bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of hyoscyamine 6β‐hydroxylase from root cultures of Hyoscyamus niger L.
Authors: HASHIMOTO Takashi , YAMADA Yasuyuki
Published: March 2005

Hyoscyamine 6β‐hydroxylase, a 2‐oxoglutarate‐dependent dioxygenase that catalyzes the hydroxylation of l‐hyoscyamine to 6β‐hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619–625] was purified 310‐fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8. l‐Hyoscyamine and 2‐oxoglutarate are required for the enzyme activity, with respective Km values of 35 μM and 43 μM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2‐Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested.

The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o‐dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4‐dicarboxylate and 3,4‐dihydroxybenzoate are competitive inhibitors with respect to 2‐oxoglutarate with the respective Ki values of 9 μM and 90 μM.

Several alkaloids with structures similar to l‐hyoscyamine were hydroxylated by the enzyme at the C‐6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7‐dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 μM). This epoxidation reaction required the same co‐factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6β‐hydroxylase.
Studies on the in vivo transfer to retinoids from parenchymal to stellate cells in rat liver
Authors: BLANER William S. , DIXON Joseph L. , MORIWAKI Hisataka , MARTINO Rosemary A. , STEIN Olga , STEIN Yechezkiel , GOODMAN DeWitt S.
Published: March 2005

Studies were conducted to examine the in vivo transfer of chylomicron (dietary) retinoid from rat liver parenchymal to stellate cells. We specifically addressed the question of whether chylomieron retinyl ester is transferred directly from hepatic parenchymal to stellate cells without first undergoing hydrolysis. [14C]Retinyl palmitate and its non‐hydrolyzable ether analog, retinyl [3H]hexadecyl ether, were utilized to answer this question. Chylomicrons labeled with these retinoids were injected intravenously into rats. Liver cell fractions, highly enriched in parenchymal or in stellate cells, were isolated 0.5 h, 4.5 h and 24 h after chylomicron injection. The ratio of 3H: 14C found in parenchymal cell preparations 4.5 h after injection was 1.8 times the ratio for the injected chylomicrons, and 24 h postinjection the ratio had increased to 2.5 times that of the chylomicrons. In the stellate‐cell‐enriched preparations the 3H: 14C ratio was found to be 0.39, 0.29, and 0.23 times the ratio found in the injected labeled chylomicrons at 0.5 h, 4.5 hand 24 h after injection respectively. From the levels of 14C observed in the isolated stellate cells, it is estimated that 0.5 h, postinjection the stellate cells contained approximately 34% of the 14C (i.e. the retinol injected as chylomicron retinyl ester) present in the liver. By 4.5 h the 14C present in isolated stellate cells had risen to approximately 41% of that present in the total liver, and 24 h after injection approximately 55% of hepatic total 14C was found in the stellate cells. These findings suggest that chylomicron retinyl ester is not transferred directly from the parenchymal to stellate cells without first undergoing hydrolysis to retinol.
Nucleotide sequence of a cellulase gene of Bacillus subtilis
Authors: NAKAMURA Akira , UOZUMI Takeshi , BEPPU Teruhiko
Published: March 2005

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2‐terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a σ43‐type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was dound between B. subtilis cellulase and an alkalophilic Bacillus cellulase.
Changes in the concentration of cAMP, fructose 2,6‐bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose
Authors: FRANÇOIS Jean , ERASO Pilar , GANCEDO Carlos
Published: March 2005

Changes in the concentration of several metabolites and enzymes related to carbohydrate metabolism were measured during the growth of Saccharomyces cerevisiae on a mineral medium containing glucose as the limiting nutrient. When about 50% of the original glucose was used the exponential phase ended and the culture entered a ‘transition’ phase before the complete exhaustion of glucose. In this transition phase several metabolic changes occurred. cAMP, that decreased along growth, reached a constant value of about 0.7 nmol/g dry weight. A pronounced drop in fructose‐6‐phosphate‐2‐kinase activity and in the concentration of fructose 2,6‐bisphosphate and fructose 1,6‐bisphosphate was observed accompanied by a less marked decrease in hexose monophosphates. Trehalase activity also dropped and reached a minimal value at the onset of the stationary phase when synthesis of trehalose began. Glycogen concentration and glycogen synthase activity increased sharply during the transition phase. Plasma membrane ATPase began to increase at the middle of the exponential phase and then, coincident with the glucose exhaustion, a 90% decrease in the measurable activity was observed.
The action of the systemic fungicides tridemorph and fenpropimorph on sterol biosynthesis by the soil amoeba Acanthamoeba polyphaga
Authors: RAEDERSTORFF Daniel , ROHMER Michel
Published: March 2005

