bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and characterization of oocyte vitellin from Perinereis cultrifera (polychaete annelid)
Authors: BAERT Jean‐Luc , SAUTIERE Pierre , PORCHET Maurice 
Published: March 2005

Vitellin was identified and purified from submature oocytes of Perinereis cultrifera by concanavalin‐A–Sepharose and Ultrogel AcA 34 column chromatography. The yolk protein was defined as a glycolipoprotein with a relative molecular mass of 380000. Upon dissociation by sodium dodecyl sulphate, vitellin was shown to release five polypeptide subunits which ranged in relative molecular mass from 98000–16000. The purified vitellin was further characterized by amino acid analysis and its lipid and carbohydrate contents. The molecule contained about 5% carbohydrate and 16% lipid.Antiserum prepared against vitellin was shown to react with a component from the coelomic fluid of maturing females. Thus, it was suggested that in nereid annelids, considered phylogenetically and morphologically primitive, oocyte differentiation might depend upon the incorporation of a vitellogenin as in insects and many oviparous vertebrates.
Tyrosine modification of glucose dehydrogenase from Bacillus megaterium
Authors: FRÖSCHLE Marion , ULMER Wolfgang , JANY Klaus‐Dieter 
Published: March 2005

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation‐reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP.
The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr‐254 in the covalent structure of the enzyme.
After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer.
Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr‐254 which is essential for the catalytic activity and the other one is Tyr‐160 which seems to be located in the subunit binding area.
Regulation by adenosine of the vasopressin‐sensitive adenylate cyclase in pig‐kidney cells (LLC‐PK1L) grown in defined media
Authors: ROY Christian 
Published: March 2005

LLC‐PK1L cells, a kidney‐derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC‐PK1L cells had about 100‐times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10‐fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6‐L‐2‐phenylisopropyl‐adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high‐affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added.The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high‐affinity component of the hormonal response could be demonstrated.
Nucleotide sequence of cucumber‐mosaic‐virus RNA 2 reveals a translation product significantly homologous to corresponding proteins of other viruses
Authors: REZAIAN M. Ali , WILLIAMS Rhys H. V. , GORDON Karl H. J. , GOULD Allan R. , SYMONS Robert H. 
Published: March 2005

The nucleotide sequence of the 3035 residues of RNA 2 (Mr 1.03 × 106) of the Q strain of cucumber mosaic virus (CMV) was determined by sequencing M13 clones of the RNA 2cDNA and by dideoxy sequencing using primers prepared either from M13 clones or by chemical synthesis. A single long open reading frame starts at the second AUG from the 5′ end of RNA 2 and encodes 839 amino acids (Mr 94333). This frame has flanking regions of 92 nucleotides at the 5′ terminus and 423 nudeotides at the 3′ terminus.Computer analysis of the nucleotide sequence showed that CMV RNA 2 has a significant homology with RNA 2 of brome mosaic virus (BMV) and alfalfa mosaic virus (AMV) and also with a region for tobacco mosaic virus (TMV) RNA encoding the read‐through part of the 183‐kDa protein. About 400 amino acids in the central region of the CMV RNA 2 translation product have a striking homology with the corresponding proteins encoded by BMV and AMV and with the read‐through part of the TMV 183‐kDa protein. Hydrophobicity plots of CMV and BMV RNA translation products also had apparent similarities.It is concluded that CMV is related to BMV, AMV and TMV in order of increasing evolutionary divergence.
Sequence‐dependent structural variation in single‐helical DNA Proton NMR studies of d(T‐A‐T‐A) and d(A‐T‐A‐T) in aqueous solution
Authors: MELLEMA Jan‐Remt , PIETERS Jane M. L. , VAN DER MAREL Gijs A. , VAN BOOM Jacques H. , HAASNOOT Cornelis A. G. , ALTONA Cornelis 
Published: March 2005

