very similar proteins. Good recognition of other cytosolic glutamine synthetases from roots, root nodular tissue and seeds by barley GS1 antibodies was obtained, suggesting that they too are all quite similar proteins. In contrast, chloroplast glutamine synthetase (GS2) was considered to be a different protein in view of its low level of recognition by barley GS1 antibodies.
Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys
Authors: VACHER Jean , GROSJEAN Henri , DE HENAU Suzanne , FINELLI Jacqueline , BUCKINGHAM Richard H.
Published: March 2005

In this paper we describe the construction of a yeast tRNACys UGA suppressor. After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil. The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities. The level of aminoacylation was similar to that of the parent molecule. Readthrough of the UGA termination codon in β‐globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions. We see no post‐transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes. This may be related to the low suppressor activity; however, it is also possible that other features of tRNACys structure may be unadapted to efficient UCA anticodon function.
Study of the Zn‐containing dd‐carboxypeptidase of Streptomyces albus G by small‐angle X‐ray scattering in solution
Authors: LABISCHINSKI Harald , GIESBRECHT Peter , FISCHER Erwin , BARNICKEL Gerhard , BRADACZEK Hans , FRÈRE Jean‐Marie , HOUSSIER Claude , CHARLIER Paulette , DIDEBERG Otto , GHUYSEN Jean‐Marie
Published: March 2005

Study of the Zn2+‐containing d‐alanyl‐d‐alanine‐cleaving carboxypeptidase of Streptomyces albus G by small‐angle X‐ray scattering in solution yielded the following molecular parameters: radius of gyration R= 1.82 ± 0.05 nm; largest diameter D= 5.9 ± 0.2 nm; relative molecular mass Mr= 17000 ± 2000; volume V ∼ 35 ± 2 nm3; degree of hydration: 0.25 ± 0.02 g water/g protein. By reference to theoretical scattering curves of rigid triaxial homogeneous bodies, a model which fits all experimental data is an elliptical cylinder. Such a model is compatible with that observed in the crystal structure. At those high concentrations necessary to form inactive enzyme‐ligand associations the non‐competitive β‐lactam inhibitors, cephalothin and cephalosporin C, drastically altered the scattering behaviour of the protein.
Role of the ATPase of sugar‐cane vacuoles in energization of the tonoplast
Authors: THOM Margaret , KOMOR Ewald
Published: March 2005

Vacuoles of sugar‐cane suspension cells contained a tonoplast‐bound ATPase which was exclusively located on the cytoplasmic side of the vacuole. Vanadate and diethylstilbestrol had little effect on the vacuolar ATPase. ATP was the optimum substrate for the tonoplast ATPase, but there was also evidence for tonoplast‐bound GDP‐hydrolyzing and GTP‐hydrolyzing enzymes which can interfere with the ATPase assay. Other phosphate anhydrides and esters were not hydrolyzed.

The addition of MgATP polarized the tonoplast from about 0 mV to an interior‐positive value of about +20 mV; MgADP and MgGTP had much less effect; MgGDP and ATP (in the absence of magnesium) had no effect on the membrane potential. The polarization of the tonoplast was insensitive to valinomycin, nigericin, and inhibitors of plasmalemma ATPase, but was strongly reduced by the uncoupler carbonylcyanide m‐chlorophenyl‐hydrazone. These data are interpreted as evidence for the action of tonoplast‐bound ATPase as a pump which translocates protons into the vacuoles.

The activity of the ATPase was highly specific for MgATP2‐; the other important ionic states of ATP: ATP4−, HATP3−, MgHATP−, and Mg2ATP neither stimulated nor inhibited. The same was true for Mg2+. Since the protons were not brought to the catalytic site by protonation of the substrate, the tonoplast‐ATPase may pick up the proton for translocation from the cytoplasm. The saturation kinetics for MgATP2− hydrolysis were biphasic, the higher affinity ATPase with Km value of 0.7 mM seems to be the physiologically relevant activity.
Incorporation of 1,N6‐ethanoadenosine into the 3′ terminus of tRNA using T4 RNA ligase 1. Preparation of yeast tRNAPhe derivatives
Authors: PAULSEN Harald , WINTERMEYER Wolfgang
Published: March 2005

The 3′‐terminal A‐C‐C‐A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6‐ethenoadenosine (ɛA) or 2‐aza‐1,N6‐ethenoadenosine (aza‐ɛA). T4 RNA ligase was used to join the nucleoside 3′,5′‐bisphosphates to the 3′ end of the tRNA which was shortened by one [tRNAPhe(‐A)] or four [tRNAPhe(‐ACCA)] nucleotides. It was found that the base‐paired 3′‐terminal cytidine 72 in tRNAPhe(‐ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A‐C‐C terminus of tRNAPhe(‐A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double‐stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single‐stranded acceptors.

The replacement of the 3′‐terminal adenosine 76 with ɛA and aza‐ɛA leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu‐specific tRNAs both from yeast and Escherichia coli) are also inactivated by ɛA incorporation.