Tridemorph and fenpropimorph, two systemic fungicides known by their inhibitory effects on sterol biosynthesis in fungi and plants, were administered in vivo to the amoeba Acanthamoeba polyphaga. The compounds did not kill the cells, but modified completely their sterol pattern. Fungicide‐exposed cells accumulated cyclopropylsterols indicating a partial blockage of the cyclopropane isomerase as in higher plants and Δ8‐sterols indicating an inhibition of the Δ8→Δ7 isomerase as in fungi. Three new sterols, 4α‐methylergosta‐9(11), 24(28)‐dienol, ergosta‐6,8,22‐trienol and poriferasta‐6,8,22‐trienol were isolated and identified, the former from control cells, the two latter from fungicide‐treated cells. These results are in accordance with our previous results on the presence of cycloartenol as sterol precursor and confirm our hypothesis on a phylogenetic relationship of Acanthamoeba polyphaga with photosynthetic phyla.
Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoebae Naegleria lovaniensis and Naegleria gruberi
Authors: RAEDERSTORFF Daniel , ROHMER Michel
Published: March 2005

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniensis, it is accompanied by lanosterol and parkeol, as well as by the 24, 25‐dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4α‐Methylsterols which are present in similar amounts as those of 4,4‐desmethylsterols (3–5 mg/g, dry weight). 4α‐Methylergosta‐7,22‐dienol was identified as a new compound. Ergosterol was the major 4,4‐desmethylsterol, accompanied by small amounts of C27 and other C28 sterols.

Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4α‐methyl‐Δ7‐sterols and the appearance of the unusual Δ6,8,22‐ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the Δ8→Δ7 isomerase, the small amounts of Δ7‐sterols formed being converted into ergosterol which is still present in fenpropimorph‐exposed cells.

De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1‐14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3‐3H]squalene epoxide was efficiently cyclized by a cell‐free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non‐photosynthetic phyla (animal and fungi) and parkeol are more likely dead‐ends of this biosynthetic pathway.

The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine‐like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics. The growth of A. polyphaga containing mainly the C29 stigmasta‐5,7,22‐trienol was not affected by nystatin, and only slightly by amphotericin. Both antibiotics inhibited however the growth of the ergosterol‐containing amoeba N. lovaniensis and trypanosome Crithidia fasciculata.
Structural organization of the bovine thyroglobulin gene and of its 5′‐flanking region
Authors: de MARTYNOFF Guy , POHL Viviane , MERCKEN Luc , van OMMEN Gert‐Jan , VASSART Gilbert
Published: March 2005

The structural organization of the bovine thyroglobulin gene has been investigated by a combination of Southern genomic blotting and direct analysis of cloned gene fragments isolated from a chromosomal DNA library. The entire locus is spread over more than 200000 base pairs which makes it one of the largest eukaryotic genes studies to date. The coding information is scattered into at least 42 exons, 34 of which have been precisely identified. A different evolutionary origin of the 5′ and 3′ regions of the gene is supported by the highly different proportion of exonic material they contain (12% and 3%, respectively) and by the existence of sequence homology between the 3′ region of thyroglobulin and acetylcholinesterase. Detailed sequence analysis of the 5′ region of the gene and its flanking segment demonstrated that a significant homology exists between bovine and human thyroglobulin sequences, except for the presence within the ruminant promoter region of a 220‐base‐pair sequence belonging to the bovine monomer repeated family.
Stereospecific assignments of side‐chain protons and characterization of torsion angles in Eglin c
Authors: HYBERTS Sven G. , MÄRKI Walter , WAGNER Gerhard
Published: March 2005

Stereospecific assignments were obtained in the protein Eglin c for β‐methylene protons of 9 out of the 24 residues with AMX symmetry, for three residues with long side chains and for the γ‐methyl groups of 8 out of the 11 valines. This was achieved by analyzing the cross‐peak multiplet structures in two‐dimensional correlated spectra with spectral simulations and peak fitting, and by quantitative measurements of intraresidue nuclear Overhauser enhancements. In addition to the stereospecific assignments, information on the torsion angels φ and X1 was obtained. To estimate inferences of internal motions on this analysis, the effect of uniform averaging within a certain range of the torsion angle X1 on cross‐peak multiplet structures and on relative intraresidue nuclear Overhauser enhancement is discussed.
Changes in the quaternary structure of phosphoenolpyruvate carboxylase induced by ionic strength affect its catalytic activity
Authors: WAGNER Richard , GONZALEZ Daniel H. , PODESTA Florencio E. , ANDREO Carlos S.
Published: March 2005

