bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotonin organelles of rabbit platelets contain synaptophysin
Authors: BÄHLER Martin , CESURA Andrea M. , FISCHER Günther , KUHN Herbert , KLEIN Ronald L. , Da PRADA Mosé
Published: March 2005

Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno‐electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10–15‐fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (∼ 38 kDa) and the extent of glycosylation of platelet‐derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno‐electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin‐like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle‐associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.
The isolation and characterization of a barley 1,3‐1,4‐β‐glucanase gene
Authors: LITTS James C. , SIMMONS Carl R. , KARRER Erik E. , HUANG Ning , RODRIGUEZ Raymond L.
Published: March 2005

The barley gene encoding isozyme I of 1,3‐1,4‐β‐glucanase was isolated and sequenced. The 6260‐bp region sequenced included 1885 bp of the 5′‐flanking region, the entire coding region, an intron of 2490 bp, and 792 bp of the 3′‐flanking region. The 1,3‐1,4‐β‐glucanase mRNA was found to be regulated at the level of RNA accumulation by both gibberellins (positively) and abscisic acid (negatively) in barley aleurones. The mRNA for isozyme II preferentially accumulated (70%) relative to the mRNA for isozyme I (30%) in poly(A)‐rich RNA isolated from material including both the aleurone and the scutellum tissues. The gene family encoding 1,3‐1,4‐β‐glucanase enzymes in barley was found to be comprised of two closely related genes, isozymes I and II, as well as several related sequences that could be identified by Southern blot analysis. The nucleotide sequence for the 5′ untranslated leader and the coding region for the signal peptide of the isozyme II transcript were determined from a cDNA produced by the polymerase chain reaction. The structure of the protein encoded by the isozyme I gene is also discussed.
Uptake of glutamate in Corynebacterium glutamicum
Authors: KRÄMER Reinhard , LAMBERT Camille , HOISCHEN Christian , EBBIGHAUSEN Holger
Published: March 2005

The active uptake system for glutamate in Corynebacterium glutamicum is inducible by growth on glutamate as sole energy and carbon source and is also susceptible to catabolite repression by glucose. The basic level of uptake activity is low in glucose‐grown cells (1.5 nmol · mg dry mass−1· min−1), it is intermediate when acetate is the carbon source (3.8 nmol · mg dry mass−1· min−1) and becomes fully induced by glutamate (15 nmol · mg dry mass−1· min−1). In all cases the uptake has, except for different Vmax values, identical kinetic and energetic properties, and is characterized by a low apparent Km value of 0.5–1.3 μM and by high substrate specificity. The transported substrate species is the deprotonated form which can also be concluded from the extremely high pH optimum of transport above pH 9. Glutamate uptake in cells grown in media with low K+ concentration is not influenced by external Na+ but is drastically stimulated by addition of K+. Stimulation by K+ could be separated into two different mechanisms. (a) Addition of K+ increases the internal pH, thereby stimulating glutamate uptake which is regulated by the internal pH in C. glutamicum. The apparent pK of the internal ‘pH switch’ is 6.6; below this value, uptake of glutamate is inhibited. (b) Internal K+ also directly promotes glutamate uptake. Effective uptake of glutamate can be observed only when the cytosolic K+ concentration exceeds a threshold value of about 200 mM. Stimulation of glutamate uptake by external K+ is not due to functional coupling of K+ and glutamate transport but reveals the necessity to replenish the internal K+ pool.
Simian‐virus‐40 large‐T‐antigen‐catalyzed DNA and RNA unwinding reactions
Authors: SCHEFFNER Martin , KNIPPERS Rolf , STAHL Hans
Published: March 2005

Simian virus 40 large T antigen is a helicase separating the complementary strands of double‐stranded DNA in the presence of hydrolyzable ATP and of double‐stranded RNA in the presence of non‐ATP nucleotides (GTP, CTP or UTP). We have constructed partially single‐stranded nucleic acid substrates consisting of RNA or DNA strands hydrogen bonded to either RNA or DNA complements. We found that ATP is utilized as a cofactor for the T‐antigen‐catalyzed unwinding reaction when the substrates contain overhanging single‐stranded DNA, regardless of whether the double‐stranded region is DNA or hybrid DNA · RNA. Conversely, non‐ATP nucleotides are used when the overhanging single strand is RNA. Based on these and additional findings, we propose that the bound nucleic acid induces a conformational change in T antigen resulting in a proper orientation of both nucleic acid and nucleotide relative to the active center of the ATPase/helicase domain of the enzyme. The implications of our conclusion for the roles which T antigen may play in vivo are discussed.
On the mechanism of sodium ion translocation by methylmalonyl‐CoA decarboxylase from Veillonella alcalescens
Authors: HILPERT Wilhelm , DIMROTH Peter
Published: March 2005

Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7–8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl‐CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating Ψ of 60 mV, pNa+ of 50 mV, and of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions/malonyl‐CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl‐CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl‐CoA decarboxylation rate 2.8‐fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8‐fold. Methylmalonyl‐CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl‐CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl‐CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events.
Metabolism of [2‐13C]succinate in renal cells determined by 13C NMR
Authors: JANS Arnold W. H. , WILLEM Rudolph
Published: March 2005

[2‐13C]Succinate has been used to examine the metabolic carbon flux from the Krebs cycle in rat renal proximal convoluted tubular (PCT) cells under physiological and pathophysiological conditions. Therefore, we developed a mathematical model that enabled us to determine the metabolic fluxes of the Krebs cycle. A mathematical model for the calculation of flux from [2‐13C]succinate was used to determine fluxes in rat PCT cells during chronic acidosis in the presence and absence of 0.1 mM angiotensin II. The relative carbon efflux via glutamate dehydrogenase in rat renal PCT cells increases during chronic acidosis from 0.27 to 0.39, whereas this carbon flux is not affected by the presence of peptide hormone angiotensin II in the incubation medium.

The fraction of intermediate 13C‐labelled oxaloacetate transformed into the phosphoenolpyruvate and aspartate pools increases significantly from 0.41 to 0.57 in the case of chronic acidosis. The carbon efflux is not affected by angiotensin II. The 13C‐NMR data also show that the carbon efflux through phosphoenolpyruvate carboxykinase increases from 0.35 to 0.56 in rat renal PCT cells derived from chronic acidotic animals, as well as in the presence of angiotensin II. The present results indicate that angiotensin II affects only the flux through phosphoenolcarboxykinase, whereas chronic acidosis increases the flux through phosphoenolpyruvate carboxykinase as well as the gluconeogenic flux.
Failure of channelling to maintain low concentrations of metabolic intermediates
Authors: CORNISH‐BOWDEN Athel
Published: March 2005

Computer modelling has been used to investigate the effect of direct transfer of metabolites between consecutive enzymes (channelling) on the free concentrations of the channelled metabolites. When a channelled intermediate cannot participate in any other reactions, any increase in channelling tends to increase its free concentration, albeit very slightly, unless the increase in net flux brought about by the channel is compensated for by a simultaneous decrease in the activity of the route through the free intermediate, in which case channelling has no effect at all on the free steady‐state concentration of the channelled intermediate. If the free intermediate is capable of participating in side reactions, channelling can decrease these side reactions, but only slightly unless virtually all of the final product results from flux through the channel and the rate constants for the direct pathway are virtually zero. In general, channelling appears not to provide a useful mechanism for maintaining intermediate concentrations at low levels.
Isolation of schistosomin, a neuropeptide which antagonizes gonadotropic hormones in a freshwater snail
Authors: HORDIJK Peter L. , EBBERINK Rob H. M. , de JONG‐BRINK Marijke , JOOSSE Joos
Published: March 2005

The molecular mechanisms underlying parasite‐induced inhibitory effects on host reproduction were studied in the freshwater snail, Lymnaea stagnalis, infected with the schistosome parasite Trichobilharzia ocellata. This combination is used as a model system for host‐parasite interactions involved in schistosomiasis transmission. The female gonadotropic snail neuropeptide, calfluxin, was labelled with fluorescein isothiocyanate (FITC) and used as a ligand in receptor‐binding studies on membranes of its target organ, the albumen gland. The binding of calfluxin to its receptor–guanyl‐nucleotide‐binding‐protein (G‐protein) complex was inhibited in vitro in the presence of haemolymph of schistosome‐infected snails. This inhibition appeared to be established by a peptidergic factor called schistosomin. The receptor assay was used to identify schistosomin from haemolymph during subsequent purification and characterization steps. The peptide could also be purified from the central nervous systems of non infected snails, indicating that it is produced by the snail itself and released into the haemolymph as a result of infection. Analysis by plasma‐desorption mass spectrometry revealed that purified schistosomin has a molecular mass of 8780 Da.
The human leukocyte‐adhesion ligand, intercellular‐adhesion molecule 2
Authors: GAHMBERG Carl G. , NORTAMO Pekka , ZIMMERMANN Dieter , RUOSLAHTI Erkki
Published: March 2005