Replacement of adenosine 73 followed by repair of the C‐C‐A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of ɛA and aza‐ɛA at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the ɛA‐labeled tRNAPhe is complexed with phenylalanyl‐tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3′ terminus is not changed appreciably in these complexes.
Localization of Lys‐51 of L7/L12 on 50S ribosomes from Escherichia coli
Authors: MAASSEN J. Antonie , SCHOP Enno N. , MÖLLER Wim
Published: March 2005

Ribosomal protein L7/L12 from Escherichia coli was modified specifically at Lys‐51 with 4‐(6‐formyl‐3‐azido‐phenoxy)butyrimidate.

Reconstitution of ribosomal cores, lacking L7/L12, with imidate‐modified L7/L12 resulted in back formation of 50S particles which were fully active in elongation‐factor‐dependent processes. By use of the formylazidophenoxy moiety as hapten, the position of Lys‐51 of L7/L12 on the 50S ribosome was determined by immune electron microscopy. The results show that an L7/L12 dimer is present in the L7/L32 stalk in such a way that Lys‐51 is located at the far cytoplasmic end of the stalk. The experimental data are discussed in relation to a proposed model for the L7/L12 dimer.
The assignment of the 1H nuclear magnetic resonance spectrum of azurin
Authors: CANTERS Gerard W. , HILL H. Allen O. , KITCHEN Nicholas A. , ADMAN Elinor T.
Published: March 2005

A detailed assignment of the 1H nuclear magnetic resonance spectrum of azurin has been made. Resonances associated with the single tryptophan residue, all six phenylalanine residues, one of the two tyrosine residues and all four histidine residues, as well as most of the resonances from the ring‐current shifted methyl groups have been assigned. These assignments have been used to study the pH dependence of the structure of the protein and binding of analogues of redox‐active reagents to the protein.
Protein synthesis during chloroplast development in Spirodela oligorhiza
Authors: De HEIJ Hans T. , JOCHEMSEN Aart‐Gerrit , WILLEMSEN Peter Th. J. , GROOT Gert S. P.
Published: March 2005

We have studied qualitative and quantitative changes of several parameters during chloroplast development in Spirodela oligorhiza (duckweed). On a dry weight basis, the amount of protein increases from 2.5 % (w/w) in dark‐grown to 7.8 % (w/w) in light‐grown fronds. At the same time the amount of starch drops from 50 % to 27 % (w/w).

Using an immunochemical quantification method we have found that during greening of etiolated plants the amount of all subunits of the ATPase complex per frond increases 10‐fold, whereas the level of the subunits of ribulose‐1,5‐biphosphate carboxylase increases 50‐fold. Cytochrome f was found to be present in dark‐grown Spirodela and the amount of this polypeptide per frond increases about 30‐fold. The concentration of a polypeptide that possibly represents a cytochrome b6 subunit increases about 10‐fold upon greening.

The molar ratio of the CF1‐β and CF1‐γ subunits of the ATPase complex varies over 2–3, while in all stages of chloroplast development studied the molar ratio of the carboxylase subunits is about 1. As these values are in agreement with the stoichiometrical amounts in the native protein complexes, we conclude that the synthesis of CF1‐β and CF1‐γ, as well as the synthesis of the large and small carboxylase subunits, are strictly coordinated during chloroplast biogenesis in Spirodela oligorhiza.
Drastic rise of intracellular adenosine(5′)tetraphospho(5′)adenosine correlates with onset of DNA synthesis in eukaryotic cells
Authors: WEINMANN‐DORSCH Christa , HEDL Annabelle , GRUMMT Ingrid , ALBERT Waltraud , FERDINAND Franz‐J. , FRIIS Robert R. , PIERRON Gerard , MOLL Waldemar , GRUMMT Friedrich
Published: March 2005