The two deoxyribotetranucleoside triphosphates d(T‐A‐T‐A) and d(A‐T‐A‐T) were investigated in aqueous solution by one‐ and two‐dimensional proton NMR at 300 and 500 MHz. It is demonstrated that both compounds occur predominantly in the single‐helical form. Accurate coupling constants are obtained by computer simulation of several 500‐MHz spectra. The data are interpreted in terms of N and S pseudorotational ranges. The geometry of the major S‐type conformers displays a clear sequence dependence, as expressed by variation of the endocyclic backbone angle δ (C5′‐C4′‐C3′‐O3′). A simple sum rule is proposed to predict δ variation in single‐helical DNA fragments. Comparisons are made with other sequence‐dependent geometries as observed in a double‐helical B‐DNA fragment in the crystalline state.Furthermore, one‐ and two‐dimensional nuclear Overhauser effect (NOE) spectroscopy was carried out on d(T‐A‐T‐A). An inventory is made of the observed intra‐ and inter‐residue NOEs. The NOE data confirm the presence of a highly stacked single‐helical conformation of d(T‐A‐T‐A) in solution. No indications are found for the formation of a bulge‐out structure as observed for analogous alternating purine‐pyrimidine oligoribonucleotides.
Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus
Authors: ZETTLMEISSL Gerd , TESCHNER Wolfgang , RUDOLPH Rainer , JAENICKE Rainer , GÄADE Gerd 
Published: March 2005

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico‐chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable.Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 ± 1500 for both native and denatured enzyme.The unfolding transition at varying guanidine · HCl concentrations is characterized by a two‐step profile: at 0.4–0.8 M, partial unfolding is parallelled by inactivation; at 2.0–2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4–1.8 M guanidine · HCl, partial unfolding is superimposed by aggregation.The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine · HCl. Changes in the far‐ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%.Denaturation in 6 M guanidine · HCl, as monitored by the decrease of protein fluorescence, is fast (<8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (<20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long‐term denaturation, is determined by a single first‐order reaction characterized by τ= 29 ± 3 min (20°C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate‐limiting for reactivation after long‐term denaturation, are characterized by a high energy of activation (112 ± 8 kJ/mol). The slow reactivation step is compatible in rate with the first‐order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62–90].
Distinction of the roles of the two high‐affinity calcium sites in the functional activities of the Ca2+‐ATPase of sarcoplasmic reticulum
Authors: SCOTT Terrence L. , SHAMOO Adil E. 
Published: March 2005

The effects of trypsin digestion and low temperature on Ca2+ binding and on Ca2+ activation of ATP hydrolysis by the high‐affinity transport sites of the Ca2+,Mg2+‐ATPase of sarcoplasmic reticulum were examined. Sarcoplasmic reticulum vesicles contain 0.7–1.1 high‐affinity Ca2+ sites per 105 g sarcoplasmic reticulum with K= 3–5 × 105 M−1, as well as sites of lower affinity. The first cleavage of the ATPase with trypsin (TD1) has no effect on the binding properties of the high affinity sites. The second tryptic cleavage (TD2) decreases the affinity of the high sites to K= 3 × 104 M−1 with conservation of the total number of sites.The purified ATPase contains 1.6 – 2.0 high affinity Ca2+ sites per 105 g protein when measured at 23°C, while at 0–4°C there is ∼ 1 high‐affinity (K= 5 – 10 × 105 M−1) affinity site and ∼ 1 intermediate‐affinity (K= 3 × 104 M−1) site per 105 g. Trypsin digestion to the point of TD1 has no effect on either the number or the binding constants of the high‐affinity sites. Upon TD2 cleavage, one of the sites is converted to the intermediate‐affinity state, while the other remains at high affinity. After TD2 modification of the enzyme both of the sites are in the intermediate affinity state at 4°C.On the basis of the binding data, several models for the roles of the Ca2+ sites in the activation of ATP hydrolysis are derived. The results are summarized by a scheme in which the two high‐affinity Ca2+ sites are heterogeneous with respect to sensitivity to temperature and to TD2 modification. The results of this and a previous study (Scott, T. L. and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137–144] indicate that while occupation of either of the two Ca2+ sites can stimulate ATP hydrolysis, the site which is sensitive to TD2 is essential for the coupling of hydrolysis to Ca2+ transport.
Enzymatic glucosylation of dolichol monophosphate and transfer of glucose from isolated dolichyl‐D‐glucosyl phosphate to ceramides by BHK‐21 cell microsemes
Authors: SUZUKI Yasuo , ECKER Christian P. , BLOUGH Herbert A. 
Published: March 2005

