bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calcium release from calmodulin and its C‐terminal or N‐terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped‐flow fluorescence with Quin 2 and intrinsic tyrosine
Authors: SUKO Josef , WYSKOVSKY Wolfgang , PIDLICH Johann , HAUPTNER Rudolf , PLANK Brigitte , HELLMANN Gertrude 
Published: March 2005

Calcium dissociation from the C‐terminal and N‐terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped‐flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA.Calcium dissociation from the C‐terminal half of calmodulin, which contains only the two high‐affinity calcium‐binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s−1 and 12.8 s−1, respectively, in the C‐terminal half and 10.5 s−1 and 10.8 s−1, respectively, in intact calmodulin (pH 7.0, 25°C, 100 mM KCl).Alkylation of the C‐terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s−1 and 0.73 s−1 respectively; tyrosine: kobs 1.65 s−1 and 0.61 s−1 respectively). Alkylation of intact calmodulin resulted in a fourphase calcium dissociation measured with Quin 2 (Kobs 85.3 s−1, 11.1 s−1, 1.92 s−1 and 0.59 s−1); the latter two phases are assumed to represent calcium release from high‐affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s−1 and 0.53 s−1 respectively) and the rate parameters determined in the C‐terminal half. Evidently perturbation of the calcium‐binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high‐affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N‐terminal half resulted in biphasic calcium release with rates (kobs 153 s−1 and 10.9 s−1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin‐melittin or fragment‐melittin complexes, measured with Quin 2, were slower and monophasic in the C‐terminal half (kobs 1.12 s−1), biphasic in the N‐terminal half (Kobs 140 s−1 and 26.8 s−1 respectively) and triphasic in intact calmodulin (kobs 126 s−1, 12.1 s−1 and 1.38 s−1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low‐affinity sites mainly due to a reduced calcium ‘off rate’, presumably because of conformation restrictions.Phenoxybenzamine‐alkylated calmodulin and calmodulin‐melittin complex inhibit the endogenous calcium/calmodulin‐dependent protein kinase of cardiac sarcoplasmic reticulum. The alkylated calmodulin is actually an activator with lower affinity than the unmodified calmodulin suggesting a perturbation of both the calciumbinding domains as well as the enzyme‐binding domain(s).
The light‐dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center in barley
Authors: KREUZ Klaus , DEHESH Katayoon , APEL Klaus 
Published: March 2005

The light‐dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center has been studied in greening barley (Hordeum vulgare L.) seedlings.Immunoblot analysis of total cellular protein fractions and immunogold labelling of the P700 chlorophyll a protein in ultrathin sections of Lowicryl‐embedded leaf tissue revealed that the concentration of this chlorophyll‐binding protein in plastids of dark‐grown barley seedlings is below the limit of detection. Upon illumination with white light, a rapid accumulation of this protein is induced. This light effect seems not to be regulated at the level of transcription. The gene for the P700 chlorophyll a protein has been mapped within the large single‐copy region of the plastid DNA of barley. High levels of transcripts of this gene are present already in dark‐grown seedlings and remain fairly constant throughout an extended illumination period. Polysomes were isolated from etioplasts and chloroplasts. The same high relative concentration of mRNA encoding the P700 chlorophyll a protein was present in both polysome fractions. This result suggests that the light‐dependent accumulation of the P700 chlorophyll a protein during chloroplast formation in barley seedlings is regulated at the translational, or posttranslational, level.
Site specificity of casein kinase‐2 (TS) from rat liver cytosol
Authors: MARIN Oriano , MEGGIO Flavio , MARCHIORI Fernando , BORIN Gianfranco , PINNA Lorenzo A. 
Published: March 2005

The factors determining the site recognition and phosphorylation by rat liver casein kinase‐2 (CK‐2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: (a) the nature of the phosphorylatable amino acid, if any; (b) its position relative to the critically required acidic residues; (c) the extension and (d) the structure of the acidic cluster. All of them were tested as substrates and/or competitive inhibitors of CK‐2, and their kinetic and inhibition constants were determined.The results suggest the following conclusions. (a) Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. (b) In order to carry out its role of structural determinant the critical acidic cluster must be located on the C‐terminal side of the target residue, though not necessarily adjacent to it. (c) The affinity for the protein‐binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N‐terminal side. (d) An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser‐Glu‐Glu‐Ala‐Glu‐Glu having similar Km but negligible Vmax compared to Ser‐Glu‐Ala‐Glu‐Glu‐Glu and Ser‐Glu‐Glu‐Glu‐Ala‐Glu. These data provide a rationale for the substrate specificity of CK‐2 and will give a helpful insight into the structure of the protein‐binding site of this enzyme.
Studies in vitro on the flavinylation of 6‐hydroxy‐d‐nicotine oxidase
Authors: BRANDSCH Roderich , BICHLER Veronika 
Published: March 2005