An assay of adenosine(5′)tetraphospho(5′)adenosine (Ap4A), based on the luciferin/luciferase method for ATP measurement, was developed, which allows one to determine picomolar amounts of unlabeled Ap4A in cellular extracts. In eukaryotic cells this method yielded levels of Ap4A varying from 0.01 μM to 13 μM depending on the growth, cell cycle, transformation, and differentiation state of cells. After mitogenic stimulation of G1‐arrested mouse 3T3 and baby hamster kidney fibroblasts the Ap4A pools gradually increased 1000‐fold during progression through the G1 phase reaching maximum Ap4A concentrations of about 10 μM in the S phase. Quiescent 3T3 cells reach a high level of Ap4A (1 μM) in a “committed” but prereplicative state if exposed to an external mitogenic stimulant (excess of serum) and simultaneously to a synchronizer which inhibits entry into the S phase (hydroxyurea). When the block for DNA replication was removed at varying times after removal of the stimulant decay of commitment to DNA synthesis was found correlated with a shrinkage of the Ap4A pool. Cells lacking a defined G1 phase (V79 lung fibroblasts, Physarum) possess a constitutively high base level of Ap4A (about 0.3 μM) even during mitosis. From this high level, Ap4A concentration increases only about tenfold during the S phase. Temperature‐down‐shift experiments, using chick embryo cells infected with transformation‐defective temperature‐sensitive viral mutants(td‐ts), have shown that the expression of the transformed state at 35°C is accompanied by a tenfold increase of the cellular Ap4A pool. Treatment of exponentially growing human cells with interferon leads, concomitantly with an inhibition of DNA syntheses, to a tenfold decrease in intracellular Ap4A levels within 20 h. The possibility of Ap4A being a “second messenger” of cell cycle and proliferation control is discussed in the light of these results and those reported previously demonstrating that Ap4A is a ligand of mammalian DNA polymerase α, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.
Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone
Authors: CHRISTOFFERSEN Thoralf , REFSNES Magne , BRØNSTAD Gunnar O. , ØSTBY Eva , HUSE Jörgen , HAFFNER Frode , SAND Tor‐Erik , HUNT Nicholas H. , SONNE Ole
Published: March 2005

Primary monolayer cultures of rat hepatocytes were used for studies of long‐term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon‐sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine‐sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak ∼ 6 h) and a subsequent decrease in activity of both high‐Km, and low‐Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2–24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose‐dependent (1 pM– 1 μM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon‐sensitive adenylate cyclase activity occurring in vitro Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km∼ 1 and ∼ 10 μM) were decreased in the dexamethasone‐treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or β‐adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long‐term influences on the cyclic AMP system.
Complete amino acid sequence of mouse prolactin
Authors: KOHMOTO Kaoru , TSUNASAWA Susumu , SAKIYAMA Fumio
Published: March 2005

The complete primary structure of mouse prolactin has been established on the basis of tryptic peptides from cyanogen‐bromide‐treated, S‐carboxymethylated mouse prolactin and Staphylococcus‐aureus‐protease‐cleaved overlaps, which were sequenced by manual liquid‐phase and solid‐phase Edman degradation. Three disulfide bonds were assigned to Cys‐4–Cys‐9, Cys‐56–Cys‐172, and Cys‐189–Cys‐197 by digestion of intact prolactin with S. aureus protease. One of the characteristics to date is replacement of Trp‐89, which is commonly present among prolactin, growth hormone and choriomammotropin, by serine. It was suggested, by comparison with five other prolactins, five growth hormones and human choriomammotropin, that Asp‐18, His‐25, Ser‐60 and Thr‐63 are essential to lactogenic activity.
The 2‐oxoglutarate binding site of prolyl 4‐hydroxylase
Authors: MAJAMAA Kari , HANAUSKE‐ABEL Hartmut M. , GÜNZLER Volkmar , KIVIRIKKO Kari I.
Published: March 2005

The structure and function of the 2‐oxoglutarate binding site of prolyl 4‐hydroxylase was studied by assaying the inhibitory potential of 24 selected aliphatic or aromatic compounds. All except one of them inhibited the enzyme competitively with respect to 2‐oxoglutarate and noncompetitively with respect to Fe2+, the Ki values ranging from 0.8 μM to over 15 mM. The Ki values for the two most effective inhibitors, pyridine 2,5‐dicarboxylate and 2,4‐dicarboxylate, were about 0.8 μM and 2 μM, these compounds being the most potent inhibitors of prolyl 4‐hydroxylase with respect to 2‐oxoglutarate known so far. Only one of the compounds tested, 2‐oxoadipinate, was able to support hydroxylation by replacing 2‐oxoglutarate as a cosubstrate. The data suggest that the 2‐oxoglutarate binding site can be divided into three distinct subsites. Subsite I is probably a positively charged side chain of the enzyme that ionically binds the C5 carboxyl group of the 2‐oxoglutarate, subsite II consists of two cis‐positioned equatorial coordination sites of the enzyme‐bound ferrous ion and is chelated by the C1–C2 moiety, while subsite III involves a hydrophobic binding site in the C3–C4 region of the cosubstrate. The sp3 rehybridization of C2 within the dictating moiety of the cosubstrate appears to be a crucial event during decarboxylation that proceeds in the form of a ligand reaction inside the Fe2+ coordination sphere.
Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA
Authors: BÄUMEL Irmtraud , MEYER Thomas F. , GEIDER Klaus
Published: March 2005

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single‐stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme. A block in the unwinding step increases secondary initiation events in single‐stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon.
The smaller helical repeat of poly(dA) · poly(dT) relative to DNA may reflect the wedge property of the dA · dT base pair
Authors: PRUNELL Ariel , GOULET Isabelle , JACOB Yves , GOUTORBE François
Published: March 2005

