bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of a homogeneous glucosidase II from pig kidney microsomes
Authors: BRADA Daniela , DUBACH Ulrich C. 
Published: March 2005

The processing of the oligosaccharide precursor chain, (GlcNAc)2(Man)9(Gle)3, of N‐glycosylated glyco‐proteins starts with the action of glucosidase I which excises the terminal (α1–2)‐linked glucose residue. Glucosidase II removes the two inner (α1–3)‐linked glucose residues.We have purified glucosidase II to homogeneity from pig kidney microsomes. The enzyme is a glycoprotein and contains a single type of subunit of molecular mass ∼ 100 kDa. The native enzyme is probably a tetramer. It cleaves glucosidic α1–3 and α1–4, but not α1–1, α1–2 or α1–6 bonds and lacks 2‐mannosidase and glucosidase I activity. The pH optimum is between 6.0 and 7.5. Specific antibodies against the native enzyme and the denatured subunit were prepared. By activity measurements and immune blotting, a similar enzyme was found in rat liver. In the fractionated rat liver, the enzyme was localized in the lumen of the endoplasmic reticulum, probably loosely bound to the inner face of the membrane. Purified Golgi fractions contained only low levels of the enzyme.
Conformational analysis of the single‐helical DNA fragment d(T‐A‐A‐T) in aqueous solution
Authors: MELLEMA Jan‐Remt , JELLEMA August K. , HAASNOOT Cornelis A. G. , VAN BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

Proton NMR studies at 300 MHz and 500 MHz were carried out on the tetranucleoside trisphosphate d(T‐A‐A‐T). The thermodynamics of the three stacking interactions, derived from chemical shift versus temperature profiles, were used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the fully stacked forms of the molecule.The data were interpreted in terms of N and S pseudorotational ranges [Altona, C. and Sundaralingham, M. (1972) J. Am. Chem. Soc. 94, 8205–8212]. It is shown that the stacked state of the molecule cannot be described by one conformer, but consists of one major structure (60%) in which all sugar rings have S‐type geometry and another structure (30%) in which residue dT(4) has an N‐type sugar. The remainder of the stacked states eonsists of one or more conformers with two or three sugar residues in the N‐type pseudorotational range. Detailed geometrical models are proposed for the major stacked conformers encountered in aqueous solution.
Na+‐dependent, α‐adrenergic mobilization of intracellular (mitochondrial) Ca2+ in brown adipocytes
Authors: CONNOLLY Eamorin , NÅNBERG Eewa , NEDERGAARD Jan 
Published: March 2005

The existence and significance of a hormone‐sensitive, rapidly mobilizable intracellular pool of Ca2+ in hamster brown‐fat cells was investigated with 45Ca2+‐labelling techniques.It was shown that such a pool existed and was probably located within the abundant mitochondria. It was rapidly mobilized by norepinephrine (median effective concentration 50 nM) through α‐adrenergic mechanisms. The mobilization of Ca2+ from the intracellular stores (mitochondria) required the presence of extracellular Na+, but not of Ca2+, K+ or Mg2+.It is concluded that the experiments are in agreement with a hypothesis linking the mobilization of intracellular Ca2+ pools with an α‐adrenergically‐induced increase in plasma membrane Na+ permeability (observed as a membrane depolarization), and a subsequent activation of the mitochondrial Na+/Ca2+ exchange, leading to mobilization of mitochondrial Ca2+ and the mediation of α‐adrenergic effects as a result of an elevated cytosolic Ca2+ level.
Biliary transport of glutathione disulfide studied with isolated rat‐liver canalicular‐membrane vesicles
Authors: AKERBOOM Theo , INOUE Masayasu , SIES Helmut , KINNE Rolf , ARIAS Irwin M. 
Published: March 2005

Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study glutathione disulfide (GSSG) transport. GSSG is transported into an osmotically sensitive compartment. Extrapolation to an external osmolarity of infinity indicates 35% binding after 10 min of incubation, e.g. as mixed disulfides between glutathione and SH‐groups in membrane proteins.Glutathione disulfide uptake occurred with equal rates in the presence of KCl or NaCl. After equilibration, no significant GSSG concentration gradient across the canalicular membrane was found. The temperature dependence of uptake is consistent with a carrier‐mediated transport for GSSG (Q10× 1.6) and seems to exclude simple diffusion Concentration dependence of GSSG uptake shows Michaelis‐Menten kinetics at concentrations up to 1 mM; a Km of 0.4 mM and a V of 1.1 nmol × min−1× mg protein−1 is calculated. At higher concentrations a linear dependence is observed without saturation kinetics.It is concluded that transport of glutathione disulfide is mediated by a carrier present in the canalicular membrane of the hepatocyte.
Cytoplasmic mRNA‐protein complexes of chicken muscle cells and their role in protein synthesis
Authors: BAG Jnanankur 
Published: March 2005

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross‐link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA‐protein complexes(mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28000, 32000, 46000, 65000 and 150000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130000 and 43000 was higher in free mRNPs than in polysomal mRNP complexes.To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat‐shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre‐existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA‐binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes.Heat‐shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr= 81000, 65000 and 25000 (heat‐shock polypeptides). Whether this altered pattern of protein synthesis following heat‐shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat‐shock treatment. A [35S]methionine‐labeled polypeptide of Mr= 80000 was detected in mRNPs of heat‐shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80000‐Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat‐shock mRNAs.The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo. Failure to bind to regulatory protein(s) due to competition among mRNAs may be responsible for their inability to participate in protein synthesis.
Rabbit pepsinogens
Authors: KAGEYAMA Takashi , TAKAHASHI Kenji 
Published: March 2005

Six pepsinogens were purified from the gastric mucosa of adult rabbits by chromatographies on DEAE‐cellulose, DEAE‐Sephacel and Sephadex G‐150. They were designated as pepsinogen I, pepsinogens II‐1, II‐2, II‐3, and II‐4, and pepsinogen III, based on their chromatographic behaviors.Each pepsinogen was converted to pepsin at pH 2.0 and 14 °C. The resulting pepsins had roughly similar enzymatic properties. However, activation processes appeared to differ significantly among them. Pepsinogen I was converted to an active intermediate form by sequentially releasing the NH2‐terminal 25 or 26 residues. This active form was fairly stable and no further conversion occurred on further incubation for several hours. Pepsinogens II were converted to pepsins partly sequentially through intermediate forms and partly directly releasing the NH2‐terminal 44 residues as a single intact polypeptide. Pepsinogen III appeared to be converted to pepsin directly without forming intermediate species.The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences in the NH2‐terminal regions of the resulting pepsins. Five distinct sequences were identified, indicating that most of these pepsinogens are the products of different genes.
Structural organization of the 5′ region of the human thyroglobulin gene
Authors: TARGOVNIK Hector M. , POHL Viviane , CHRISTOPHE Daniel , CABRER Bartolomé , BROCAS Huguette , VASSART Gilbert 
Published: March 2005

Sequence analyses of bovine and human thyroglobulin (Tg) cDNA have demonstrated that the 5′ region of the mRNA encodes a domain responsible for thyroid hormone synthesis and exhibits striking internal repetition. Knowledge of the organization of the corresponding chromosomal DNA region would provide insight as to how such a structure has evolved.A human genomic DNA library was screened by hybridization in situ, using a bovine Tg cDNA probe corresponding to 2.8 × 103 base pairs at the 5′ end of the mRNA. Out of 3 × 105 phage plaques, four were scored as positive and yielded three different phages containing thyroglobulin sequences. Selected human Tg cDNA probes were used to order the phages and to identify overlapping regions. Electron microscopy of hybrids between human Tg mRNA and the phage DNA was performed to determine the intron/exon organization of this region. The following conclusions were reached. (a) About 4 × 104 base pairs corresponding to the 5′ region of the gene have been isolated as three overlapping recombinant phages. (b) The three phages cover altogether 2.9 × 103 base pairs of exonic sequence at the 5′ end of the mRNA. (c) Out of the 11 exons identified in this region, 9 were of a size similar to that of the 3′ exons characterized previously (≤ 200 base pairs); exons 9 (1.12 × 103 base pairs) and 10 (0.56 × 103 base pairs) were exceptions to this rule. (d) The phage nearest the 5′ end contains about 9 × 103 base pairs of sequence located upstream from the gene.The avalability of clones covering the region upstream from the thyroglobin gene will provide the basis for the identification of sequences involved in its transcriptional control by thyroid‐stimulating hormone (thyrotropin).
Occurrence of S‐adenosylhomocysteine hydrolase in prokaryote cells
Authors: SHIMIZU Sakayu , SHIOZAKI Shozo , OHSHIRO Takashi , YAMADA Hideaki 
Published: March 2005