The leukocyte cell‐adhesion receptors, complexes of the cluster of differentiation antigen 11a with cluster of differentiation antigen 18 (CD11a/CD18), cluster of differentiation antigen 11b with cluster of differentiation antigen 18 (CD11b/CD18) and cluster of differentiation antigen 11c with cluster of differentiation antigen 18 (CD11c/CD18), are of major importance in several leukocyte functions. Previously a cellular ligand named intercellular‐adhesion molecule 1 (ICAM‐1) was identified, isolated and extensively characterized. Recently a second similar molecule, intercellular‐adhesion molecule 2 (ICAM‐2), was found by a functional DNA‐cloning method. We have now synthesized the ICAM‐2DNA by the polymerase chain reaction (PCR), sequenced it, and transferred it into mammalian and bacterial expression vectors. A functional leukocyte‐binding glycoprotein was obtained by transfection of COS‐1 cells. A soluble protein‐A–ICAM‐2 fusion protein was made in Escherichia coli, purified and used for antiserum production. The antiserum precipitated a cell‐surface protein with an apparent molecular mass of 55 kDa from ICAM‐2 transfected COS‐1 cells, leukocytes and endothelial cells, and inhibited leukocyte binding to transfected COS‐1 cells. The bacterial fusion protein, lacking carbohydrate, specifically bound to leukocyte receptors.
Rapid characterization of asparagine‐linked oligosaccharides isolated from glycoproteins using a carbohydrate analyzer
Authors: ANUMULA Kalyan Rao , TAYLOR Paul B.
Published: March 2005

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo‐complex and high‐mannose) oligosaccharides released from glycoproteins with peptide N‐glycosidase F, endo‐β‐N‐acetylglucosaminidase F and endo‐β‐N‐acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion‐exchange column HPIC‐AS6/CarboPac PA‐1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1–0.3 μA output with suitable sensitivity (< 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients.

Oligosaccharides released from heat‐denatured and trypsin‐treated glycoproteins were purified initially from large‐scale digestions (> 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ionexchange chromatography of the aqueous phase. Oligosaccharides isolated from the peptide N‐glycosidase digests of bovine fetuin, human transferrin and α1‐acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono‐, di‐, tri‐ and tetra‐sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high‐mannose and complex types were poorly resolved. The high‐mannose peaks were eliminated specifically from complex oligosaccharides by digesting with α‐mannosidase. Treatment with β‐galactosidase, βN‐acetylglucosaminidase and α‐mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with α‐l‐fucosidase, endo‐β‐N‐acetylglucosaminidase F and endo‐β‐N‐acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue.

Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endo‐and exo‐glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.
Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins
Authors: MOLLNER Stefan , SIMMOTEIT Robert , PALM Dieter , PFEUFFER Thomas
Published: March 2005

Monoclonal antibodies against partially purified adenylyl cyclase from bovine brain cortex were raised in mice. Three types of antibody were obtained. Type I was specific for the calmodulin‐sensitive enzyme. Type II also recognized this enzyme, but recognized the calmodulin‐insensitive enzymes from a variety of species and tissues as well. Type I antibodies precipitated their antigens in both the native and denatured forms, while type II strongly favored the denatured forms. Type III antibodies precipitated adenylyl cyclase activity, but as shown by Western blot analysis, were directed against 38‐kDa and 45‐kDa glycoproteins. The 38‐kDa protein was identified as synaptophysin.
Sensory transduction in eukaryotes
Authors: Van HAASTERT Peter J. M. , JANSSENS Pim M. W. , ERNEUX Christophe
Published: March 2005

The organization of multicellular organisms depends on cell–cell communication. The signal molecules are often soluble components in the extracellular fluid, but also include odors and light. A large array of surface receptors is involved in the detection of these signals. Signals are then transduced across the plasma membrane so that enzymes at the inner face of the membrane are activated, producing second messengers, which by a complex network of interactions activate target proteins or genes [1].

Vertebrate cells have been used to study hormone and neurotransmitter action, vision, the regulation of cell growth and differentiation. Sensory transduction in lower eukaryotes is predominantly used for other functions, notably cell attraction for mating and food seeking. By comparing sensory transduction in lower and higher eukaryotes general principles may be recognized that are found in all organisms and deviations that are present in specialised systems. This may also help to understand the differences between cell types within one organism and the importance of a particular pathway that may or may not be general. In a practical sense, microorganisms have the advantage of their easy genetic manipulation, which is especially advantageous for the identification of the function of large families of signal transducing components.
Molecular cloning and chromosomal localization of a human peripheral‐type benzodiazepine receptor
Authors: RIOND Joelle , MATTEI Marie Genevieve , KAGHAD Mourad , DUMONT Xavier , GUILLEMOT Jean Claude , LE FUR Gerard , CAPUT Daniel , FERRARA Pascual
Published: March 2005

The sequencing of endopeptidase‐generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2‐terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h‐pPBS11, is 831 nucleotides and contains a full‐length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.
Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells
Authors: KUMAR Rajeswari V. , PANNIERS Richard , WOLFMAN Alan , HENSHAW Edgar C.
Published: March 2005

Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2+ might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 μM calmidazolium, 12 μM N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W7) and 17.5 μM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80S monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell‐free protein‐synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 μM, 125 μM, 300 μM and 500 μM, respectively. A weak analogue of W7 inhibited only 20% at 1000 μM. Inhibition in the cell‐free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43S complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43S complexes.