The gene of 6‐hydroxy‐D‐nicotine oxidase (6‐HDNO), a flavoenzyme from Arthrobacter oxidans with covalently bound FAD, was expressed with the aid of an expression vector in a cell‐free coupled transcription‐translation system derived from Escherichia coli MZ9. Ultraviolet irradiation of the E. coli extract did not affect synthesis of the 6‐HDNO polypeptide nor total protein synthesis but enzymatic 6‐HDNO activity could not be detected. Addition of FAD to the irradiated cell extract restored the capability of the transcription‐translation assays to synthesize enzymatically active 6‐HDNO. However, enzymatic activity could not be restored on addition of FAD plus cell‐free extract to the ultraviolet‐inactivated assays after completion of apo‐6‐HDNO synthesis (60 min) nor to immunoprecipitates thereof. Under similar conditions, addition of [14C]FAD did not increase the protein‐bound radioactivity.These results indicate that under conditions of limited FAD supply in the in vitro system a flavinless apo‐6‐HDNO‐polypeptide was synthesized. It was, however, not possible to bind the cofactor to the completed polypeptide chain. These findings argue for a cotranslational cofactor binding.
Simple‐kinetic descriptions of alcohol dehydrogenase after immobilization on tresyl‐chloride‐activated agarose
Authors: BILLE Vincent , REMACLE José 
Published: March 2005

Yeast alcohol dehydrogenase was successfully immobilized on tresyl‐chloride‐activated agarose; the optimized conditions allowed an enzyme activity recovery of over 90%.Comparison of free and immobilized enzyme properties showed an unchanged intrinsic activation energy of the reaction and a shift of optimum activity to a higher pH medium after immobilization. Comparison of the kinetic parameters for both substrates of the reaction showed that the Michaelis‐Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell‐Chance model was more appropriate. These results are discussed taking into consideration the factors affecting the immobilized enzyme. Finally, we discuss the possibilities of cofactor regeneration with this immobilized alcohol dehydrogenase.
Isolation of a mutant LLC‐PK1 cell line defective in hormonal responsiveness
Authors: JANS David A. , RESINK Therese J. , WILSON Lynette E. , REICH Edward , HEMMINGS Brian A. 
Published: March 2005

A mutant LLC‐PK1 cell line, M18, was isolated after a single treatment of the parent culture with N‐methyl‐N′‐nitro‐N‐nitroso‐guanidine. In contrast to LLC‐PK1 cells, the mutant did not exhibit production of urokinasetype plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor‐independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8‐bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP‐dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non‐hydrolyzable GTP analogue guanosine 5′‐[β,γ‐imino]triphosphate, but were unresponsive to calcitonin or vasopressin.The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC‐PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.
Methylation in vivo of elongation factor EF‐Tu at lysine‐56 decreases the rate of tRNA‐dependent GTP hydrolysis
Authors: VAN NOORT Johannes M. , KRAAL Barend , SINJORGO Karin M. C. , PERSOON Niek L. M. , JOHANNS Earl S. D. , BOSCH Leendert 
Published: March 2005

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys‐56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine.We have undertaken an extensive comparison between the properties of the highly methylated EF‐Tu and unmodified EF‐Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF‐Tu (site I) also appears not to be affected by the methylation of the protein. tRNA binding at site II is. Although the apparent affinity of tRNA for site II remains unaltered upon methylation of EF‐Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys‐81 are significantly less stimulated by the tRNA when EF‐Tu is methylated.A possible physiological implication of this phenomenon is discussed.
Light‐modulated ADP‐ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes
Authors: BENTROP Joachim , PAULSEN Reinhard 
Published: March 2005

