bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary structure of chicken skeletal muscle and fibroblast α‐actinins deduced from cDNA sequences
Authors: ARIMURA Chieko , SUZUKI Tadaaki , YANAGISAWA Masashi , IMAMURA Michihiro , HAMADA Yoshio , MASAKI Tomoh
Published: March 2005

The complete 897‐amino‐acid sequence of chicken skeletal muscle α‐actinin and the 856‐amino‐acid sequence (97% of the entire sequence) of chicken fibroblast α‐actinin have been determined by cloning and sequencing the cDNAs. Genomic Southern analysis with the cDNA sequences shows that skeletal and fibroblast α‐actinins are encoded by separate single‐copy genes. RNA blot analyzes show that the skeletal α‐actinin gene is expressed in the pectoralis muscle and that the fibroblast gene is expressed in the gizzard smooth muscle as well as in the fibroblast. The deduced skeletal α‐actinin molecule has a calculated Mr of 104 × 103, and each α‐actinin can be divided into three domains: (1) the NH2‐terminal highly conserved actin‐binding domain, which shows similarity to the product of the Duchenne's muscular dystrophy locus; (2) the middle rod‐shaped dimer‐forming domain, which contains the spectrin‐type repeat units; and (3) the COOH‐terminal two EF‐hand consensus regions. Comparison of the skeletal α‐actinin sequence with the fibroblast and smooth muscle α‐actinin sequences demonstrated that the EF‐hand structure was conserved in all of these α‐actinin sequences, despite the reported variability of the Ca2+ sensitivities of the actin‐gelation by various α‐actinin isoforms.
Heat‐resistant inhibitors of protein kinase C from bovine brain
Authors: PRIBILLA Iris , KRÜGER Helmut , BUCHNER Klaus , OTTO Henning , SCHIEBLER Werner , TRIPIER Dominique , HUCHO Ferdinand
Published: March 2005

Bovine brain cytosol is shown to contain two heat‐resistant inhibitors of protein kinase C, with the following characteristics:

One protein kinase C inhibitor can be easily purified to homogeneity. Evidence is presented that this polypeptide of Mr 19 000 is calmodulin. It inhibits protein kinase C with an EC50 of about 2.5 μM and the inhibition is Ca2+‐independent. It inhibits only intact protein kinase C. Removal of the regulatory domain of protein kinase C, by limited proteolysis with trypsin, abolishes the inhibition.

Another protein kinase C inhibitory activity has been partially purified. Its Mr 600–700, as estimated by gel chromatography). It is not digested by proteases, is hydrophilic, acid‐ and alkali‐resistant, acts Ca2+‐independently, and, in contrast to calmodulin, inhibits even the catalytic fragment of protein kinase C after removal of the regulatory domain by limited proteolysis. This inhibition is, at least partially, due to a competition with ATP. Besides protein kinase C, calcium/calmodulin‐dependent protein kinase II is inhibited to a similar extent. cAMP‐dependent protein kinase is not affected.
Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release
Authors: MAISEY E. Anne , WADSWORTH Jonathan D. F. , POULAIN Bernard , SHONE Clifford C. , MELLING Jack , GIBBS Paul , TAUC Ladislav , DOLLY J. Oliver
Published: March 2005

The abilities of botulinum neurotoxins, types A and B (single and two‐chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic‐nerve‐diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay.

Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly.

Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking the evoked release of acetylcholine when bath‐applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter.

When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide‐linked two‐chain species.

A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively. No blockade was observed when the order of application of the two chains was reversed.

These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.
A novel human myosin alkali light chain is developmentally regulated
Authors: ARNOLD Hans‐Henning , LOHSE Peter , SEIDEL Ulla , BOBER Eva
Published: March 2005