The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium‐binding protein in protein synthesis.
Isolation and structure elucidation of a novel adipokinetic hormone (Lom‐AKH‐III) from the glandular lobes of the corpus cardiacum of the migratory locust, Locusta migratoria
Authors: OUDEJANS Rob C. H. M. , KOOIMAN Frank P. , HEERMA Wigger , VERSLUIS Cees , SLOTBOOM Arend J. , BEENAKKERS Ad M. T.
Published: March 2005

A new adipokinetic hormone (named Lom‐AKH‐III) was isolated from the glandular lobes of the corpora cardiaca of Locusta migratoria. At the N‐terminus it is blocked by a 5‐oxoproline (pyroglutamic acid) residue (< Glu). After enzymatic deblocking, the amino acid sequence of the N‐terminus was partly established by automatic Edman degradation to be [< Glu]‐Leu‐Asn‐Phe‐Thr‐Pro‐. Fast‐atom‐bombardment spectrometry (FAB‐MS) revealed that the new hormone is an octapeptide, which is amidated at the C‐terminus, and has a relative molecular mass of 1072. Based on the FAB‐MS data the complete sequence is < Glu‐Leu‐Asn‐Phe‐Thr‐Pro‐Trp‐Trp‐NH2, which was confirmed by chemical synthesis. All characteristics from HPLC, FAB‐MS and biological activity of the natural hormone and the synthetic peptide appeared to be identical. Although the structure of this new hormone resembles that of Lom‐AKH‐I (< Glu‐Leu‐Asn‐Phe‐Thr‐Pro‐Asn‐Trp‐Gly‐Thr‐NH2), its amino acid sequence points to a completely different route for its biosynthesis, involving a third prohormone. High‐[K+]‐containing media can cause release of all three adipokinetic hormones in vitro. Interestingly, the new hormone is absent in another locust species, Schistocerca gregaria. Based on in vitro biosynthesis experiments the turnover for this hormone is very high, suggesting an important physiological function. Locusta migratoria is the first insect species in which three different adipokinetic hormones have been demonstrated.
cDNA sequence and deduced amino acid sequence of the precursor of the 37‐kDa inner envelope membrane polypeptide from spinach chloroplasts
Authors: DRESES‐WERRINGLOER Ute , FISCHER Karsten , WACHTER Elmar , LINK Thomas A. , FLÜGGE Ulf‐Ingo
Published: March 2005

We present the nucleotide sequence and the deduced amino acid sequence of a cDNA clone that encodes the entire precursor of the 37‐kDa inner envelope membrane protein from spinach chloroplasts. The precursor protein consists of 344 amino acids (Mr 38976). In vitro processing followed by radiosequence analysis of the in vitro transcribed and translated precursor protein revealed that its transit peptide consists of only 21 amino acid residues. The transit peptide has the potential to form an amphiphilic α‐helix with a strong hydrophobic moment. It is speculated that this structural element represents an ancesteral envelope‐targeting domain. The in vitro synthesized precursor protein is directed to the chloroplasts and it is inserted into the envelope membrane in an ATP‐dependent manner. The mature protein (323 amino acid residues, Mr 36830) has a moderate hydrophobicity and contains only one membrane‐spanning segment which is located at the C‐terminus and possibly anchors the protein within the envelope membrane.
Purification and properties of an aryl‐alcohol dehydrogenase from the white‐rot fungus Phanerochaete chrysosporium
Authors: MUHEIM Andreas , WALDNER Roland , SANGLARD Dominique , REISER Jakob , SCHOEMAKER Hans E. , LEISOLA Matti S. A.
Published: March 2005

An intracellular aryl‐alcohol dehydrogenase (previously referred to as aryl‐aldehyde reductase) was purified from the white‐rot fungus Phanerochaete chrysosporium. The enzyme reduced veratraldehyde to veratryl alcohol using NADPH as a cofactor. Other aromatic benzaldehydes were also reduced, but not aromatic ketones. Methoxy‐substituted rings were better substrates than hydroxylated ones. The enzyme was also able to reduce a dimeric aldehyde (4‐benzyloxy‐3‐methoxybenzaldehyde). The highest reduction rate was measured when 3,5‐dimethoxybenzaldehyde was used as a substrate. On SDS/PAGE the purified enzyme showed one major band with a molecular mass of 47 kDa, whereas gel filtration suggested a molecular mass of 280 kDa. Polyclonal antibodies raised against the gel purified 47‐kDa protein were able to immunoprecipitate the aryl‐alcohol dehydrogenase indicating that its activity possibly resides entirely in this protein fragment. The pI of the enzyme was 5.2 and it was most active at pH 6.1. The aryl‐alcohol dehydrogenase was partially inhibited by typical oxidoreductase inhibitors.
Kinetic characterisation of the enzymatic activity of the eEF‐2‐specific Ca2+‐ and calmodulin‐dependent proteinkinase III purified from rabbit reticulocytes
Authors: NILSSON Anders , CARLBERG Ulf , NYGÅRD Odd
Published: March 2005