Rhodopsin (P, λmax 480 nm) of blowfly photoreceptors R1–6 is converted by light into a thermally stable metarhodopsin (M, λmax 565 nm). In isolated blowfly rhabdoms photoconversion of P to M affects bacterial toxin‐catalyzed ADP‐ribosylation of a 41‐kDa protein, activates phosphorylation of opsin and induces the binding of a 48‐kDa phosphoprotein to the rhabdomeric membrane.ADP‐ribosylation of the 41‐kDa protein is catalyzed by cholera toxin and is inhibited by P → M conversion. The 41‐kDa protein might represent the α‐subunit of the G‐protein, proposed to be part of the phototransduction mechanism [Blumenfeld, A. et al. (1985) Proc. Natl Acad. Sci. USA 82, 7116–7120].P → M conversion leads to phosphorylation of opsin at multiple binding sites: up to 4 mol phosphate are bound/mol M formed. Dephosphorylation of the phosphate binding sites is induced by photoconversion of M to P. High levels of calcium (2 mM) inhibit phosphorylation of M and increase dephosphorylation of P.Protein patterns obtained by sodium dodecyl sulfate gel electrophoresis of irradiated retina membranes show an increased incorporation of label from [γ‐32P]ATP also into protein bands of 48 kDa, 68 kDa and 200 kDa. Binding studies reveal that in the case of the 48‐kDa protein this effect is primarily due to a light‐induced binding of the protein to the photoreceptor membrane. The binding of the 48‐kDa phosphoprotein is reversible: after M → P conversion the protein becomes extractable by isotonic buffers. These data suggest that in rhabdomeric photoreceptors of invertebrates light‐activation of rhodopsin is coupled to an enzyme cascade in a similar way as in the ciliary photoreceptors of vertebrates, although there may be differences, e.g. in the type of G‐protein which mediates between the activated state of metarhodopsin and a signal‐amplifying enzyme reaction.
Binding of pig plasma gelsolin to F‐actin and partial fractionation into calcium‐dependent and calcium‐independent forms
Authors: POPE Brian , WEEDS Alan G. 
Published: March 2005

The interaction of pig plasma gelsolin with F‐actin has been studied by a sedimentation assay using 125I‐gelsolin in a Beckman Airfuge. Over 90% of the gelsolin bound to F‐actin in 0.1 mM CaCl2 in experiments using 24 μM actin and 2–10 nM 125I‐gelsolin, but only 40–50% bound in 1 mM EGTA. Addition of more F‐actin to the EGTA supernantant does not sediment this gelsolin. Demonstration of this partial calcium sensitivity depends critically on the use of F‐actin that has been prepared in the absence of calcium ions. F‐actin prepared from G‐actin in calcium or pretreated with calcium, binds 125I‐gelsolin more completely in EGTA. This suggests that gelsolin activity is influenced by transient exposure of actin to calcium.Further evidence for partial calcium sensitivity in the interactions between gelsolin and F‐actin has been obtained by other methods, including viscometry and electron microscopy.The gelsolin present in the EGTA supernatant is complexed to G‐actin, predominantly as binary complexes. Very low concentrations of these complexes reduce the viscosity of F‐actin in calcium but not in EGTA. Whether this effect is due to severing activity, or capping with consequent depolymerization to establish the new critical concentration, is uncertain.The results suggest the presence of two types of gelsolin, one that requires micromolar concentrations of calcium for binding to F‐actin and one that does not. Both bind to G‐actin. Partial separation has been achieved using actin‐Sepharose.Pig plasma gelsolin is heterogeneous on isoelectric focussing gels in urea, but the two types of gelsolin separated on actin‐Sepharose do not correspond to specific isoelectric species.
Mucopolysaccharidases from Pseudomonas sp.
Authors: HORTON Denise S. P. Q. , MICHELACCI Yára M. 
Published: March 2005

Four constitutive enzymes, capable of degrading keratan sulfate, were isolated from Pseudomonas sp.: a particulate endoglycosidase, a soluble endoglycosidase, a soluble exo‐β‐D‐galactosidase and a soluble exo‐β‐D‐N‐acetylglucosaminidase. The endoglycosidase were shown to act only upon keratan sulfate forming β‐D‐2‐acetamido‐2‐deoxy‐6‐O‐sulfoglucosyl‐(1 → 3)‐D‐galactose, as the main product. This result indicates that the enzyme catalyses the hydrolysis of β‐D‐galactose‐(1 → 4)‐N‐acetylglucosamine linkages. It was also shown that this monosulfated disaccharide inhibits the particulate keratan sulfate endoglycosidase. The bovine nucleus pulposus keratan sulfate is depolymerized at a lower rate and extent when compared to the corneal keratan sulfate. The soluble endoglycosidase is very labile, in contrast to the particulate enzyme, which has been stored at −20°C or at 4°C for at least 12 months with no loss in activity. The particulate endoglycosidase and the soluble exo‐β‐D‐galactosidase and exo‐β‐D‐N‐acetylglucosaminidase are induced when the bacteria is grown in adaptative media containing either 0.1% keratan sulfate or 0.1% chondroitin sulfate. Furthermore, particulate forms of the exoenzymes were detected. The soluble endoglycosidase specific activity, in contrast, is approximately the same in extracts of cells grown in glucose, kertan sulfate or chondroitin sulfate.A chondroitin sulfate lyase was also identified in the soluble extracts of Pseudomonas sp. cells. This enzyme depolymerizes chondrotin 4‐sulfate, chondroitin 6‐sulfate and hyaluronic acid forming unsaturated disaccharides as main products. It is also active upon the glucuronic‐acid‐containing regions of the dermatan sulfate molecules.The properties of the soluble enzymes, further purified by ion‐exchange chromatography, and of the particulate keratan sulfate endoglycosidase are presented.
Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes
Authors: FLOCKERZI Veit , OEKEN Hans‐Jochem , HOFMANN Franz 
Published: March 2005