We have isolated cDNA recombinant phages encoding the embryonic isoform of the myosin alkali light chain (MLC1emb) from a human fetal skeletal muscle library. The cDNA clones were detected by their weak cross‐hybridization to a human MLC1F and MLC3F cDNA clone. Nucleotide sequence analysis of the complete cDNA (GT14) revealed an open reading frame for 197 amino acids. The derived protein sequence constitutes the first structural information on this myosin isoform of any organism. Remarkable structural similarities to other alkali MLC polypeptides, particularly to those of the slow‐muscle type, are evident. Under conditions of high stringency, the GT14 clone hybridized to an abundant mRNA species in fetal ventricular muscle and adult atrial muscle, whereas in fetal skeletal muscle only a very weakly hybridizing mRNA component was detected. These mRNAs were indistinguishable by size and the thermal stability of their hybrids formed with the DNA insert of clone GT14. We therefore conclude that identical mRNA is expressed in these tissues, presumably transcribed from the same gene. According to its pattern of mRNA expression, the novel MLC isoform described here was designated as “embryonic and atrial myosin light chain” (MLC1emb/A) in reference to its developmental stage‐specific and tissue‐specific appearance in embryonic skeletal muscle, fetal ventricle and adult atrium.
The K+‐translocating Kdp‐ATPase from Escherichia coli
Authors: SIEBERS Annette , ALTENDORF Karlheinz
Published: March 2005

The Kdp system from Escherichia coli is a derepressible high‐affinity K+ ‐uptake ATPase. Its membrane‐bound ATPase activity was approximately 50 μmol g−1 min−1.

The Kdp‐ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE‐Sepharose CL‐6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW‐65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 μM) and Mg2+‐ATP (Km= 80 μM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki= 1.5 μM), fluorescein isothiocyanate (Ki= 3.5 μM), N,N′‐dicyclohexylcarbodiimide (Ki= 60 μM) and N‐ethylmaleimide (Ki= 0.1 mM).

The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild‐type enzyme exhibited an increased Km value for K+ of 6 mM and a 10‐fold lowered sensitivity for vanadate.

Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio‐Gel P‐100 in the presence of SDS.

Both the native Kdp‐ATPase and the SDS‐denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti‐KdpABC and anti‐KdpB sera impaired ATPase activity in the membrane‐bound as well as in the purified state of the enzyme. In contrast, the anti‐KdpC serum did not inhibit enzyme activity.
Monoclonal antibody recognizes a conformational epitope in a random coil protein
Authors: SAAD Bashar , CORRADIN Gianpietro , BOSSHARD Hans Rudolf
Published: March 2005

The antigenic determinants for two monoclonal antibodies directed against horse apo‐cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme‐linked immunoassay, and differential acetylation of free and antibody‐bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen‐antibody complex is compared to the accessibility in the free antigen.

The two antibodies against the heme‐free protein do not recognize intact native cytochrome c, but they crossreact with the heme‐containing peptides 1–38 and 1–65 of cytochrome c. The antigenic determinant recognized by monoclonal antibody SJL 2–4 is conformational and discontiguous, it is composed of residues close to the N‐terminus and around position 25. The other monoclonal antibody, Cyt‐1–59, seems to recognize a contiguous epitope close to the N‐terminus.

The present results show that even a seemingly disordered protein which is conventionally classified as a random coil may feature subtle spatial regularities. The presence of ordered conformational elements in apocytochrome c may be important for the enzyme‐catalyzed covalent attachment of the heme and the import of cytochrome c into mitochondria. A discontiguous determinant for SJL 2–4 is particularly interesting because this antibody inhibits the proliferation of a T‐cell clone specific for apo‐cytochrome c [Corradin & Engers (1984) Nature (Lond.) 308, 547–548].
Purification of the yeast mitochondrial methionyl‐tRNA synthetase
Authors: SCHWOB Etienne , SANNI Ambaliou , FASIOLO Franco , MARTIN Robert P.
Published: March 2005

Yeast‐mitochondrial methionyl‐tRNA synthetase was purified 1060‐fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four‐step procedure based on affinity chromatography (heparin‐Ultrogel, tRNAMet‐Sepharose, Agarose‐hexyl‐AMP) to yield a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl‐tRNA synthetase (Mr 85000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNAMet are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNAMet. Furthermore, polyclonal antibodies raised against the two proteins did not show any cross‐reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl‐tRNA synthetases are different proteins coded for by two distinct nuclear genes.