The Ca2+ ‐ and calmodulin‐dependent protein kinase III, which specifically phosphorylates the eukaryotic elongation factor 2 (eEF‐2), has been purified to apparent homogeneity from the post‐ribosomal fraction of rabbit reticulocytes by an efficient four‐step method. The method results in a more than 4000‐fold purification of the enzyme. SDS‐gel electrophoresis showed that the purified kinase contained only one polypeptide with the apparent molecular mass of 90 kDa. The kinase activity was associated with the 90‐kDa protein as shown by analyzing the phosphorylating activity of SDS gel electrophoretically purified protein electroblotted to nitrocellulose membranes. The purified kinase was dependent on Ca2+, Mg2+ and calmodulin for activity. Kinetic analysis of the phosphorylation reaction indicates that the turnover number of the kinase was approximately 1 s−1. The Km for the two substrates ATP and eEF‐2 was calculated to be approximately 100 μM and 10 μM, respectively. The activity of the kinase was competitively inhibited by cAMP. The inhibition constant Ki (0.5 mM) was found to be in the same order of magnitude as that calculated for the competitive product inhibition caused by ADP. GTP was ten‐times less efficient as competitor, indicating that the kinase had a preference for adenosine nucleotides. Phosphorylation of eEF‐2 did not interfere with the diphtheria‐toxin‐catalysed ADP‐ribosylation of the factor nor did ADP‐ribosylation inhibit phosphorylation.
Carbohydrate microheterogeneity of rat serotransferrin
Authors: SPIK Geneviève , CODDEVILLE Bernadette , STRECKER Gérard , MONTREUIL Jean , REGOECZI Erwin , CHINDEMI Paul A. , RUDOLPH John R.
Published: March 2005

A previously established procedure [Regoeczi, E., Chindemi, P. A., Rudolph, J. R., Spik, G. & Montreuil, J. (1987) Biochem. Cell Biol. 65, 948 – 954] was used to isolate from three DEAE‐cellulose chromatographic fractions of diferric rat serotransferrin (rTf) subpopulations having discernible affinities for concanavalin A (ConA). These entities are designated rTf‐1 (not retarded by ConA column), rTf‐2 (retarded) and rTf‐3 (bound). Each rTf type was found to be endowed with carbohydrate sufficient to account for a single diantennary glycan/protein molecule. Glycan structures were determined on the glycopeptides by employing GLC/MS and 400‐MHz 1H‐NMR spectroscopy.

All glycans possessed a common, trimannosyl‐N,N′‐diacetylchitobiose core with or without one L‐fucose α‐1,6‐linked to the Asn‐linked GlcNAc. However, there were differences in the antennae. Thus, in rTf‐3, both antennae were of the disialylated diantennary N‐acetyllactosamine type which is frequently encountered in other plasma glycoproteins. However, the α‐1,3‐Man‐linked antenna in rTf‐1 as well as rTf‐2 had the sequence: Neu5Ac(α2–3)Gal(β1–3)[Neu5Ac(α2–6)]GlcNAc(β1–2)Man.

In addition, the α‐1,6‐Man‐linked antenna deviated in rTf‐2 from the standard structure by having the sequence: Neu5Ac(α2–3)Gal(β1–3)GlcNAc(β1–2)Man. The possible relevance of the above structures to the ConA binding of rTf is discussed. A further preparation, obtained from the most anionic DEAE‐cellulose fraction (peak V) of rTf contained several tetrasialylated diantennary glycans whose precise structures remain to be established in future studies.
Purification, biosynthesis and cellular localization of a major 125‐kDa glycophosphatidylinositol‐anchored membrane glycoprotein of Saccharomyces cerevisiae
Authors: FANKHAUSER Christoph , CONZELMANN Andreas
Published: March 2005

The yeast Saccharomyces cerevisiae has been shown to contain a major 125‐kDa membrane glycoprotein which is anchored in the lipid bilayer by a glycophosphatidylinositol anchor. This protein was purified to near homogeneity and was used to raise a rabbit antibody. Biosynthesis of the 125‐kDa protein was studied by immunoprecipitation of 35SO4‐labeled material from wild‐type cells or a secretion mutant (sec18) in which the vesicular traffic from the endoplasmic reticulum (ER) to the Golgi is blocked. The 125‐kDa protein is first made in the ER as a 105‐kDa precursor which already contains a glycophosphatidylinositol anchor and which is slowly transformed into the 125‐kDa form upon chase (t1/2∼ 10–15 min). The 105‐kDa precursor can be reduced to an 83‐kDa form by the enzymatic removal of N‐glycans. The removal of N‐glycans from the mature 125‐kDa protein yields a 95‐kDa species. Thus, removal of the N‐glycans does not reduce the ER and mature forms to the same molecular mass, indicating that not only elongation of N‐glycans but also another post‐translational modification takes place during maturation. Selective tagging of surface proteins by treatment of 35SO4‐labeled cells with trinitrobenzene sulfonic acid at 0°C followed by immunoprecipitation of the tagged proteins shows that the 125‐kDa protein, but not the 105‐kDa precursor, becomes transported to the cell surface. This tagging of cells after various lengths of chase also shows that the surface appearance of the protein is biphasic with about one half of the mature 125‐kDa protein remaining intracellular for over 2 h.