Phosphoenolpyruvate carboxylase from maize leaves dissociated into dimers and/or monomers when exposed to increasing ionic strength (e.g. 200–400 mM NaCl) as indicated by gel filtration experiments. Changes in the oligomerization state were dependent on pH, time of preincubation with salt and protein concentration. A dissociation into dimers and monomers was observed at pH 8, while at pH 7 dissociation into the dimeric form only was observed. Exposure of the enzyme to higher ionic strength decreased the activity in a time‐dependent manner. Turnover conditions and glucose 6‐phosphate protected the carboxylase from the decay in activity, which was faster at pH 7 than at pH 8. The results suggest that changes in activity of the enzyme, following exposure to high ionic strength, are the consequence of dissociation. Tetrameric and dimeric forms of the phosphoenolpyruvate carboxylase seemingly reveal different catalytic properties. We suggest that the distinct catalytic properties of the different oligomeric species of phosphoenolpyruvate carboxylase and changes in the equilibrium between them could be the molecular basis for an effective regulation of metabolite levels by this key enzyme of C4 plants.
Anion exchanger is present in both luminal and basolateral renal membranes
Authors: TALOR Zvi , GOLD Robert M. , YANG Wu‐Chang , ARRUDA Jose A. L.
Published: March 2005

Binding of the anion‐exchange inhibitor 3H2‐labeled 4,4′‐diisothiocyano‐2,2′‐stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 μM and 19.3 μM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward‐directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion‐dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion‐dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3‐dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 μM. The similar vaiues of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward‐directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na‐dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well‐known Na‐dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.
A high‐performance liquid chromatography procedure for recovering subnanomole amounts of protein from SDS‐gel electroeluates for gas‐phase sequence analysis
Authors: SIMPSON Richard J. , MORITZ Robert L. , NICE Edouard E. , GREGO Boris
Published: March 2005

A high‐performance liquid chromatographic procedure for recovering subnanomole amounts of protein from SDS/polyacrylamide gel electroeluates in a form suitable for gas‐phase sequence analysis has been developed. By a judicious choice of reversed‐phase column packing, proteins can be retained at high concentrations of n‐propanol (90–100%) where sodium dodecylsulfate and acrylamide gel‐related contaminants are washed through the column. Retained proteins can be recovered from the column in high yield (>90%) by the simultaneous adding of an ion‐pairing reagent into the mobile phase and elution with a gradient of decreasing n‐propanol concentration (i.e. an ‘inverse or negative gradient’). Furthermore, by using a steep gradient (e.g. 50%/min) at a low flow rate (20–200 μl/min) the proteins can be recovered in less than 100 μl and can be used for gas‐phase sequence analysis without further manipulation. This procedure is independent of sodium dodecylsulfate concentration (up to 1.2% w/v) in sample loading volumes of up to 1.5 ml. Microbore columns (2.1 mm internal diameter) have been employed for recovering small amounts of protein (1–100 μg) from electroeluates of protein‐containing gel spots while conventional columns (4.6 mm internal diameter) were used for isolating larger amounts of protein (>500 μg) from electroeluates of preparative gel bands. The general utility of this inverse‐gradient high‐performance liquid chromatography procedure has been demonstrated by its successful application in recovering a wide variety of proteins from sodium dodecylsulfate gel electroeluates in a form suitable for N‐terminal sequence analysis in the 10–500 pmol range.
Inhibition of human vitamin‐K‐dependent protein‐S‐cofactor activity by a monoclonal antibody specific for a Ca2+‐dependent epitope
Authors: MALM Johan , PERSSON Ulla , DAHLBÄCK Björn
Published: March 2005