Like the yeast cytoplasmic aminoacyl‐tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.
Quantification and characterization of regulin, a Mr‐230000 highly elongated protein of rabbit reticulocytes
Authors: ZARDENETA Gustavo , KRAMER Gisela , HARDESTY Boyd
Published: March 2005

Procedures are described by which regulin in rabbit reticulocytes was quantified and isolated in relatively large amounts. In these cells the protein occurs at a ratio of about 1.1–1.6 regulin monomers/spectrin tetramer, corresponding to 80000–100000 molecules of Mr‐230000 regulin/cell. Erythrocytes contain less than 12% of the amount of regulin in reticulocytes and the protein has not been detected in non‐erythroid cells. Regulin was found primarily in the cytosolic fraction of lysed reticulocytes. It appears to be unusually sensitive to proteolysis by Ca2+‐activated thiol proteases. Isolation of Mr‐230000 undegraded regulin was accomplished by the use of protease inhibitors including N‐ethylmaleimide. A striking characteristic of regulin is its tendency to aggregate in neutral solution of low ionic strength. Physical studies of the isolated protein indicate that it has a highly elongated form in solution. The protein has no known enzymatic activity but was shown previously to interact with and increase the enzymatic activity of a protein phosphatase. The properties of regulin suggest that it may have a structural function but it appears to be physically and immunologically distinct from known proteins. It is suggested that regulin may contribute to a gel matrix within the cytoplasm of reticulocytes.
Folding of collagen IV
Authors: DÖLZ Reinhard , ENGEL Jürgen , KÜHN Klaus
Published: March 2005

Collagen IV dimers of two collagen IV molecules connected by their C‐terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth‐Holm‐Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N‐terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44°C with a midpoint at 37°C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non‐collagenous interruptions in the triple‐helical domain of collagen IV. Refolding at 25°C following complete unfolding at 50°C was monitored by circular dichroism, selective proteolytic digestion of non‐refolded segments and by a newly developed method in which the recovered triple‐helical segments were visualized by electron microscopy. Triple‐helix formation was found to proceed in a zipper‐like fashion from the C‐terminal NC1 domains towards the N‐terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple‐helix growth was of comparable magnitude to that of collagen III, demonstrating that the non‐collagenous interruptions do not slow down the refolding process where the rate‐limiting step is the cis‐trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple‐helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N‐terminal region of the triple‐helical domain were intact, was refolding from the N‐ towards the C‐terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple‐helix folding.
Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin
Authors: CHRIST Bruno , NATH Annegret , BASTIAN Helge , JUNGERMANN Kurt
Published: March 2005

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon.

The mRNA of PEPCK was induced maximally 2–3 h after addition of 10 nM glucagon, as detected by Northern‐blot analysis after hybridization with a biotinylated antisense RNA of PEPCK.

The synthesis rate of PEPCK increased maximally 2–3 h after application of glucagon as revealed by pansorbin‐linked immunoprecipitation of [35S]methionine‐labelled PEPCK.

The enzyme amount and activity was maximally induced 4 h after glucagon application.

The mRNA of PEPCK was half‐maximally induced by 0.1 nM and maximally by 1 nM and 10 nM glucagon. The half‐maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin.

The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon‐dependent increase in mRNA and enzyme‐synthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1–1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range with glucagon being the dominant hormone.
Fidelity of aminoacylation by rat‐liver tyrosyl‐tRNA synthetase
Authors: TAKAHASHI Ryoya , GOTO Sataro
Published: March 2005

The possibility of change in the rate of misrecognition of amino acids by rat liver tyrosyl‐tRNA synthetase during aging was investigated. Frequency of misrecognition of phenylalanine vs tyrosine was determined at two temperatures by competitive assay using partially purified enzymes. At 25°C, the error frequencies were 5.17 × 10−8 and 8.24 × 10−8 in young and old animals, respectively. These values are much below the reported error frequencies for the prokaryotic enzymes: i.e. ∼ 5 × 10−6. Although the fidelity of tyrosyl‐tRNA synthetase from old animals appeared to be slightly lower, the difference was not statistically significant. At 37°C, the error frequencies were increased 1.3–1.5‐fold, but again the difference between young and old animals was not significant.

To our knowledge, this is the first report in which fidelity of aminoacyl‐tRNA synthetase of animals of various ages has been compared using natural amino acids.
Panulirus interruptus hemocyanin
Authors: JEKEL Peter A. , BAK Henk J. , SOETER Nell M. , VEREIJKEN Johan M. , BEINTEMA Jaap J.
Published: March 2005

The primary structure of subunit b of Panulirus interruptus hemocyanin has been derived from two digests (trypsin and CNBr) and, in some cases, with aid from the similarity with the sequence of subunit a. Differences between the amidation states of Asx and Glx residues in subunit b relative to a were investigated more thoroughly. When compared to the sequence of subunit a, 18 differences (2.7%) were found and certain heterogeneities, indicating the presence of a minor subunit b′, were observed.