The occurrence of S‐adenosylhomocysteine hydrolase (EC 3.3.1.1) was found in a variety of prokaryotes. These prokaryotes did not exhibit any activities of S‐adenosylhomocysteine nucleosidase (EC 3.2.2.9) and S‐ribosyl‐homocysteine hydrolase (EC 3.3.1.3), which had been the generally accepted prokaryote enzymes for the regeneration of free homocysteine from S‐adenosylhomocysteine in the activated methyl cycle. In these prokaryotes S‐adenosylhomocysteine hydrolase was suggested to be the only enzyme functioning for the regeneration of free homocysteine by enzymological and immunochemical studies.
S‐Adenosylhomocysteine hydrolase was purified and crystallized from cells of a prokaryote, Alcaligenes faecalis. The purified enzyme was found to be homogenous on ultracentrifugation and gel electrophoresis. Its relative molecular mass is approximately 280000 and it is composed of six identical subunits with a Mr of approximately 48000. The NH2‐terminal and COOH‐terminal amino acids are lysine and tyrosine respectively. The enzyme contains 6 ma1 NAD/mol. Some nucleosides, such as formycin A, nebularine, adenosine N1 oxide and so on, are able to substitute for adenosine yielding the corresponding S‐nucleosidylhomocysteine congeners. Modification of the 5′‐hydroxymethyl group in adenosine leads to the most potent inhibition of the thioether formation of homocysteine with adenosine.
The enzyme from A. faecalis has some immunological similarities to other prokaryote S‐adenosylhomocysteine hydrolases, but is different from the enzymes of animal sources.
Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308
Authors: NIMMO Gillian A. , BORTHWICK Andrew C. , HOLMS W. Henry , NIMMO Hugh G. 
Published: March 2005

Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000‐fold from Escherichia coli ML308 by a procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE‐cellulose, blue‐dextran‐Sepharose and Sephadex G150.
The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D. C. and Koshland, D. E. (1982) Nature (Lond.) 300, 458–460] that a single protein bears both activities.
Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation.
Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine.
Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P‐phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions.
The active and inactive forms of isocitrate dehydrogenase can be resolved by non‐denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.
Immunochemistry, genuine size and tissue localization of collagen VI
Authors: VON DER MARK Helga , AUMAILLEY Monique , WICK Georg , FLEISCHMAJER Raul , TIMPL Rupert 
Published: March 2005

Collagen VI was solubilized with pepsin from human placenta and used for preparing rabbit antisera. Major antigenic determinants were located in the central region of the antigen including triple‐helical and globular structures. Antisera prepared against a constituent chain showed preferential reactions with unfolded structures. Antibodies were purified by affinity chromatography and failed to cross‐react with other collagen types I–V and with fibronectin. These antibodies demonstrated intracellular and extracellular collagen VI in fibroblast and smooth muscle cell cultures. Immunoblotting identified a disulfide‐bonded constituent chain about twice as large as those of the pepsin fragments in both cell cultures and tissue extracts. Rotary shadowing electron microscopy indicated that the increase in mass is due to larger globular domains present at both ends of collagen VI monomers.Indirect immunofluorescence demonstrated a wide occurrence of collagen VI in connective tissue particularly of large vessels, kidney, skin, liver and muscle. Collagen VI is apparently not a typical constituent of cartilage or of basement membranes. Ultrastructural studies using the immunoferritin technique showed collagen VI along thin filaments or in amorphous regions of aortic media or placenta but not in association with thick, cross‐striated collagen fibrils or elastin. This supports previous suggestions that collagen VI is a constituent of microfibrillar structures of the body.
The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: BOSMA Hans J. , DE KOK Arie , WESTPHAL Adrie H. , VEEGER Cees 
Published: March 2005