Protein S is an anticoagulant vitamin‐K‐dependent plasma protein functioning as a cofactor to activated protein C in the degradation of factors Va and VIIIa. A murine monoclonal antibody, HPS 7, specific for a calcium‐stabilized epitope in human protein S, is described. The epitope was available in intact protein S, both in its free form and when protein S was bound to C4b‐binding protein. It disappeared upon reduction of disulfide bridges and also after thrombin of chymotrypsin cleavage of protein S. Thrombin cleaves protein S close to the calcium‐binding region containing γ‐carboxyglutamic acid (Gla). The cleaved protein still contains the Gla region, linked by a disulfide bridge, but it has a lower affinity for calcium and no protein C cofactor activity. The thrombin‐mediated cleavage of protein S could be inhibited by HPS 7. The Ka for the interaction between protein S and the monoclonal was estimated to be approximately 0.7 × 108 M−1. Half‐maximal binding between HPS 7 and protein S was observed at a calcium concentration of 0.50 mM, indicating that saturation of the Gla region with calcium was required for the interaction. The recently reported Gla‐independent high‐affinity calcium binding did not induce the epitope. The calcium‐dependent binding of protein S to phospholipid vesicles as well as the protein C cofactor activity was inhibited by HPS 7. The data suggests that the epitope for HPS 7 is located in the Gla region of protein S or in the closely positioned thrombin‐sensitive region.
Application of a reference convolution method to tryptophan fluorescence in proteins
Authors: VOS Kees , van HOEK Arie , VISSER Antonie J. W. G.
Published: March 2005

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p‐terphenyl, p‐bis[2‐(5‐phenyloxazolyl)]benzene and N‐acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p‐terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p‐terphenyl (20°C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile‐up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile‐up effects play a role at this frequency.

The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple‐exponential fluorescence decay. The anisotropy decay of albumin was found to be mono‐exponential with a rotational correlation time of 26 ns (20°C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for λex= 295 nm and 36 ns for λex= 300 nm at 20°C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple‐exponential fluorescence decay. A single‐exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.
Functional molecular mass of binding sites for [3H]dihydrotetrabenazine and [3H]reserpine and of dopamine β‐hydroxylase and cytochrome b561 from chromaffin granule membrane as determined by radiation inactivation
Authors: GASNIER Bruno , ELLORY J. Clive , HENRY Jean‐Pierre
Published: March 2005

The monoamine transporter of chromaffin granule membrane has two distinct high‐affinity binding sites for tetrabenazine and reserpine, which can be assayed by [3H]dihydrotetrabenazine and [3H]reserpine binding, respectively. The functional molecular mass of the components bearing these sites has been investigated by the radiation inactivation technique. The decline of [3H]dihydrotetrabenazine binding activity with increasing radiation doses followed a single exponential, from which a functional molecular mass of 68 kDa was derived for tetrabenazine binding sites. [3H]Reserpine binding activity declined in a more complex way; however, under conditions where high‐affinity reserpine binding sites were specifically assayed, the decline was also exponential, corresponding to a functional molecular mass of 37 kDa for these sites. The figures obtained for high‐affinity tetrabenazine and reserpine binding sites are consistent with previous values obtained by photoaffinity of tetrabenazine and serotonin binding sites, respectively. It is thus concluded that the monoamine transporter has an oligomeric structure.

By the radiation inactivation technique, cytochrome b561 and dopamine β‐hydroxylase have functional molecular masses of 25 and 123 kDa, respectively. The latter value might be attributed to the dimeric form of the enzyme.
Vascular localization of bovine pancreatic trypsin‐inhibitor‐related molecular forms in bovine spleen
Authors: BUSINARO Rita , FIORETTI Evandro , FUMAGALLI Lorenzo , CITRO Gennaro , DE RENZIS Gabriella , ASCOLI Franca
Published: March 2005

Bovine spleen proteic inhibitors of serine proteases, belonging to the bovine pancreatic trypsin inhibitor (BPTI or aprotinin) family, have been localized, using immunocytochemical techniques, in the smooth muscle cells of some bovine spleen blood vessels. This vascular localization also occurs in a variety of bovine organs and differs from that of BPTI itself which is found exclusively in bovine mast cells, in agreement with previous reports. These data would be in favour of a possible involvement of one or more BPTI‐type inhibitors in vascular processes by acting at the level of the smooth muscle cells, the tissue responsible for vasodilation/vasoconstriction events.
Nucleotide sequence of barley chymotrypsin inhibitor‐2 (CI‐2) and its expression in normal and high‐lysine barley
Authors: WILLIAMSON Martin S. , FORDE Janice , BUXTON Bernadette , KREIS Martin
Published: March 2005