The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200–110 to an apparent density of 1.5–2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200–110. cAMP‐dependent protein kinase phosphorylated preferentially the 142‐kDa band up to a stoichiometry of 0.82 ± 0.07 (15) mol phosphate/mol peptide. The 56‐kDa band was phosphorylated only in substoichiometric amounts.[3H]PN 200–110 bound at 4°C to one site with apparent Kd and Bmax values of 9.3 ± 1.7 nM and 2.2 ± 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200–110 in an apparent allosteric manner. (–)Desmethoxyverapamil inhibited the binding of [3H]PN 200–110 at 37°C and stimulated it at 18°C. In agreement with these results, (–)desmethoxyverapamil increased the dissociation rate of [3H]PN 200–110 from 0.29 min−1 to 0.38 min−1 at 37°C and decreased it threefold from 0.046 min−1 to 0.017 min−1 at 18°C. The (+)isomer of desmethoxyverapamil inhibited PN 200–110 binding at all temperatures tested. d‐cis‐Diltiazem stimulated the binding of [3H]PN 200–110 at 37°C with an apparent EC50 of 1.4 μM and decreased the dissociation rate from 0.29 min−1 to 0.11 min−1. The stimulatory effect of d‐cis‐diltiazem was temperature‐dependent and was seen only at temperatures above 18°C.These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane‐bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.
Purification and properties of guanylate kinase from bovine retinas and rod outer segments
Authors: HALL Scott W. , KÜHN Hermann 
Published: March 2005

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside‐diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3).The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP ⇌ GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20–23 kDa for both native enzyme and dodecylsulfate‐denatured polypeptide), Km values (13 μM for GMP and 430 μM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s−1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.
Kinetics of cadmium and terbium dissociation from calmodulin and its tryptic fragments
Authors: MARTIN Stephen R. , LINSE Sara , BAYLEY Peter M. , FORSÉN Sture 
Published: March 2005

The kinetics of cadmium and terbium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by stopped‐flow fluorescence methods, using the calcium indicator Quin 2.Studies of the tryptic fragments TR1C and TR2C, comprising the N‐terminal or C‐terminal half of calmodulin, have clearly identified cadmium binding sites I and II as the low‐affinity (rapidly dissociating) sites and sites III and IV as the high‐affinity (slowly dissociating) sites. Thus the site preference of cadmium is the same as that of calcium. For terbium, however, sites I and II are the high‐affinity sites and sites III and IV are the low‐affinity sites. Thus, the site preference or terbium is not the same as that of calcium and cadmium.In contrast to previous studies with calcium, we observe two kinetic precesses for dissociation from sites III and IV for experiments with both cadmium and terbium. Possible models for the binding of metal ions are discussed.
In vitro competition between adenosine(5′)tetraphospho(5′)adenosine and deoxyribonucleic acid in the reaction with diamminedichloroplatinum(II)
Authors: BACHMAIER Annelies , JUST Gerald , HOLLER Eggehard 
Published: March 2005

Competition between adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and DNA for the synthesis of adducts with the cis or trans isomer of diamminedichloroplatinum(II) was measured in the presence and absence of magnesium and spermidinium ions. Reaction products were analysed by circular dichroism, poly(ethyleneimine) thin‐layer chromatography and reversed‐phase chromatography. Competition was affected by the oligovalent cations that bound specifically to the dinucleotide. Platination of DNA was favoured under all conditions. Chromatin was less competitive. The mechanism was kinetic competition, DNA reacting considerably faster than Ap4A. Platinum(II) did not exchange between adducts and free DNA and Ap4A, respectively. On that basis only low amounts of Ap4A adducts were estimated to be formed under conditions of clinical chemotherapy. The cis and trans isomers of diamminedichloroplatinum(II) were equally effective.Platinum(II) adducts of Ap4A were neither degraded by Ap4A‐specific pyrophosphohydrolases nor by phosphodiesterase nor in the presence of unfractionated extract of calf thymus. Unphysiologically high concentrations of Crotalus durissus phosphodiesterase I were required for hydrolytic splitting, the amount of which was similar for both platinum(II) isomer adducts. The results suggest that Ap4A platinum(II) adducts might accumulate during chemotherapy of cancer treatment.