Several differences in properties between subunits a and b, including their anomalous behaviour on SDS/ polyacrylamide gel electrophoresis, could be explained by amino acid replacements.
NMR studies of the conformations of leghemoglobins from soybean and lupin
Authors: NARULA Surinder S. , DALVIT Claudio , APPLEBY Cyril A. , WRIGHT Peter E.
Published: March 2005

Phase‐sensitive two‐dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X‐ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein‐heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.
Yeast adenylate kinase is active simultaneously in mitochondria and cytoplasm and is required for non‐fermentative growth
Authors: BANDLOW Wolfhard , STROBEL Gertrud , ZOGLOWEK Cornelia , OECHSNER Ulrich , MAGDOLEN Viktor
Published: March 2005

Displacement of the single copy structural gene for yeast adenylate kinase (long version) by a disrupted nonfunctional allele is tolerated in haploid cells. Since adenylate kinase activity is a pre‐requisite for cell viability, the survival of haploid disruption mutants is indicative of the presence of an adenylate kinase isozyme in yeast, capable of forming ADP from AMP and, thus, of complementing the disrupted allele. The phenotype of these disruption mutants is pet, showing that complementation occurs only under fermentative conditions. Even on glucose, growth of the disruption mutants is slow. Adenylate kinase activity is found both in mitochondria and cytoplasm of wild type yeast. The disruption completely destroys the activity in mitochondria, whereas in the cytoplasmic fraction about 10% is retained. An antibody raised against yeast mitochondrial adenylate kinase recognizes cross‐reacting material both in mitochondria and cytoplasm of the wild type, but fails to do so in each of the respective mutant fractions. The data indicate that yeast adenylate kinase (long version, AKY2) simultaneously occurs and is active in mitochondria and cytoplasm of the wild type. Nevertheless, it lacks a cleavable pre‐sequence for import into mitochondria. A second, minor isozyme, encoded by a separate gene, is present exclusively in the cytoplasm.
Isolation of a mutant from Salmonella typhimurium producing acyl‐deficient lipopolysaccharides
Authors: LEHMANN Volker , BENNINGHOFF Bernd
Published: March 2005

The present paper describes the isolation and characterization of a mutant (mutant Ts7) of Salmonella typhimurium that is conditionally defective in the incorporation of dodecanoic and tetradecanoic acid into lipopolysaccharide precursor structures. Enrichment of mutant Ts7 was achieved by free‐flow electrophoresis and was based on a previous observation that at least some Salmonella mutants conditionally blocked in the synthesis of the 3‐deoxy‐D‐manno‐octulosonic acid (dOclA)–lipid‐A region exhibit higher electrophoretic mobilities than cells with intact dOclA–lipid‐A regions. Under nonpermissive conditions (42°C) mutant Ts7 accumulates at least two incomplete dOclA–lipid‐A structures. One is made up of glucosamine, phosphate, dOclA, and 3‐hydroxytetradecanoic acid in a molar ratio 1.0:1.3:1.0:2.2 and is devoid of dodecanoic and tetradecanoic acid. The other structure has the same basic structure but contains hexadecanoic acid.
Domain preference in iron removal from human transferrin by the bacterial siderophores aerobactin and enterochelin
Authors: FORD Steven , COOPER Ronald A. , EVANS Robert W. , HIDER Robert C. , WILLIAMS Peter H.
Published: March 2005

The ability of the siderophores aerobactin and enterochelin to remove iron from transferrin is reported. Aerobactin removes iron from both high‐affinity sites on the transferrin molecule, but shows a marked preference for the C‐terminal site. This preference is different to that of many iron chelators. Enterochelin removes iron perferentially from the N‐terminal site. No evidence for synergism between aerobactin and bidentate ligands could be detected.
Erythrocyte adducin
Authors: WASEEM Ahmad , PALFREY H. Clive
Published: March 2005