A simple geometric approach is described which suggests that the smaller helical repeat of the homopolymer poly(dA)·poly(dT) relative to DNA (10.1 against 10.6 base pairs/turn) quantitatively reflects the property of the dA·dT base pair to behave like a wedge, of angle 11°, pointing towards dA.
Structures of Miltenberger class I and II specific major human erythrocyte membrane sialoglycoproteins
Authors: DAHR Wolfgang , NEWMAN Roland A. , CONTRERAS Marcela , KORDOWICZ Maria , TEESDALE Phyllis , BEYREUTHER Konrad , KRÜGER Jürgen
Published: March 2005

The N‐terminal structures of the Miltenberger (Mi‐) blood group class I and II specific human MN erythrocyte membrane sialoglycoproteins were determined by manual sequencing of tryptic glycopeptides and various secondary fragments. The Mi‐I and Mi‐II active glycoproteins were found to exhibit a threonine → methionine and threonine → lysine exchange, respectively, at position 28 which prevents N‐glycosylation of asparagine 26. Due to the absence of the N‐glycosidic oligosaccharide chain, the monomeric form of the Mi‐I and Mi‐II specific glycoproteins possesses a slightly increased sodium dodecyl sulfate/polyacrylamide gel electrophoretic mobility, in comparison to its normal counterpart. Serological studies suggest that antibodies, specific for Mi‐I or Mi‐II red cells, react with the structurally altered region of the MN glycoprotein.
Supercoiling and the mechanism of restriction endonucleases
Authors: SNOUNOU Georges , MALCOLM Alan D. B.
Published: March 2005

We have used topoisomerase I in the presence of netropsin and ethidium bromide to generate DNA molecules of varying superhelical density.

Digestion by endonuclease EcoRI is sensitive to supercoiling, being maximal for the relaxed form. Endonucleases AvaI and BamHI, by contrast, are relatively unaffected.

The results are interpreted in terms of the base composition of the DNA in the vicinity of these sites. dA + dT‐rich regions are more susceptible to deformation than are dG + dC‐rich ones.

Analysis of the rates of disappearance of linear molecules confirms a two‐step mechanism for EcoRI cleavage but suggests that BamHI and AvaI cleave both strands simultaneously.
On the metal‐ion coordinating properties of the 5′‐monophosphates of 1, N6‐ethenoadenosine (ɛ‐AMP), adenosine and uridine
Authors: SIGEL Helmut , SCHELLER Kurt H.
Published: March 2005

The concentration dependence of the chemical shifts of the protons H‐2, H‐8, H‐10, H‐11, and H‐1′ of 1,N6‐ethenoadenosine 5′‐monophosphate (ɛ‐AMP2−) has been measured. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K= 2.5 ± 0.3 M−1, is within experimental error identical to the value determined earlier for AMP2−,K= 2.1 ± 0.4 M−1. The conditions for the potentiometric pH titrations, used to determine the acidity constants of H2(ɛ‐AMP), H2(AMP), and H(UMP)− and the stability constants of the metal ion (M2+) complexes of the corresponding nucleoside 5′‐monophosphates (NMP), were chosen so that the ligands were present in the monomeric form. The stabilities of Mg(ɛ‐AMP) and Mg(AMP) are similar; however, the stabilities of the Mn2+, Cu2+ and Zn2+ complexes of ɛ‐AMP2− are much larger (in the case of Cu2+ by a factor of 700) than those of AMP2−. This is due to the much larger metal ion affinity of the ɛ‐adenosine moiety compared to that of the parent adenosine residue. As the uridine moiety does not participate in complex formation, the stability constants of M(UMP) have been used to evaluate the extent of macrochelation (i.e. the simultaneous coordination of M2+ to the base moiety and the phosphate group) in the ɛ‐AMP and AMP complexes: the concentration of the macrochelated isomer is considerably larger for M(ɛ‐AMP) than for M(AMP). A comparison with previous results for the complexes with ADP3− and ATP4− indicates the order, M(AMP)cl < M(ADP)−cl > M(ATP)2−cl for the tendency to form macrochelates (cl). Due to the relatively high affinity of the ɛ‐adenosine moiety towards Mn2+, Cu2+ and Zn2+, the phosphate‐monoprotonated complexes M(H ·ɛ‐AMP)+ also become important; the corresponding complexes play only a minor role in the M2+/AMP systems. Intramolecular aromatic‐ring stacking occurs in the ternary Cu(2,2′‐bipyridyl)(NMP) complexes: about 80% of Cu(Bpy)(AMP) and Cu(Bpy)(ɛ‐AMP) exist as the stacked isomer in aqueous solution; for the former system it has been shown in a previous X‐ray study that the intramolecular ligand‐ligand interaction occurs also in the solid state [Aoki, K. (1978) J. Am. Chem. Soc. 100, 7106]. Overall, the results emphasize that great care should be exercised in drawing conclusions based on studies of metal‐ion‐containing enzymic systems in which the natural adenine nucleotide cofactors have been replaced by the corresponding 1,N6‐etheno derivatives.
Purification and characterization of 3′‐phosphoadenylylsulfate: N‐desulfoheparan sulfate sulfotransferase from arterial tissue
Authors: GÖHLER Dieter , NIEMANN Reinhard , BUDDECKE Eckhart
Published: March 2005

A 3′‐phosphoadenylsulfate: N‐desulfoheparan sulfate sulfotransferase (EC 2.8.2.12) was purified 450‐fold from the microsomal fraction of calf arterial tissue and separated from 3′‐phosphoadenylylsulfate: chondroitin sulfotransferase (EC 2.8.2.5) activity.