cDNA clones for chymotrypsin inhibitor‐2 (CI‐2) have been isolated from an endosperm‐specific library of barley using a synthetic oligonucleotide probe. The nucleotide sequences of several of the cDNAs predict an open reading frame (beginning with an ATG codon) which encodes a protein of 84 residues (Mr 9380). In the longest clone another ATG codon is present, a further 69 nucleotides upstream. The nucleotide sequence between these two ATG codons predicts an amino acid sequence with the characteristics of a signal peptide, as found in other cloned plant protease inhibitors. However, it contains an in‐frame TAA stop codon, which is also present in all of the shorter cDNAs which extend into this region. From in vitro translation experiments, using mRNAs synthesized from cDNAs, we conclude that, in vitro, translation of all or the vast majority of CI‐2 mRNAs begins at the second ATG codon, 31 nucleotides downstream from the ochre stop codon.

Southern blotting of genomic DNA shows that CI‐2 is encoded by a small multigene family, while sequence analysis of the cDNAs shows that at least two sub‐families of mRNAs, which are more than 90% homologous, are present in the endosperm. Northern blotting analysis shows that related but different sequences are present in leaf and shoot RNA populations.

Further Northern blot hybridizations using RNA from the normal line, Sundance, and the high‐lysine barley mutant, Hiproly, show that endosperms of the latter contain greatly increased levels of CI‐2 mRNA. This correlates with the increased amount of CI‐2 protein deposited in Hiproly, and demonstrates that the differential expression of CI‐2 in the two genotypes is controlled at the level of transcription and/or stability of the mRNA. In contrast, the abundance of CI‐2 mRNAs in leaves and shoots is not affected.
Characterization and regulation of somatomedin‐C/insulin‐like growth factor I (Sm‐C/IGF‐I) receptors on cultured pig Leydig cells
Authors: PERRARD‐SAPORI Marie‐Hélène , CHATELAIN Pierre G. , JAILLARD Christine , SAEZ José M.
Published: March 2005

We have characterized somatomedin‐C/insulin‐like growth factor I (Sm‐C/IGF‐I) receptors in cultured pig Leydig cells by binding and cross‐linking affinity experiments. At equilibrium the dissociation constant and the number of binding sites per cell were 1.8 ± 0.2 × 10−9 M and 12200 ± 3200 respectively. Under reducing conditions, disuccinimidyl suberate cross‐linked 125I‐Sm‐C to one receptor complex with apparent Mr= 120000. Continuous treatment of Leydig cells with increasing concentrations of human chorionic gonadotropin (hCG) for 48 h resulted in a dose‐dependent (ED50 0.05 nM) increment in IGF type I receptors (2.5–3‐fold increase). Conversely, treatment of Leydig cells for 48 h with increasing concentrations of Sm‐C/IGF‐I produced a 3–4‐fold increase in hCG receptors. This effect was dose‐dependent (ED50= 7 ng/ml). Sm‐C/IGF‐I treatment also enhanced Leydig cell responsiveness to hCG for both cAMP (6‐fold) and testosterone (8‐fold). Moreover the stimulatory effects of Sm‐C/IGF‐I on cells cultured either in the absence or presence of 5% human serum from an hypopituitary patient were inhibited by anti‐(Sm‐C/IGF‐I) antibodies. Taken together these results not only show that Leydig cells must be considered as targets for Sm‐C/IGF‐I, but also lend support to the possibility that Sm‐C/IGF‐I plays a role in the regulation of Leydig cell function. Moreover, they suggest that Sm‐C/IGF‐I might be responsible for the delayed puberty observed in some patients with isolated growth hormone deficiency.
The effect of monovalent and divalent cations on the ATP‐dependent Ca2+‐binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+‐transport ATPase
Authors: MEDDA Pankaj , FASSOLD Elisabeth , HASSELBACH Wilhelm
Published: March 2005

The coupling of Ca2+ movements and phosphate fluxes as well as the time‐dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg‐dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 μM does not allow more than one reaction cycle to occur. The respective fractions of ADP‐sensitive and ADP‐insensitive phosphoenzyme have been determined. The chosen experimental conditions (0–1 C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively.

It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P ⇀ E2P transition, E2P hydrolysis, and E2⇀ E1 transformation). Mg2+ represents the sole ionic co‐factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P ⇀ E2P transition rate (including Ca2+ translocation and Ca2+ release from low‐affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded.