Two major substrates for human erythrocyte protein kinase C (PK‐C) of Mr 120 000 and 110 000, previously named PKC‐1 and PKC‐2 [Palfrey, H. C. & Waseem, A. (1985) J. Biol. Chem. 260, 16021–16029] have been found to be identical to CaM‐BP103/97 or ‘adducin’, recently described by K. Gardner and V. Bennett [(1986) J. Biol. Chem. 261, 1339–1348; (1987) Nature (Lond.) 328, 359–362]. These proteins have been purified from the membrane skeleton by high‐salt extraction, ion‐exchange and gel filtration chromatography. The two proteins co‐fractionate in a ratio of ∼ 1:1 under a number of conditions suggesting that they exist as a complex. Physicochemical data indicate that the native adducin complex is probably an asymmetric heterodimer of α and β subunits. Adducin binds to a calmodulin (CaM) affinity matrix in a Ca2+‐dependent manner and is specifically eluted with EGTA. Fingerprinting of the iodinated peptides derived from the α and β subunits using three different proteases yields 16–37% overlapping peptides, indicating limited similarity between the two polypeptides. Affinity‐purified polyclonal antibodies against each protein show little or no cross‐reactivity with the other, indicating that the β subunit is not derived from the α subunit or vice versa. Proteins reactive with both anti‐(α‐adducin) and anti‐(β‐adducin) antibodies are found in erythrocytes from rat, rabbit, pig, ferret and duck. Immunoblots of adducin after non‐ionic detergent extraction of ghosts reveal that a significant fraction of the protein may associate with non‐skeleton membrane components.

The phosphorylation of adducin is stimulated by both phorbol esters and cAMP analogues in intact erythrocytes. Fingerprinting suggests that protein kinase C preferentially phosphorylates four distinct sites on the two proteins. Phosphopeptide maps of α‐adducin are virtually identical to those of β‐adducin after phorbol ester stimulation of intact cells, or after PK‐C‐catalyzed phosphorylation of the purified protein, indicating strong local similarities in the two proteins. Such maps also suggest that cAMP‐dependent protein kinase (cAMP‐PK) modifies adducin at some similar and some distinct sites as those modified by PK‐C. In vitro phosphorylation of isolated adducin by purified PK‐C results in rapid incorporation of phosphate to a final level of ∼ 1.5 mol/mol in both α and β subunits. Phosphorylation by the catalytic subunit of cAMP‐PK is equally rapid, with α‐ adducin being preferentially phosphorylated to a level of ∼ 1.8 mol/mol. Increased phosphorylation enhances the extraction of adducin from the membrane by non‐ionic detergent.

The multivalent properties of this protein complex suggest that it may play a role in mediating the interactions of components of the membrane skeleton with the inner surface of the membrane and that CaM and/or phosphorylation may modulate this function.
Altered expression of myosin light‐chain isoforms in chronically stimulated fast‐twitch muscle of the rat
Authors: BÄR Angelika , SIMONEAU Jean‐Aimé , PETTE Dirk
Published: March 2005

Fast‐twitch tibialis anterior muscle of the rat was chronically stimulated for periods of 18 days, 28 days and 56 days. Changes in the myosin light‐chain (LC) pattern consisted in an increase in LC1f, concomitant with a decrease in LC3f. In contrast to previous findings in chronically stimulated fast‐twitch tibialis anterior muscle of the rabbit, no substantial increases occured in the slow myosin light‐chain isoforms. In vivo labeling using [35S]methionine incorporation revealed differences in relative turnover between the fast myosin light chains. The relative turnover of the fast myosin light chains appeared to increase in normal muscle in the order LC2f<LC1f<LC3f. As judged from [35S]methionine incorporation, the changes in light‐chain tissue content mainly resulted from altered synthesis rates. However, in the case of LC3f the decrease in protein content could not only be explained by a reduced synthesis, but, additionally, appeared to be due to enhanced degradation. Parvalbumin, which was included in the present study, was also found to decrease in the stimulated muscle. However, its decrease appeared to result primarily from reduced synthesis.
Structural studies on the neutral polysaccharide of Bacillus subtilis AHU 1219 cell walls
Authors: IWASAKI Hiroyoshi , ARAKI Yoshio , KAYA Shunji , ITO Eiji
Published: March 2005

A neutral and an acidic polysaccharide with molecular masses of about 22 kDa and 45 kDa, respectively, were isolated from the N‐acetylated cell walls of Bacillus subtilis AHU 1219 by heating at pH 2.5, followed by separation of the water‐soluble product by ion‐exchange chromatography and gel chromatography. The neutral polysaccharide, accounting for 40% of the mass of the cell walls, contained glucose, N‐acetylglucosamine, N‐acetylgalactosamine and N‐acetylmannosamine in a molar ratio of 1:2:1:1. The minor, acidic polysaccharide contained glucuronic acid, glucose, galactose, L‐serine and L‐threonine in an approximate molar ratio of 1:1:1:0.5:0.5. Lysozyme digestion of the N‐acetylated cell walls gave a polymer containing the neutral polysaccharide and glycopeptide components and another polymer which contained the acidic polysaccharide components together with small proportions of the neutral polysaccharide and glycopeptide components. Thus, the neutral and acidic polysaccharide chains seem to be attached to peptidoglycan through acid‐labile linkages in the cell walls of this strain.