Glycosylation and/or glycophosphatidylinositol anchor addition is important for the stability of the 125‐kDa protein since the protein remains undetectable in sec53, a temperature‐sensitive mutant which does not make GDP‐mannose at 37°C and does not add glycophosphatidylinositol anchors at 37°C.
Determination of the covalent structure of an N‐ and C‐terminally blocked glycoprotein from endocuticle of Locusta migratoria
Authors: TALBO Gert , HØJRUP Peter , RAHBEK‐NIELSEN Henrik , ANDERSEN Svend Olav , ROEPSTORFF Peter
Published: March 2005

The complete structure of a protein isolated from endocuticle of sexually mature locusts, Locusta migratoria, has been determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The protein is extensively post‐translationally modified. The N‐terminal is 5‐oxoproline (pyroglutamic acid) and the C‐terminal proline residue is amidated. Furthermore, the protein is glycosylated by a single N‐acetyl‐galactosamine residue at one, two or three threonines. The N‐terminal sequence was obtained by analysing the N‐acetylated N,O‐permethylated derivative using plasma desorption mass spectrometry. The position and type of carbohydrate were determined by combining an HPLC‐based carbohydrate analysis with the peak pattern of the phenylthiohydantoin derivative in automatic sequencing and with mass information on peptides.

The protein has pronounced similarity to cuticular proteins from larvae of diptera and lepidoptera, but only slight resemblance to the previously sequenced locust exocuticular proteins. This indicates a similarity between soft larval cuticles and locust endocuticle, a similarity which may extend to their mechanical properties.
S= 9/2 EPR signals are evidence against coupling between the siroheme and the Fe/S cluster prosthetic groups in Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase
Authors: PIERIK Antonio J. , HAGEN Wilfred R.
Published: March 2005

Sulfite reductases contain siroheme and iron‐sulfur cluster prosthetic groups. The two groups are believed to be structurally linked via a single, common ligand. This chemical model is based on a magnetic model for the oxidized enzyme in which all participating iron ions are exchange coupled. This description leads to two serious discrepancies. Although the iron‐sulfur cluster is assumed to be a diamagnetic cubane, [4Fe–4S]2+, all iron appears to be paramagnetic in Mössbauer spectroscopy. On the other hand, EPR spectroscopy has failed to detect anything but a single high‐spin heme. We have re‐addressed this problem by searching for new EPR spectroscopic clues in concentrated samples of dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). We have found several novel signals with effective g values of 17, 15.1, 11.7, 9.4, 9.0, 4. The signals are interpreted in terms of an S= 9/2 system with spin‐Hamiltonian parameters g= 2.00, D=−0.56 cm−1, |E/D|= 0.13 for the major component. In a reductive titration with sodium borohydride the spectrum disappears with Em=−205 mV at pH 7.5. Contrarily, the major high‐spin siroheme component has S= 5/2, g= 1.99, D=+9 cm−1, |E/D|= 0.042, and Em=−295 mV. The sum of all siroheme signals integrates to 0.2 spin/half molecule, indicating considerable demetallation of this prosthetic group. Rigorous quantification procedures for S= 9/2 are not available, however, estimation by an approximate method indicates 0.6 S= 9/2 spin/half molecule. The S= 9/2 system is ascribed to an iron‐sulfur cluster. It follows that this cluster is probably not a cubane, is not necessarily exchange‐coupled to the siroheme, and, therefore, is not necessarily structurally close to the siroheme. It is suggested that this iron‐sulfur prosthetic group has a novel structure suitable for functioning in multiple electron transfer.
Interaction of guanine‐nucleotide‐binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL‐60 cells
Authors: TOHKIN Masahiro , MORISHIMA Naoki , IIRI Taroh , TAKAHASHI Katsunobu , UI Michio , KATADA Toshiaki
Published: March 2005