We find Ca2+ release to occur time‐coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low‐affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19–49, Academic Press, Orlando].
Regulation of the cardiac calcium channel by protein phosphatases
Authors: HESCHELER Jürgen , KAMEYAMA Masaki , TRAUTWEIN Wolfgang , MIESKES Gottfried , SÖLING Hans‐Dieter
Published: March 2005

The calcium current (ICa) through the L‐type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285–293]. We investigated the possible contribution of the ‘catalytic subunits’ of protein phosphatase 1 and 2A in the down‐regulation of the cardiac calcium channel. Single guinea‐pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 μM) as well as 2A (2.3 μM) completely abolished the increase of ICa induced by isoprenaline (0.05 μM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor‐2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash‐out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor‐2. (4) Perfusion of the myocyte under basal conditions with adenosine 5′‐[γ‐thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 μM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min.

From these results we make the following conclusions. (1) The calcium channel from guinea‐pig myocytes is regulated by phosphorylation‐dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down‐regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.
Metabolism of glyphosate in an Arthrobacter sp. GLP‐1
Authors: PIPKE Rüdiger , AMRHEIN Nikolaus , JACOB Gary S. , SCHAEFER Jacob , KISHORE Ganesh M.
Published: March 2005

The metabolism of glyphosate [N‐(phosphonomethyl)glycine] in a bacterium tentatively identified as an Arthrobacter sp., capable of growth on this herbicide as its sole phosphorus source, has been investigated using solid‐state NMR techniques as well as radiotracer analysis. The pathway involves the conversion of glyphosate to glycine, a C1 unit and phosphate. The phosphonomethyl carbon is specifically incorporated into the amino acids serine, cysteine, methionine, and histidine, as well as into purine bases and thymine, indicating the involvement of tetrahydrofolate in single‐carbon transfer reactions. Glycine derived from glyphosate is utilized in purine and protein biosynthesis. This pathway for glyphosate degradation in a gram‐positive bacterium is similar to that previously reported for Pseudomonas sp. PG2982 [Jacob et al. (1985) J. Biol. Chem. 260, 5899–5905] and is distinct from that reported for soil metabolism of glyphosate where aminomethylphosphonic acid has been shown to be a major metabolite. Preliminary evidence is presented which indicates that the conversion of glyphosate to glycine and the C1 unit involves the intermediate formation of sarcosine. Thus, the primary event in glyphosate degradation by Arthrobacter sp. GLP‐1 is the cleavage of its C‐P bound. This report constitutes the first demonstration of the metabolism of glyphosate in a gram‐positive bacterium.
d‐Galactofuranose in the N‐linked sugar chain of a glycopeptide from Ascobolus furfuraceus
Authors: GROISMAN José F. , de LEDERKREMER Rosa M.
Published: March 2005

Two types of linkages between the carbohydrate and the peptide moiety in the glycopeptide from Ascobolus furfuraceus are described. Treatment with mild alkali produced β‐elimination of a small oligosaccharide. Evidence for the O‐glycosidic linkage was provided by (a) increase in absorbance at 240 nm, (b) decrease in threonine and serine content after the alkaline treatment and (c) detection of tritiated oligosaccharide following alkaline NaB3H4 reduction. Mannose is the sugar involved in the O‐glycosidic linkage. The remaining glycopeptide was branched by galactofuranose units, which were selectivity released by mild acid hydrolysis. The N‐glycosidic linkage of the sugar chain was conclusively proved by cleavage with endo‐β‐N‐acetyl‐glucosaminidase. Sequential NaB3H4 reduction and acid hydrolysis gave [3H]glucosaminitol. The structure of the sugar chain was studied by 13C NMR spectroscopy and by methylation analysis.
The peptidyltransferase centre of the Escherichia coli ribosome
Authors: TATE Warren P. , SUMPTER Vicki G. , TROTMAN Clive N. A. , HEROLD Marzel , NIERHAUS Knud H.
Published: March 2005

Modification of the Escherichia coli 50S ribosomal subunit with histidine‐specific diethyl pyrocarbonate affects peptide bond formation and release‐factor‐dependent peptidyl‐tRNA hydrolysis.

Unmodified L16 can restore activity to a split protein fraction from the altered subunit but other proteins of the core also contain histidine residues important for the activity of the peptidyltransferase centre.