Structural analysis of the neutral‐polysaccharide preparation, involving 1H‐NMR and 13C‐NMR measurement. methylation and Smith degradation, led to the most likely structure, →6)[Glc(β1 → 3)]GalNAc(α1 → 4)‐[GlcNAc(β1 → 3)]ManNAc(β1 → 4)GlcNAc(β1 →, for the repeating units of this polysaccharide.
Tropomyosin in the sea urchin egg cortex
Authors: MAEKAWA Shohei , TORIYAMA Masaru , SAKAI Hikoichi
Published: March 2005

Tropomyosin was purified from the Triton‐treated cortex fraction of fertilized sea urchin egg. Egg tropomyosin showed characteristics typical of nonmuscle tropomyosins such as low molecular mass, short periodicity of Mg2+‐ paracrystals, low lysine/arginine ratio, high Mg2+‐ requirement in binding to F‐actin, in addition to the properties of all tropomyosins, namely, stability to high temperature, anomalous migration of SDS/urea gel, dissociation from F‐actin under high ionic conditions and very acidic isoelectric point. Co‐sedimentation assay of egg tropomyosin with actin in the presence of the previously purified high‐molecular‐mass actin binding protein (260–kDa protein) showed that these two proteins bind to actin filaments in a non‐competitive manner. This suggested that both the proteins play a cooperative role in the formation of actin‐filament‐based cytoskeletal structure in the cortex.
Regulation of gene expression at the translational level
Authors: GANOZA M. Clelia , KNAUBER Donna C. , MURPHY Brian , FALLAVOLITA Dino , CHUNG Dae‐Gyun
Published: March 2005

Extracts of the conditionally‐lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non‐permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non‐permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read‐through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis.

Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine‐puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.
Interactions of concanavalin A with asparagine‐linked glycopeptides
Authors: BHATTACHARYYA Lokesh , BREWER C. Fred
Published: March 2005

We have recently demonstrated that certain oligomannose and bisected hybrid‐type glycopeptides are bivalent for concanavalin A (ConA) binding and that they can precipitate the lectin [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P. & Brewer, C. F. (1987) J. Biol. Chem. 262, 1288–1293]. Two protein‐binding sites on each glycopeptide were identified: one on the α(1–6) arm of the core β‐mannose residue which binds with high affinity (primary site); the other on the α(1–3) arm of the core β‐mannose residue which binds with lower affinity (secondary site). In the present study, we have investigated the relationship between the structures of the primary sites of oligomannose‐type glycopeptides and their affinities for ConA. Two mechanisms of binding at the primary sites of oligomannose‐type glycopeptides have been identified which account for the 3000‐fold increase in affinity of a Man9 glycopeptide relative to that of methyl α‐D‐mannopyranoside. Changes in the structures and affinities of both the primary and secondary sites are observed to influence the precipitation activities of the glycopeptides. These findings have important consequences for the specificity of ConA binding in solutions containing mixtures of the carbohydrates.
In vitro synthesis of colominic acid by membrane‐bound sialyltransferase of Escherichia coli K‐235
Authors: ORTIZ Ana I. , REGLERO Angel , RODRÍGUEZ‐APARICIO Leandro B. , LUENGO José M.
Published: March 2005

The membrane‐bound sialyltransferase obtained from Escherichia coli K‐235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half‐life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33°C and pH 8.3) hyperbolic behaviour with respect to CMP‐Neu5Ac (Km 250 μM) and a transition temperature at 31.3°C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein‐complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 μM. Incubations of protein‐[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP‐[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP‐[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.
Regulation of ornithine decarboxylase in murine erythroleukemic cells by N,N′‐diacetyl‐1,6‐hexanediamine and N‐acetyl‐1,6‐hexanediamine
Authors: PAPAZAFIRI Panagiota , OSBORNE Howard Beverley
Published: March 2005