An improved purification procedure of the pyruvate dehydrogenase complex of Azotobacter vinelandii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15–19 U/mg and an overall yield of 40%.The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:1:0.5 for E1:E2:E3 respectively. Based on E2 this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 ± 50 kDa, it is concluded that the complex as isolated from A. vinelandii is based on a tetramer of E2 chains.The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E2E3 subcomplexes with E1 resulted in maximum complex activity at an E1/E2 ratio of 1.5–1.6. Similar titrations of E1E2 subcomplexes with E3 resulted in maximum activity at an E3/E2 ratio of 0.45–0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three E1 dimers, one E2 tetramer and one E3 dimer. With excess of either E1 or E3 a decrease in activity is observed which indicates competition between these components for binding sites on E2.As shown before [Bosma, H. J., de Kok, A., Markwijk, B. W., and Veeger, C. (1984) Eur. J. Biochem. 140, 273–280], the isolated E2 component is composed of 32 peptide chains of 66 kDa each. Upon addition of E1 or E3, E2 dissociates into tetramers. Dissociation is complete upon the addition of four E1 dimers of four E3 dimers per E2 tetramer. Addition of E1 to saturated E2E3 subcomplex or E3 to saturated E1E2 subcomplex did not result in extra binding but rather in displacement of bound E3 or E1 respectively. It is therefore concluded that the binding sites of E1 and E3 to the E2 chains are either identical or so closely spaced that steric hindrance prevents simultaneous binding of both components.A model is presented based on the cubic structure of the isolated E2 component. In this model the 32 E2 peptide chains are arranged in tetramers in the corners of the cube. This model is discussed in connection with the existing model for the Escherichia coli complex.
Anion‐induced increases in the affinity of colcemid binding to tubulin
Authors: RAY Kunal , BHATTACHARYYA Bhabatarak , BISWAS Birendra B. 
Published: March 2005

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37°C the association rate constant for colcemid binding is 1.88 × 106 M−1 h−1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high‐affinity site (Ka= 0.7 × 105 M−1 at 37°C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low‐affinity site (Kb= 1.2 × 104 M−1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high‐affinity site) and the reaction is entropy driven (163 J K−1 mol−1).Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower‐affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower‐affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine.We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B‐ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher‐affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.
Proteinase‐treated photoreceptor discs
Authors: BAYRAMASHVILI Dmitry I. , DRACHEV Alexander L. , DRACHEV Lel A. , KAULEN Andrey D. , KUDELIN Andrey B. , MARTYNOV Vladimir I. , SKULACHEV Vladimir P. 
Published: March 2005

Photoreceptor discs from rod outer segments of cattle retina were treated with (a) papain, (b) thermolysin or (c) trypsin, the procedures resulting in the cleavage of the rhodopsin polypeptide chain between (a) 323 and 324, 236 and 237, 241 and 242, (b) 327 and 328, 240 and 241, or (c) 339 and 340 amino acid residues, respectively.In all the cases, partially digested rhodopsins proved to be competent in generating photoelectric potential and increasing membrane conductance of the discs adsorbed onto phospholipid‐impregnated collodion film. The kinetics of generation and dissipation of photopotential as well as of formation of metarhodopsin II and of the light‐induced rhodopsin protonation were found to be the same in the partially digested preparations and in the intact one.Incubation of papain‐treated or thermolysin‐treated discs at pH 6.0 induced formation of inside‐out vesicles which, when incorporated into the collodion film, generated an oppositely directed photopotential. Treatment of such vesicles with papain gave rise to further cleavages of the polypeptide localized between 30 and 31, 186 and 187 amino acid residues. One more proteinase‐sensitive site, localized between 104 and 105 residues, has been discovered in the inside‐out vesicles treated with thermolysin. This fact consistent with the scheme of the ‘seven column’ arrangement of the visual rhodopsin [Ovchinnikov, Yu. A. (1982) FEBS Lett. 148, 179–191].Rhodopsin, when treated with papain on both sides, was deprived of sixty amino acid residues being split in two sites in the middle part of the polypeptide, but was still active as a photoelectric energy transducer.The main specific feature inherent in the photoelectric response of the papain‐treated or thermolysin‐treated rhodopsin and absent from the native protein is that the former survives addition of long trains of saturating flashes when the response of the intact preparation becomes negligible. This effect was shown to be due to conversion of partially digested rhodopsin to a photolytic product that at room temperature lived for minutes even in the presence of NH2OH. A 532‐nm laser flash effectively converted this product back to rhodopsin.