When isolated and purified by centrifugation, particles reconstituted with unmodified proteins and modified L16 do not retain the altered L16. The modified protein does mediate the partial restoration of peptide bond formation and release‐factor‐2 activities to these particles. It must be exerting its effect during the assembly of the peptidyltransferase centre in the reconstituted particle.

A particle could be reconstituted which lacks L16 and has significant activity in peptide bond formation and peptidyl‐tRNA hydrolysis. L16 stimulates these activities.

A tighter ribosomal binding of the release factor 2, dependent upon the absence of protein L11, can in part compensate for the loss of activity of the peptidyltransferase centre when it is assembled with either modified L16 or in the absence of L16. The protein and its histidine residue seem important, therefore, for the peptidyltransferase centre to be formed in the correct conformation but not essential for activity once the centre is assembled.
Characterisation of a type‐I collagen trimeric cross‐linked peptide from calf aorta and its cross‐linked structure
Authors: HENKEL Werner , GLANVILLE Robert W. , GREIFENDORF Dieter
Published: March 2005

A collageneous trimeric cross‐linked peptide has been isolated from the insoluble matrix of calf aorta, using trypsin solubilisation, and purified by gel filtration, cation‐exchange chromatography and reversed‐phase HPLC. Molecular mass and amino acid composition indicated that the C‐terminal, non‐helical region of type I collagen in its dimer form, designated as [Colc(I)]2, is cross‐linked to a tryptic peptide TN(I) from the N‐terminal helical cross‐link region of an adjacent type I molecule, forming the cross‐linked peptide [Colc(I)]2× TN(I).

Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross‐linking domains ColC(I) and TN(I) and furnished the first direct chemical evidence for the 4D staggered arrangement of type I molecules within native fibers.

The trifunctional cross‐linking amino acid pyridinoline was shown to occur in the peptide, confirming the peptides three‐chain structure. Pyridinoline was isolated from the cross‐linked peptide by preparative amino acid analysis and reversed‐phase HPLC and identified by (a) its ultraviolet absorption spectra, (b) its fluorescence excitation and emission spectra and, for the first time, (c) its time‐of‐flight secondary ion‐mass spectrum. The high sensitivity of the latter method, exceeding that of fast‐atom‐bombardement mass spectroscopy by three orders of magnitude, allowed detection of pyridinoline in the picomole range.

The occurrence of pyridinoline in non‐stoichiometric amounts, the presence of hydroxylysine in hydrolysates of all cross‐linked peptides and the finding that hydrolysates also contained an unidentified component indicated that there is at least one cross‐link form that is different from pyridinoline and is hydrolysable.
Immunological evidence for the existence of H1‐like histone in yeast
Authors: SREBREVA Ljuba , ZLATANOVA Jordanka , MILOSHEV George , TSANEV Roumen
Published: March 2005

In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt.

Perchloric‐acid‐extracted proteins from whole cells contain only two fractions which comigrate with ‘authentic’ yeast high‐mobility‐group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross‐reaction with anti‐(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1.

The isolation of ‘authentic’ yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti‐HMG antiserum whereas the richer preparation also gives considerable cross‐reaction with the anti‐H1 antiserum. Immunoblotting analysis performed on the salt‐extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti‐H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.
Inhibition of leukotriene D4 catabolism by D‐penicillamine
Authors: HUBER Michael , KEPPLER Dietrich
Published: March 2005

Inhibition of the catabolism of the most biologically potent cysteinyl leukotriene, LTD4, was studied in rat hepatoma cells in vitro and in the rat in vivo. LTD4 dipeptidase, an ectoenzyme on the surface of AS‐30D hepatoma cells, exhibited an apparent Km value of 6.6μM for LTD4. D‐Penicillamine and L‐penicillamine inhibited this enzyme activity with apparent Ki values of 0.46 mM and 0.21 mM respectively. Bestatin, an inhibitor of the aminopeptidase activity of hepatoma cells, did not affect LTD4 hydrolysis at concentrations as high as 5 mM, indicating that the aminopeptidase did not contribute to LTD4 catabolism.

In the rat in vivo, D‐penicillamine also inhibited LTD4 catabolism. After intravenous injection of [3H]LTC4 an accumulation of [3H]LTD4 and a retarded formation of [3H]LTE4 were observed in the circulating blood after D‐penicillamine pretreatment. Within 1 h after intravenous [3H]LTC4 injection, about 80% of the administered radioactivity was recovered in bile. After D‐penicillamine pretreatment [3H]LTD4 was the major biliary leukotriene metabolite, whereas in untreated controls leukotriene metabolites more polar than LTC4 predominated in bile.