The enzyme has optimal activity at neutral pH, requires divalent cations (Mn2+, Mg2+, Ca2+) for maximal activity and exhibits specificity towards N‐desulfoheparan sulfate, N,O‐desulfoheparan sulfate and oligosaccharides derived therefrom. N,O‐desulfoheparan sulfate tetrasaccharides serve as acceptor substrates only if the nonreducing terminus is occupied by glucuronic acid (not iduronic acid).

The N,O‐desulfoheparan sulfate sulfotransferase transfers [35S]sulfate from 3′‐phosphoadenylyl[35S]sulfate to the 2‐amino groups and to the 6‐hydroxy groups of glucosamine units of the acceptor substrates. The ratio of N/O‐sulfation ranged between 3:1 and 2:1.

O‐[35S]Sulfated unsaturated disaccharides were obtained from enzymatically labelled [35S]N‐desulfoheparan sulfate by heparitinase degradation and subsequent deamination. Evidence for the O‐sulfation at C‐6 of the glucosamine units was provided by isolation of anhydromannose [35S]monosulfate, which was formed from uronosylanhydromannose [35S]monosulfate by β‐glucuronidase treatment.

An N‐desulfo‐N‐[1‐14C]acetylheparan sulfate deacetylase activity was copurified with the N‐desulfoheparan sulfate sulfotransferase.
Detection of type‐2 casein kinase and its endogenous substrates in the components of the microsomal fraction of rat liver
Authors: MEGGIO Flavio , BRUNATI Anna Maria , DONELLA‐DEANA Arianna , PINNA Lorenzo A.
Published: March 2005

Rat liver type‐2 casein kinase‐TS (Ck‐TS) is unevenly distributed among the different components of the rat liver post‐mitochondrial particulate fraction: while it accounts for the whole casein kinase activity of protein‐glycogen particles, it is absent in the microsomal membranes (exhibiting exclusively casein kinase activity of type‐1) and coexists with type‐1 casein kinase in ribosomes.

At least seven proteins whose phosphorylation is promoted by Ck‐TS have been detected in these fractions. The only important target of Ck‐TS in protein‐glycogen particles is a rather heterogeneous protein of Mr around 85000 which has been identified as glycogen synthase. Two polypeptides of Mr about 16000, possibly identifiable with acidic proteins P1 and P2, and an unidentified protein of Mr about 35000 are the main ribosomal substrates of Ck‐TS. Three protein bands of Mr 52000, 79000 and 91000 are also very efficiently phosphorylated whenever the microsomal membranes, devoid of intrinsic casein kinase‐2, are incubated with cytosolic Ck‐TS. These membrane‐bound radiolabeled proteins require deoxycholate for their solubilization: they are very acidic, according to their high affinity for DEAE‐cellulose, and give rise to partially superimposable [32P]peptide maps, suggesting extensive homologies among them. They also exhibit a low affinity Ca2+ binding activity comparable to that of calsequestrin.
Region X, the cell‐wall‐attachment part of staphylococcal protein A
Authors: GUSS Bengt , UHLÉN Mathias , NILSSON Björn , LINDBERG Martin , SJÖQUIST John , SJÖDAHL Jörgen
Published: March 2005

The sequence of region X of staphylococcal protein A has been determined. The hypothesis has been put forward that this region spans the Staphylococcus aureus cell wall and is responsible for the binding to the peptidoglycan. The primary amino acid sequence of region X was determined for two strains exhibiting cell‐wall‐bound protein A, Cowan I and 8325–4. The sequence determination of the Cowan I material is partial and was performed by Edman degradation, in contrast to the sequence of the 8325–4 material which was completely analyzed by nucleotide sequencing of the corresponding gene. The region consists of two structurally different domains, a highly repetitive region (Xr), with an octapeptide structure repeated approximately 12 times, and a C‐terminal domain (Xc) with an unique sequence. A comparison between the two strains reveals a high mutual homology as well as a high internal homology between the octapeptide structures. Six out of eight amino acids are identical in the repetition of this structure throughout region Xr in both proteins and the other two are changed in a rather regular pattern.
Isozymes of myosin in growing and regenerating rat muscles
Authors: MARÉCHAL Georges , SCHWARTZ Ketty , BECKERS‐BLEUKX Godelieve , GHINS Etienne
Published: March 2005