The levels at which the ornithine decarboxylase gene is regulated in murine erythroleukemic cells treated with N,N′‐hexamethylene bisacetamide (N,N′diacetyl‐1,6‐hexanediamine) and with N‐acetyl‐1,6‐hexanediamine, the monoacetylated catabolite of this inducer of erythrodifferentiation, have been investigated. Although these two molecules are structurally related and both cause a decrease in ornithine decarboxylase activity, they did not similarly affect ornithine decarboxylase mRNA. In the presence of 4 mM N,N′‐hexamethylenebisacetamide, at the concentration generally used to induce differentiation in these cells, a decreased steady‐state level of ornithine decarboxylase mRNA, due to decreased gene transcription, was observed. In the presence of N‐acetyl‐1,6‐ hexanediamine the decrease in enzyme activity was shown to be associated with a decrease in the half‐life of the enzyme in the absence of a change in gene transcription. These results show that in proliferating cultured cells changes in ornithine‐decarboxylase‐gene transcription can be uncoupled from changes in cell growth and that N‐acetyl‐1,6‐hexanediamine only regulates ornithine decarboxylase expression post‐transcriptionally.
Sequence analysis and expression of the arginine‐deiminase and carbamate‐kinase genes of Pseudomonas aeruginosa
Authors: BAUR Heinz , LUETHI Ernst , STALON Victor , MERCENIER Annick , HAAS Dieter
Published: March 2005

The arcABC operon of Pseudomonas aeruginosa encodes arginine deiminase, catabolic ornithine carbamolytransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and arcC genes. The arcA open reading frame specifies a polypeptide of 46.3 kDa. The same molecular mass was obtained for the subunit of purified arginine deiminase after electrophoresis under denaturing conditions. The N‐terminal amino acid sequence of arginine deiminase was in agreement with the corresponding nucleotide sequence. The native arginine deiminase had an estimated molecular mass of 175–180 kDa, suggesting a tetrametric structure. The enzyme was activated by Mg2+ or Mn2+ and strongly inhibited by Zn2+. The apparent Km for L‐arginine was 0.04 mM in the presence of Mg2+ and 0.47 mM without Mg2+. The arcC open reading frame codes for a 33‐kDa protein, confirming the molecular mass previously reported for the subunit of carbamate kinase. The translation‐initiation site of arcC was determined by deletion mapping. Two regions of dyad symmetry found between arcA and arcC might stabilize the putative arcABC transcript in the upstream (arcA) region; this might contribute to the high level of arcA expression as compared to the moderate level of arcC expression.

Carbamate kinase had 37% sequence similarity (and 13.5% identity) with the C‐terminal part of carbamoylphosphate synthetase (large subunit) from Escherichia coli. Arginine deiminase had no apparent similarity with argininosuccinate lyase. Thus, the arcA and arcC genes do not appear to be closely related to arginine biosynthetic genes, whereas it had previously been shown that the arcB gene has a high degree of identity with the arginine biosynthetic argF genes of P. aeruginosa and E. coli.
Molecular cloning of rat human type IX collagen cDNA and localization of the α1(IX) gene on the human chromosome 6
Authors: KIMURA Tomoastsu , MATTEI Marie‐Geneviève , STEVENS Jeff W. , GOLDRING Mary B. , NINOMIYA Yoshifumi , OLSEN Bjorn R.
Published: March 2005

Type IX collagen is found in hyaline cartilage, where it is asociated with type II collagen lin quarter‐stagered collagen firils. Chicken type IX collagen has been extensively characterized and shown to contain molecules with three tripole‐helical domains, interspersed with non‐triple‐helical sequences. The molecule contains three, genetically disstinct, subunits and one of these subunits carries a covalently bound glycosaminoglycan side chain.

In the present report, we describe for the first time the primary structure of mammalian type IX collagen chains, based on cloning and sequencing of cDNA from rat and human cDNA libraries. The results suggest that mammalian α1(IX) chains have the same multi‐domain structure as the avian protein. We also demonstrate, by in situ hybridization of chromosome spreads, that the human α1(IX) collagen gene is located on the long arm of chromosome 6. The cloning of human type IX collagon cDNA provides a probe for molecular studies of human chondrodysplasias that may involve abnormalities in this extracellular collagen‐proteoglycan.
Chloroplast ATP synthase contains one single copy of subunit δ that is indispensable for photophosphorylation
Authors: ENGELBRECHT Siegfried , SCHÜRMANN Karin , JUNGE Wolfgang
Published: March 2005

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit δ, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast δ, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the lieterature, we reinvestigated two further aspects related to subunit δ, namely (a) its stoichiometry in CF0CF1 and (b) whether or not δ is required for photophosphorylation.