Human leukemic HL‐60 cells were differentiated into neutrophil‐like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2′‐dibutyryladenosine 3′,5′‐phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM, N‐formylmethionine) and guanine‐nucleotide‐binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet‐activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high‐affinity binding to vesicle preparations from the Me2SO‐ and Bt2cAMP‐induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP‐sensitive G proteins in each of the two vesicle types. However, the high‐affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP‐induced cells than in those from Me2SO‐induced cells, although the amount of IAP‐substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi‐2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi‐2 from rat brain was reconstituted into the two IAP‐treated vesicles, high‐affinity fMLF binding was restored in a similar manner in both. IAP‐substrate G proteins partially purified from the two differentiated HL‐60 cells were also effective in restoring high‐affinity fMLF binding to the IAP‐treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G‐protein‐rich fraction from Bt2cAMP‐induced cells than with that from Me2SO‐induced cells, with each of the two IAP‐treated vesicle types. These results suggest that the different high‐affinity binding of fMLF observed in the two differentiated HL‐60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi‐2.
Tissue‐specific and time‐coordinated hormone regulation of plasminogen‐activator‐inhibitor type I and tissue‐type plasminogen activator in the rat ovary during gonadotropin‐induced ovulation
Authors: LIU Yi‐Xun , PENG Xiao‐Rong , NY Tor
Published: March 2005

The plasminogen‐activator system provides proteolytic activity in many biological processes. The regulation of plasminogen activation may occur at many levels including the synthesis and secretion of plasminogen activators (PA) and the specific inhibition of PA activity by inhibitors. PA‐inhibitor type‐1 (PAI‐1) is an efficient inhibitor of tissue‐type PA (tPA) and urokinase‐type PA (uPA) that may therefore be instrumental for the control of plasminogen activation. To investigate if coordinated regulation of PA and PA inhibitors take place in vivo in response to physiological signals, we have examined the regulation of PAI‐1 and tPA in the ovary during gonadotropin‐induced ovulation. We found that PAI‐1, as well as tPA activity and mRNA levels, were coordinately regulated by gonadotropins in a time‐dependent and cell‐specific manner, such that a surge of PA‐activity was obtained just prior to ovulation. Both theca‐interstitial and granulosa cells synthesized PAI‐1, but their maximal PAI‐1 expression occurred at different times during the periovulatory period, ensuring inhibition of proteolytic activity in ovarian extra cellular compartments both before and after ovulation. The coordinated regulation of tPA and PAI‐1 in the ovary may fine‐tune the peak of PA activity which may be important for the regulation of the ovulatory process.
Requirement of down‐regulation of NAD+ ADP‐ribosyltransferase for the interferon‐γ‐induced activation process of murine macrophage tumor cells
Authors: TANIGUCHI Taketoshi , TAKAHASHI Seiichi , YAMAMOTO Hiroshi , FUJIMOTO Shigeyoshi , OKOYAMA Hiroto
Published: March 2005

Expression of the NAD+ ADP‐ribosyltransferase gene is depressed during interferon‐γ‐induced activation of murine macrophage P388D1 tumor cells [Taniguchi, T., Yamauchi, K., Yamamoto, T., Tokushima, K., Harada, N., Tanaka, H., Takahashi, S., Yamamoto, H. & Fujimoto, S. (1988) Eur. J. Biochem. 171, 571–575]. In order to study the role(s) of NAD+ ADP‐ribosyltransferase in interferon‐γ‐induced activation of P388D1 cells, we transfected an cloned synthetase gene into P388D1 cells and examined the effect of exogenous transferase gene expression on the induction of the Ia antigen, one of the major histocompatibility gene products, by interferon‐γ. The transferase activity of the transfected cells was twice that of control cells, and Southern blot analysis revealed that characteristic restriction sizes of cDNA were detected in the clones. RNA blot analysis using a cDNA for the transferase as a probe showed that the level of mRNA for the transferase in transfected cells was higher than that in control cells, and mRNA for the exogenous transferase was still detectable 2 days after the transfected cells were treated with interferon‐γ. This indicates that the exogenous transferase gene was expressed in transfected cells. RNA blot analysis with a cDNA for the Ia antigen and flow‐cytometric analysis showed that the Ia antigen was induced much less in the transfected cells by interferon‐γ, in terms of the mRNA and the Ia antigen. The results suggest that down‐regulation of the transferase is required for the induction of the Ia antigen in P388D1 cells by interferon‐γ.
Structures of regulatory regions in the human cytochrome P‐450scc (desmolase) gene
Authors: INOUE Hideshi , WATANABE Nobuaki , HIGASHI Yujiro , FUJII‐KURIYAMA Yoshiaki
Published: March 2005