Native myosin isozymes of rat muscles have been isolated by electrophoreses in non‐dissociating conditions. Their mobilities were measured, using taenia coli myosin as an internal standard and their relative concentrations were determined by computer planimetry of the electrophoretograms. Three isozymes were observed in extensor digitorum longus (EDL), two in soleus (SOL), four in neonatal muscles and four in muscles three days before birth. Regenerates of minced EDL or SOL muscles in adult animals had no native myosin the third day after surgery; they were similar to neonatal muscles 15 days after surgery and to adult muscles 60 days after surgery.
The variable relation between myosin light‐chain phosphorylation and actin‐activated ATPase activity in chicken gizzard smooth muscle
Authors: MERKEL Linda , MEISHERI Kaushik D. , PFITZER Gabriele
Published: March 2005

Using pure myosin light‐chain kinase and myosin from chicken gizzard and rabbit skeletal actin, we have established the relationship between myosin phosphorylation and actin‐activated ATPase activity by varying the concentration of Ca2+ or calmodulin (CaM). By increasing Ca2+ or CaM the extent of myosin phosphorylation increased from 10% to about 100%, the requirements for half‐maximal activation being 0.17 μM Ca2+ and 1 nM CaM. ATPase was activated only when a phosphorylation ‘threshold’ of about 60 % was surpassed. By increasing phosphorylation from 60% to 100%, ATPase activity was further stimulated, the relationship between myosin phosphorylation and ATPase activity being curvilinear. Addition of 1 μM tropomyosin lowered the Ca2+ requirement for half‐maximal ATPase activity in the prescence of 0.05 μM CaM from 0.4 μM to 0.2 μM but did not affect the maximal ATPAse activity. In addition, the CaM requirement of ATPase activity was also lowered by gizzard tropomyosin at 9 μM Ca2+. The Ca2+/CaM requirement for myosin phosphorylation remained unaffected by tropomyosin.

Thus, tropomyosin altered the relationship between myosin phosphorylation and ATPase activity: the phosphorylation ‘threshold’ was lowered from 60% to about 10%, hence causing a leftward shift of the relationship which becomes similar to that in the case of native actomyosin.
Glutathione conjugation by isolated lung cells and the isolated, perfused lung
Authors: DAWSON Janet R. , VÄHÄKANGAS Kirsi , JERNSTRÖM Bengt , MOLDÉUS Peter
Published: March 2005

Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1‐chloro‐2,4‐dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH‐transferase activity.

Conjugation of 1‐chloro‐2,4‐dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium.

The results indicate the ability of rat lung to utilize extracellular GSH.
Synthesis in vitro of precursor‐type carnitine acetyltransferase with messenger RNA from Candida tropicalis
Authors: UEDA Mitsuyoshi , TANAKA Atsuo , HORIKAWA Saburo , NUMA Shosaku , FUKUI Saburo
Published: March 2005

Carnitine acetyltransferase was synthesized in vitro in the mRNA‐dependent reticulocyte system with mRNA from alkane‐grown or propionate‐grown cells of Candida tropicalis. The protein synthesized in vitro was isolated by immunoprecipitation with antibody against peroxisomal or mitochondrial carnitine acetyltransferase and was compared with peroxisomal carnitine acetyltransferase (Mr of subunits, 64000 and 57000) and the mitochondrial enzyme (Mr of subunits, 64000 and 52000) of C. tropicalis by electrophoresis in the presence of sodium dodecyl sulfate. Nascent carnitine acetyltransferase prepared in vitro showed a hetero‐oligomeric property, like the peroxisomal and mitochondrial enzymes isolated from C. tropicalis. The molecular weights of the subunits of nascent carnitine acetyltransferase were estimated to be 71000 and 57000, indicating the existence of the precursor form of the enzyme. By sucrose density gradient centrifugation of total mRNA, these two subunit proteins were shown to be synthesized with respective mRNAs of different sizes. The same precursor‐type of carnitine acetyltransferase was obtained with the mRNAs from the alkane‐grown cells and the propionate‐grown cells. The results obtained suggest that a common precursor will be post‐translationally modified to form the peroxisomal and mitochondrial enzymes.
Structure of ascorbic acid and its biological function
Authors: LOHMANN Wolfgang , PAGEL Detlef , PENKA Volker
Published: March 2005

The four O‐H bands of ascorbic acid could be assigned by means of infrared investigations. It could be shown by electron spin resonance and nuclear magnetic resonance measurements that the radical sodium ascorbate is formed by a cyclic side‐chain structure resulting in a loss of C(6)‐OH and C(3)‐OH. The C(2) = C(3) double bond is still maintained as could be shown by infrared and ultraviolet absorption spectroscopy. In the case of complete oxidation of ascorbic acid to dehydroascorbic acid, C(6)‐OH is reestablished (indicating the reopening of the furanoid ring), while C(2)‐OH as well as the C(2) = C(3) double bond have disappeared due to the deprotonation of C(2)‐OH and C(3)‐OH. In the case of isoascorbic acid and its radical potassium isoascorbate similar results are obtained with one distinct difference: in the case of isoascorbic acid, C(2)‐OH does not appear while C(3)‐OH exhibits a shoulder.
Nuclear‐magnetic‐resonance investigation of 15N‐labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin
Authors: FRANKEN Hans‐Dieter , RÜTERJANS Heinz , MÜLLER Franz
Published: March 2005