After stimulation of endogenous leukotriene production in vivo by platelet‐activating factor, N‐acetyl‐LTE4 was the major cysteinyl leukotriene detected in bile. D‐Penicillamine treatment prior to platelet‐activating factor resulted in the accumulation of LTD4, which under these circumstances was the major endogenous leukotriene metabolite detected in bile.
DNA methylation in wheat
Authors: THEISS Gudrun , SCHLEICHER Roland , SCHIMPFF‐WEILAND Gabriele , FOLLMANN Hartmut
Published: March 2005

The origin and function of the large amount of 5‐methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300‐fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE‐cellulose, Sephadex G‐75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in double‐stranded DNAs isolated from wheat, maize, calf thymus or bacteria using S‐adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de novo and in maintenance methylation. A relative molecular mass of 50000–55000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of Mr= 50000 and one other component (Mr= 35000).

The preference for endogenous, double‐stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA.
The γ‐aminobutyric‐acid/benzodiazepine‐receptor protein from rat brain
Authors: STAUBER Gregory B. , RANSOM Richard W. , DILBER Aydin I. , OLSEN Richard W.
Published: March 2005

The γ‐aminobutyric‐acid‐receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high‐titer rabbit antiserum. High concentrations of Triton X‐100 detergent plus KCl solubilized about 90% of the membrane‐bound γ‐aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column‐absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor‐like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600‐fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168–171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble γ‐aminobutyric‐acid/benzodiazepine‐receptor‐binding activities and stain the receptor subunits (principally 52‐kDa band) on Western blots.
Candida L‐norleucine, leucine: 2‐oxoglutarate aminotransferase
Authors: DER GARABEDIAN P. Arsène , VERMEERSCH Jacqueline J.
Published: March 2005

A new enzyme which catalyzes the transamination of L‐norleucine (2‐aminohexanoic acid) and L‐leucine with 2‐oxoglutarate was purified to homogeneity from cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 100000. The transaminase behaved as a dimer which consists of two subunits identical in molecular mass (Mr 51000). The enzyme has a maximum activity in the pH range of 8.0–8.5 and at 55°C. 2‐Oxoglutarate, and to a lesser extent pyridoxal 5′‐phosphate, were effective protecting agents against increasing temperature. The enzyme exhibits absorption maximum at 330 nm and 410 nm. L‐Norleucine, and L‐leucine to a lesser extent, are the best amino donors with 2‐oxoglutarate as amino acceptor. The Km values for L‐norleucine, L‐leucine and 2‐oxoglutarate determined from the Lineweaver‐Burk plot were 1.8 mM, 6.6 mM and 2.0 mM respectively. A ping‐pong bi‐bi mechanism of inhibition with alternative substrates is found when the enzyme is in the presence of both L‐norleucine and L‐leucine. The inhibitory effect of various amino acid analogs on the transamination reaction between L‐norleucine and 2‐oxoglutarate was studied and Ki values were determined.
Mitochondrial phosphate carrier
Authors: LIGETI Erzsébet , FONYÓ Attila
Published: March 2005

1. The phosphate carrier of isolated rat liver mitochondria was studied.

2. To investigate the effect of the substrate on the external side of the transport protein, a technique was required by which the extramitochondrial phosphate concentration could be changed without any effect on either the phosphate content or the pH value in the intramitochondrial compartment. This was found when non‐respiring mitochondria were incubated with the ionophore nigericin.

3. Using this method variation of the phosphate concentration outside the mitochondria did not protect the essential SH groups located at the external surface of the phosphate carrier.

4. Interrelations within the phosphate carrier unit were investigated in mitochondria in which only one of the two essential thiol groups of the carrier unit was free. These mitochondria were however able to carry out phosphate transport.

5. Alterations of the intramitochondrial pH‐induced modification of the position of the single free sulfhydryl group at the external surface of the membrane similarly to the changes which occur in intact mitochondria where both SH groups are free [E. Ligeti and A. Fonyó (1984) Eur. J. Biochem. 139, 279–285].

6. It is suggested that the sulfhydryl groups essential for the transport function do not participate in the formation of the substrate binding site of the carrier protein. They probably have a role in the conformational change necessary for the translocation step. The possibility of two equivalent but independently functioning parts within the carrier unit is raised.