The mechanism of regulatory expression of human cytochrome P‐450scc gene by cAMP was investigated in a transient expression system using Y‐1 cells (mouse adrenal tumor cell line) and a chimeric DNA composed of the structural gene for bacterial chloramphenicol acetyltransferase and the 5′ flanking upstream sequence of the cytochrome P‐450scc (cholesterol desmolase) gene which was revealed to contain a DNA element(s) responsive to cAMP [Inoue, H. et al. (1988) Eur. J. Biochem. 171, 435–440]. Introduction of deletions and point mutations in the upstream regulatory sequence demonstrated that three regions were mainly required for response to cAMP. These regions contained a short similar sequence. All of them have a 5‐bp motif GTCAT (or ATGAC) in common, and have at least two motifs which conserve four out of five base pairs of the consensus sequence of the cAMP‐responsive element (CRE), CGTCA (or TGACG). They are all apparently necessary for regulation by cAMP. Gel mobility shift assays suggested that a binding factor(s) to these regions was present in the nuclear extracts of Y‐1 cells and adrenal cortex tissues and appeared to be different from the somatostatin CRE‐binding protein. Deletion analysis also suggested that the region around ‐44 was essential to the basal transcriptional activity. This region shows some similarity to the CTF/NF‐1 binding site [Johnson and McKnight (1989) Annu. Rev. Biochem. 58, 799–839].
Myosin heavy‐chain‐based isomyosins in developing, adult fast‐twitch and slow‐twitch muscles
Authors: TERMIN Angelika , PETTE Dirk
Published: March 2005

A modified method of electrophoresis under nondenaturing conditions made it possible to separate rat muscle extracts of defined myosin heavy chain (HC) and light chain (LC) composition into subsets of developmental, fast and slow myosin heavy‐chain‐based isomyosins. The fastest migrating isomyosins were the neonatal isomyosins (nM1, nM2, nM3), followed by the slightly slower migrating embryonic isomyosins (eM1, eM2, eM3, eM4). Of the nine adult fast isomyosins, the HCIIb‐based isomyosins (FM1b, FM2b, FM3b) were the fastest migrating. These were followed by the HCIId‐based isomyosins (FM1d, FM2d, FM3d). The HCIIa‐based isomyosins (FM1a, FM2a, FM3a) were the slowest. Our results suggest that FM3a is identical with the so‐called intermediate isomyosin (IM) described in the literature. The slow myosin heavy‐chain‐based isomyosins (SM1, SM2, SM3) migrated far behind the fast isomyosins. Whereas the gross electrophoretic mobilities of each of these isomyosin triplets is determined by the specific heavy chain complement, the different mobilities of the bands within each triplet result from different alkali light chain combinations. Thus, the fastest triplet bands of the neonatal (nM1) and adult fast isomyosins (FM1b, FM1d, FM1a) represent the LC3f homodimers, the slowest (nM3, FM3b, FM3d, FM3a) the LC1f homodimers, and the intermediate bands (nM2, FM2b, FM2d, FM2a) the LC1f/LC3f heterodimers. Different proportions of the adult fast isomyosin triplet bands indicate that the affinity for LC3f decreases in the order HCIIb, HCIId, HCIIa. The three slow isomyosins represent LC1sa (SM1) and LC1sb (SM3) homodimers and a LC1sa/LC1sb heterodimer (SM2). Circumstantial evidence suggests an inverse order in rabbit muscle where SM1 and SM3 most likely represent LC1sb and LC1sa homodimers, respectively.
Cuttlefish sperm protamines
Authors: MARTIN‐PONTHIEU Annie , WOUTERS‐TYROU Danièle , BÉLAÏCHE Denise , SAUTIÈRE Pierre , SCHINDLER Patrick , VAN DORSSELAER Alain
Published: March 2005

The amino acid sequences of two cuttlefish protamine variants Sp1 and Sp2 have been established from automated sequence analysis and mass spectrometry data. Sp1 (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Sp1; 13 and 35 in Sp2). With an arginine content of about 77%, cuttlefish protamine is one of the most basic proteins which have ever been characterized and the first typical protamine sequenced in invertebrates. It is closely similar to sperm basic proteins identified in squids but strongly differs from the protamine‐like components isolated from the sperm of bivalve molluscs.
Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII Δ II
Authors: KRISHNAN Sivadasan , KOLBE Hanno V. J. , LEPAGE Pierre , FAURE Therese , SAUERWALD Raymonde , de la SALLE Henri , MULLER Claudine , BIHOREAU Nicolas , PAOLANTONACCI Philippe , ROITSCH Carolyn , MEULIEN Pierre , PAVIRANI Andrea
Published: March 2005

Factor VIII Δ II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771 – Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N‐terminal amino acid sequencing. Thrombin first cleaves factor VIII Δ II at Arg740‐Ser741 to generate the 90‐kDa heavy chain and an 80‐kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73‐kDa light chain. The 90‐kDa fragment is further cleaved, giving rise to 50‐kDa and 40‐kDa fragments while the 80‐kDa fragment generates a 71/73‐kDa doublet. The 71/73‐kDa doublet, 50‐kDa and 40‐kDa fragments were further analysed by N‐terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII Δ II, the essential structural elements required for thrombin activation are conserved.