Flavin derivatives, enriched with 15N (≊95%) at the four nitrogen atoms of the isoalloxazine ring, have been investigated in the oxidized and the two‐electron reduced state by the 15N nuclear magnetic resonance technique. The measurements were conducted with aqueous and chloroform solutions of flavin. A comparison of the chemical shifts of the N(1) and N(5) atoms of oxidized flavin in the two solvents revealed that these atoms are sensitive indicators for possible hydrogen‐bridge formation to these atoms. The N(5) atom of oxidized flavin resonates at low field and shifts about 300 ppm upfield upon reduction.

A pKa of 6.8 was determined from pH‐dependent 15N NMR measurements of the two‐electron reduced flavin molecule. In addition it is also shown that reduced flavin in aqueous solution possesses a more coplanar structure than in chloroform solution.

The 15N chemical shifts of flavin bound to Megasphaera elsdenii apoflavodoxin indicate that various hydrogen bridges are formed between the prosthetic group and the apoprotein. Especially the N(1) atom of the prosthetic group in the oxidized state seems to form a strong hydrogen bond with the apoprotein. In the reduced state the prosthetic group is bound in the anionic form and possesses an almost coplanar structure. These results are in agreement with published crystallographic data on the related flavodoxin from Clostridium M P.

Where possible 15N‐1H, 15N‐15N and 13C‐15N coupling constants were determined. Some of the coupling constants are useful parameters for the elucidation of the planarity of free and protein‐bound flavin and for the evaluation of the interaction between flavin and apoprotein.

Spin‐lattice relaxation measurements show that the relaxation of the 15N(3)H group of flavin is predominantly determined by dipole‐dipole interaction. The calculated rotational correlation times of flavin in two different solvents were determined and are in good agreement with published results.
Glutathione transport across hepatocyte plasma membranes
Authors: INOUE Masayasu , KINNE Rolf , TRAN Thao , ARIAS Irwin M.
Published: March 2005

Transport of reduced glutathione (GSH) and its derivatives was studied in rat‐liver sinusoidal plasma‐membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited transport of GSH into an osmotically active intravesicular space. Equilibrium uptake of vesicle‐associated GSH was similar to that of free ligands which can be trapped by the intravesicular space of sinusoidal membrane samples. Kinetic analysis of the transport process revealed that the sinusoidal membrane vesicles have high‐affinity and low‐affinity GSH transport systems; the former has an apparent Km of 0.34 mM for GSH and V of 1.4 nmol × mg protein−1× 20 s−1, and the latter has an apparent Km of 3.3 mM and V of 3.9 nmol × mg protein−1× 20 s−1. Both Km values are lower than intrahepatic GSH levels, indicating that GSH transport across the sinusoidal membrane occurs via carrier‐mediated mechanism and argues simple diffusion mechanism. The presence of oxidized glutathione (GSSG) or S‐benzylglutathione inhibited GSH transport by the vesicles; the low‐affinity transport system was inhibited more markedly than the high‐affinity transport system. This suggests that these glutathione derivatives interacted preferentially with the low‐affinity transport system for GSH. S‐Dinitrophenylglutathione was also transported by the vesicles by a process which was inhibited by GSH and GSSG. The vesicles also transported GSSG, and this transport was markedly inhibited by S‐benzylglutathione or GSH.

The transport systems in sinusoidal plasma membranes may function in vivo in translocating GSH and its derivatives from hepatocytes into plasma and play an important role in inter‐organ metabolism of these compounds.
Synthesis of pigment‐binding protein in toluene‐treated Rhodopseudomonas capsulata and in cell‐free systems
Authors: DIERSTEIN Roland
Published: March 2005

Pigment‐binding protein of the facultatively phototrophic bacterium Rhodospeudomonas capsulata could be selectively synthesized in toluene‐treated cells as well as in homologous and heterologous cell‐free translation systems by isolated polysomes. It is shown that the pigment‐binding polypeptides of the light‐harvesting complexes are encoded by messenger RNA of extreme longevity. The dependence of their synthesis on the concomitant synthesis of tetrapyrroles was demonstrated in the toluene‐treated cells.