Enzymatic glucosylation of dolichol monophosphate (dolichol‐P) from UDP‐D‐[3H]glucose was studied using the microsomal fraction of BHK‐21 cells. The reaction product was separated by preparative thin‐layer chromatography, further purified by DEAE‐cellulose acetate column chromatography, and characterized as dolichyl‐β‐D‐glucosyl phosphate (Dol‐P‐Glc). The microsomal fraction of BHK cells catalyzed the incorporation of glucose from UDP‐[3H]glucose into ceramides (endogenous and exogenous) and Dol‐P; both reactions required Mn2+. Maximal glucosylation of Dol‐P was achieved at pH 5.6–5.8 in the presence of a non‐ionic detergent, Zonyl A. Glucosylation of exogenous Dol‐P, from UDP‐Glc, was non‐competitively inhibited by exogenous ceramides.Incubation of Dol‐P‐[3H]Glc or Dol‐P‐[14C]Glc with liposomes (containing ceramides) and the microsomal fraction of BHK‐21 cells resulted in the formation of a radioactive glucolipid which comigrated with the same RF value as glucosylceramide (Glc‐Cer) on silica gel thin‐layer chromatography. Transfer of [14C]glucose from Dol‐P‐[14C]Glc to exogenous ceramides was confirmed by double‐labeling techniques. The pH dependence for transfer of radio‐labeled glucose from Dol‐P‐[3H]Glc to ceramides was multi‐phasic (optima at pH 4.0 and 7.0); glycosylation occurred within 5 min and Zonyl A was absolutely essential for the transfer reaction. These results indicate that Dol‐P‐Glc may also participate in the synthesis of ceramide hexosides.
Mechanism of hepatic megamitochondria formation by ammonia derivatives
Authors: WAKABAYASHI Takashi , HORIUCHI Michio , SAKAGUCHI Mikako , MISAWA Kazuko , ONDA Hiroyuki , HJIMA Moritake , ALLMANN David W. 
Published: March 2005

Correlation between the chemical structure and the ability to induce hepatic megamitochondria formation was studied by feeding mice and rats diets containing a wide spectrum of ammonia derivatives.

Ammonia derivatives with electron‐releasing groups, such as hydrazine, phenylhydrazine, hydroxylamine and aniline were effective in inducing megamitochondria.
Ammonia derivatives with electron‐withdrawing groups, such as formamide, sulfamic acid, acetamide were ineffective in inducing megamitochondria.
Inducibility of ammonia derivatives with electron‐releasing groups plus electron‐withdrawing groups for the megamitochondria formation was dependent upon nucleophilicity of the chemical: 2,4‐dinitrophenylhydrazine induced megamitochondria, while acetanilide did not induce megamitochondria.
The megamitochondria formation induced by ammonia derivatives was a reversible process.
Freeze‐fracture studies on megamitochondria indicated that megamitochondria were formed by the fusion of adjacent mitochondria.
Phosphorylating capacity of megamitochondria (hydrazine‐induced megamitochondria, for example) were normal despite morphological changes.

These data might suggest that the nucleophilicity of chemicals plays a key role in the induction of hepatic megamitochondria. These data might also suggest that the phenomenon is an adaptive process to changes of intracellular milieu.
Methylamine‐induced conformational change of α2‐macroglobulin and its zinc(II) binding capacity
Authors: ÖSTERBERG Ragnar , MALMENSTEN Bo 
Published: March 2005

Methylamine induces a conformational change of α2‐macroglobulin which is very similar to that obtained by proteinase reaction and binding. This was shown by small‐angle X‐ray scattering at 21 °C in 0.03 M Hepes buffer of pH 8.0 containing 0.15 M NaCl and 0.3 mM EDTA. When α2‐macroglobulin reacts with methylamine the side maximum virtually disappears from the X‐ray scattering curve and the radius of gyration decreases from 7.8 nm to 7.2 nm.The X‐ray data of α2‐macroglobulin are consistent with an open shape model similar to that deduced via electron micrographs [Schramm, H. J. and Schramm, W. (1982) Hoppe‐Seyler's Z. Physiol. Chem. 363, 803–812]; one projection of the model resembles the letter H; the four subunits are mainly represented as elliptical cylinders which are connected via a central, quite flat cylinder.Zinc(II) ions cause aggregation of α2‐macroglobulin even at such a low total zinc concentration as 12.5 μM; for 25 μM zinc(II) concentration, the average molecular mass indicates that the aggregation goes beyond the dimeric stage. Monomeric species of α2‐macroglobulin appear to have the capacity specifically to bind 8.0 zinc(II) ions per molecule, which corresponds to two zinc(II) ions per subunit.
A kinetic study of the oxidative deamination of L‐glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes
Authors: HORNBY David P. , ENGEL Paul C. 
Published: March 2005