By rocket immunoelectrophoresis of thylakoid membrances and calibration against purified δ, we confirmed a stoichiometry of one δ per CF0CF1. In CF1‐depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit δ but, surprisingly, also by CF1(−δ). We found that the latter was attributable to a contamination of CF1(−δ) preparations with integral CF1. To Lesser extent CF1(−δ) acted by complementary rebinding to CF0 channels that were closed because they contained δ [CF0(+δ)]. This added catalytic capacity to proton‐tight thylakoid vesicles.

The ability of subunit δ to control proton flow through CF0 and the absolute requirement for δ in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: δ may be part of the couping site between electrochemical, conformational and chemical events in this enzyme.
Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes
Authors: van SCHAFTINGEN Emile , VANDERCAMMEN Annick
Published: March 2005

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2‐3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2–4‐fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half‐maximal stimulatory effect was observed at about 50 μM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D‐Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritaiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acylCoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1‐phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long‐chain acyl‐CoAs.
Molecular cloning, sequencing and expression in Escherichia coli of the 25‐kDa growth‐related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins
Authors: GAESTEL Matthias , GROSS Burckhard , BENNDORF Rainer , STRAUSS Michael , SCHUNK Wolf‐Hagen , KRAFT Regine , OTTO Albrecht , BÖHM Hans , STAHL Joachim , DRABSCH Heinz , BIELKA Heinz
Published: March 2005

The growth‐related 25‐kDa protein (p25) of Ehrlich ascites tumor (EAT) has been characterized by molecular cloning and sequencing of cDNA clones detected by hybridization with oligonucleotide probes synthesized according to the amino acid sequence of a tryptic peptide of p25. Detection of p25 mRNA in EAT of the exponential growth phase and of the stationary phase using cDNA‐derived RNA probes demonstrated that the abundance of p25 mRNA is also growth‐related. High‐level expression of p25 in Escherichia coli has been established by oligonucleotide‐directed mutagenesis of cDNA and insertion of the mutated cDNA into a T7‐promoter expression vector. Recombinant p25 from the expressed cDNA sequence has been shown to comigrate with EAT p25 in electrophoresis and to react with antibodies against the EAT p25. On the amino acid level, p25 shows about 80% sequence homology to the human stress protein hsp27. Furthermore, p25 has similar isoforms of phosphorylation as demonstrated for small mammalian stress proteins from rat and human. From the results obtained, it is concluded that p25 is a mammalian stress protein, the abundance of which is related to growth characteristics of the Ehrlich ascites tumor.
Specific S‐thiolation of a 30‐kDa cytosolic protein from rat liver under oxidative stress
Authors: ROKUTAN Kazuhito , THOMAS James A. , SIES Helmut
Published: March 2005

Thin‐gel isoelectric focusing (IEF) is a simple and sensitive method of quantifying S‐thiolation of individual proteins (protein mixed‐disulfide formation). IEF of rat liver cytosol identified one major protein (pI 7.0) which underwent S‐thiolation with glutathione disulfide to produce two acidic bands with pIs 6.4 and 6.1. The S‐thiolated forms of the protein were purified by preparative isoelectric focusing. An apparent molecular mass of 30 kDa was determined by SDS/polyacrylamide gel electrophoresis. The 30‐kDa protein amounted to 7 ± 2% of the total cytosolic protein on IEF.

The most abundant soluble protein of freshly isolated hepatocytes, with an identical isoelectric point to the liver 30‐kDa protein, was modified in a similar manner in response to oxidative stress induced by model compounds. Addition of 50 μM tert‐butyl hydroperoxide, 50 μM diamide [1,1‐azobis(N,N′‐dimethylformamide)] or 20 μM menadione (2‐methyl‐1,4‐naphthoquinone) initiated the S‐thiolation within less than 2 min in the hepatocytes. These compounds, at the concentrations employed, did not result in cell death. Menadione produced slowly progressive S‐thiolation of the protein, while tert‐butyl hydroperoxide or diamide produced rapid S‐thiolation that decreased quickly after 2 min.