The large Mr‐28000 polypeptide of the reaction center and the Mr‐10000 pigment‐binding polypeptide of the light‐harvesting complex II were found to be synthesized by free (water‐soluble) polysomes without a cleavable ‘leader’ or ‘signal’ peptide [reviewed by W. Wickner (1979) Annu. Rev. Biochem. 48, 23–45]. The Mr‐10000 polypeptide, as synthesized in vitro, was studied in more detail. Unlike the membrane‐assembled polypeptide in vivo it was insoluble in an organic solvent mixture (chloroform/methanol 1:1, v/v). After detergent denaturation in the presence of membrane isolated from the organism it became organic‐solvent‐soluble. Obviously the polypeptide could be induced to assume alternative conformations in which its apolar residues were either exposed to the solvent or buried within. These findings, in agreement with Wickner's hypothesis, indicate that the Mr‐10000 polypeptide may enter the lipid bilayer by a ‘membrane‐triggered’ conformational change.
Identification of an essential carboxylate group at the active site of lacZβ‐galactosidase from Escherichia coli
Authors: HERRCHEN Monika , LEGLER Günter
Published: March 2005

[3H] Conduritol C cis‐epoxide (1,2‐anhydro‐epi‐inositol, I) was synthesized as an active‐site‐directed inhibitor for lacZβ‐galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH4 of the p‐benzoquinone‐derived precursor for I.

Complete loss of β‐galactosidase activity occurred on incorporation of 4 mol I/mol β‐galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH < 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo‐inositol. The stereochemistry of the expoxide reaction (trans‐diaxial ring opening) is thus the same as for β‐glucosidases with the corresponding epoxides.

The binding site for I was identified as Glu‐461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the N‐terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified γ‐carboxyl group of its N‐terminal Glu‐461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions.

The participation of the carboxylate of Glu‐461 in catalysis is discussed.
Studies on reconstituted partially purified glycerophosphate acyltransferase from Escherichia coli
Authors: KESSELS Joop M. M. , OUSEN Harry , VAN DEN BOSCH Henk
Published: March 2005

Solubilized glycerophosphate acyltransferase from Escherichia coli was reconstituted in small unilamellar vesicles consisting of phosphatidylcholine/phosphatidylglycerol in a molar ratio of 4:1. Glycerol 3‐phosphate, trapped inside these vesicles, cannot be acylated by the enzyme upon addition of extra‐vesicular palmitoyl‐CoA. Thus, substrate‐binding sites and active sites are asymmetrically oriented in the model membrane.

When up to 10mol/100mol lysophosphatidic acid was incorporated in the vesicles a decrease in glycerophosphate acyltransferase activity is observed at amounts exceeding 1 mol% lysophosphatidate. Similar experiments, using lysophosphatidylcholine and phosphatidic acid, suggest the decrease to result from an increase in negative surface charge. Reconstituted glycerophosphate acyltransferase exhibits a preference for palmitoyl‐CoA over oleoyl‐CoA. This preference increases considerably at elevated temperatures. The glycerophosphate acyltransferase could, therefore, participate in the temperature‐dependent changes in the fatty acid composition of the phospholipids in E. coli.
Glycosyl transferases of baby hamster kidney cells and ricin‐resistant mutants
Authors: STOJANOVIC Dagmar , VISCHER Peter , HUGHES R. Colin
Published: March 2005

Extracts of baby hamster kidney (BHK) cells catalyzed the incorporation of N‐acetylgalactosamine from UDP‐N‐acetyl[14C]galactosamine into myelin basic protein and an acylated tetrapeptide, N‐acetylthreonyl‐triproline, based on the threonine residue 98, glycosylated in myelin basic protein. The incorporated N‐acetylgalactosamine residues were shown to be in α linkage to the peptide moieties. Several ricin‐resistant BHK cell lines contained enhanced (approximately twofold) levels of the transferase activity. Apomucins obtained from bovine submaxillary gland mucin by chemical or enzymic degradation were relatively poor acceptors.

Using asialomucin as acceptor, galactosyl transferase activities and a weak sialyl transferase activity were detected in BHK cell extracts. Galactose transfer occurred at two sites: to peptide‐linked N‐acetylgalactosamine residues to form the linkage, galactosyl‐(β1→3)‐N‐acetylgalactosamine and to terminally linked N‐acetylglucosamine residues that exist as a minor constituent in bovine submaxillary mucin O‐glycans, to form a galactosyl N‐acetylglucosamine linkage. This reaction was not inhibited by ovalbumin, an efficient acceptor of the β1→4 galactosyl transferase involved in N‐glycan assembly.

Incorporation of galactose and N‐acetylgalactosamine into endogenous proteins of BHK cell extracts was also detected. Sialic acid, fucose and N‐acetylglucosamine residues were not incorporated. The incorporated N‐acetylgalactosamine residues were shown to be in α linkage to polypeptide, and galactose incorporation represented synthesis of the galactosyl‐(β1→3)‐N‐acetylgalactosamine sequence linked to polypeptide. The major endogenous protein labelled by either sugar had a molecular weight of approximately 80000. A BHK‐cell‐associated glycoprotein, analogous to the urinary Tamm‐Horsfall glycoprotein of molecular weight similar to the major endogenous acceptor of glycosylation, was not glycosylated in the experiments in vitro.