1. The NAD+ ‐specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows Michaelis‐Menten kinetics in contrast to the enzyme from several other sources, and thus gives linear double‐reciprocal plots of initial‐rate data.2. The initial‐rate parameters have been determined for this bacterial glutamate dehydrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition.3. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6–9.4. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.
Actinomycin D, 1H NMR studies on intramolecular interactions and on the planarity of the chromophore
Authors: JURETSCHKE Hans Paul , LAPIDOT Aviva 
Published: March 2005

The conformation of actinomycin D in acetone and chloroform solution at different temperatures has been studied by 1H NMR spectroscopy.At lower temperature the resonances due to the two chromophoric amino protons were observed. These signals exhibit very different resonance positions indicating a severely hindered rotation of the 2‐amino group and the presence of a hydrogen‐bond connecting the 2‐amino and the 1‐carbonyl groups.In 1H NMR spectra of partially 15N‐enriched actinomycin D, the 1JN‐H coupling constants at the 2‐amino group were determined and a strong sp2 character for the 2‐amino nitrogen was deduced. The strong amide character of the 2‐amino group is caused by mesomerism involving the 1‐carbonyl group.The amino proton signals are sensitive indicators for differences in the spatial relationship of the diverse parts of the actinomycin molecule.At lower temperatures a simultaneous and selective broadening of the α ring threonine and valine amide proton signals as well as of the 2‐amino group resonance was observed, indicating the presence of one dynamic process in the molecule which slows down upon temperature reduction. A swinging motion of the N(10) nitrogen through the chromophore plane would explain this observation.The interpretation of these results requires the presence of a non‐planar chromophoric system in the actinomycin molecule in acetone and chloroform solution. The possible implications of this non‐planarity for the intercalation process and for the biological activity of the drug are discussed.
Magnetic resonance studies on the interaction of metal‐ion and nucleotide ligands with brain hexokinase
Authors: JARORI Gotam K. , MEHTA Alka , KASTURI Sitapati R. , KENKARE Umakant W. 
Published: March 2005

Our previous studies have shown that one manganous ion binds tightly to bovine brain hexokinase, with a Kd= 25 ± 4 μM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (ɛb) is 3.5 at 9 MHz and 23°C [Jarori, G. K. Kasturi, S. R., and Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258–268]. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E · Mn binary complex, we now show the formation of a nucleotide‐bridge ternary complex, enzyme · nucleotide · Mn. Addition of glucose 6‐phosphate to enzyme · ATP · Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E · ADP · Mn · Glc‐6‐P appears to be formed when both the products are present.β, γ‐Bidentate Cr(III)ATP has been used to elucidate the role of direct binding of Mn(II) in catalysis, and the stoichiometry of metal‐ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(III)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron‐spin resonance studies on the binding of Mn(II) to the enzyme in the presence of Cr(III)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis.Experiments carried out with hexokinase spin‐labeled with 3‐(2‐iodo‐acetamido)‐2,2,5,5‐tetramethyl 1‐pyrrolidinyloxyl clearly showed that the direct‐binding Mn site on the enzyme is distinctly located from its ATP Mn binding site.
The nature of the decreased activity of glycogen synthase phosphatase in the liver of the adrenalectomized starved rat
Authors: BOLLEN Mathieu , DOPERE Frans , GORIS Jozef , MERLEVEDE Wilfried , STALMANS Willy 
Published: March 2005

We have investigated the nature of the decrease in synthase phosphatase activity which occurs progressively in the livers of adrenalectomized rats that are starved for 48h. No evidence could be found for the accumulation of an inhibitor. Addition of the heat‐stable deinhibitor protein, which antagonizes the effects of thermostable inhibitor proteins (inhibitor‐1 and modulator), did not affect the activity of synthase phosphatase in gel‐filtered liver extracts from normal or adrenalectomized starved rats; it did, however, increase the activity of phosphorylase phosphatase about fivefold in either condition. The restoration of synthase phosphatase activity by cortisol in vivo was prevented by actinomycin D.Further evidence concerning the nature of the missing protein came from a comparison of synthase phosphatase activities in liver homogenates from control and adrenalectomized starved rats, with the use of three distinct synthase b substrates. The apparent loss of synthase phosphatase activity in the deficient homogenates varied between 30% and 90% according to the type of substrate. The magnitude of this decrease corresponds to the degree of dependence of these substrates on the G‐component of synthase phosphatase for efficient conversion to the a‐form. No G‐component could be isolated from livers of adrenalectomized starved rats. Cross‐combination of subcellular fractions from control and deficient livers revealed an almost total loss of G‐component, with little loss of S‐component. This specific loss of functional G‐component is identical to the deficiency previously observed in the livers of rats with severe chronic alloxan‐diabetes.