bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glycosyl transferases of baby hamster kidney cells and ricin‐resistant mutants
Authors: STOJANOVIC Dagmar , VISCHER Peter , HUGHES R. Colin 
Published: March 2005

Extracts of baby hamster kidney (BHK) cells catalyzed the incorporation of N‐acetylgalactosamine from UDP‐N‐acetyl[14C]galactosamine into myelin basic protein and an acylated tetrapeptide, N‐acetylthreonyl‐triproline, based on the threonine residue 98, glycosylated in myelin basic protein. The incorporated N‐acetylgalactosamine residues were shown to be in α linkage to the peptide moieties. Several ricin‐resistant BHK cell lines contained enhanced (approximately twofold) levels of the transferase activity. Apomucins obtained from bovine submaxillary gland mucin by chemical or enzymic degradation were relatively poor acceptors.
Using asialomucin as acceptor, galactosyl transferase activities and a weak sialyl transferase activity were detected in BHK cell extracts. Galactose transfer occurred at two sites: to peptide‐linked N‐acetylgalactosamine residues to form the linkage, galactosyl‐(β1→3)‐N‐acetylgalactosamine and to terminally linked N‐acetylglucosamine residues that exist as a minor constituent in bovine submaxillary mucin O‐glycans, to form a galactosyl N‐acetylglucosamine linkage. This reaction was not inhibited by ovalbumin, an efficient acceptor of the β1→4 galactosyl transferase involved in N‐glycan assembly.
Incorporation of galactose and N‐acetylgalactosamine into endogenous proteins of BHK cell extracts was also detected. Sialic acid, fucose and N‐acetylglucosamine residues were not incorporated. The incorporated N‐acetylgalactosamine residues were shown to be in α linkage to polypeptide, and galactose incorporation represented synthesis of the galactosyl‐(β1→3)‐N‐acetylgalactosamine sequence linked to polypeptide. The major endogenous protein labelled by either sugar had a molecular weight of approximately 80000. A BHK‐cell‐associated glycoprotein, analogous to the urinary Tamm‐Horsfall glycoprotein of molecular weight similar to the major endogenous acceptor of glycosylation, was not glycosylated in the experiments in vitro.
Reconstitution of Na+ transport from purified methylmalonyl‐CoA decarboxylase and phospholipid vesicles
Authors: HILPERT Wilhelm , DIMROTH Peter 
Published: March 2005

Methylmalonyl‐CoA decarboxylase from Veillonella alcalescens which was isolated by affinity chromatography on avidin‐Sepharose was incorporated into phospholipid vesicles by the detergent dilution method with octyl glucoside as the detergent. By this procedure the Na+ pump activity was reconstituted. The optimal octyl glucoside concentration for reconstitution was about 2.8%. The activity of reconstituted Na+ transport increased with the amount of enzyme present during reconstitution until a plateau was reached at about 7 μg enzyme/mg phospholipid. All four polypeptides of the decarboxylase were incorporated into the proteoliposomes but the relative amounts of α and γ subunits were considerably reduced.The reconstitution process was highly asymmetric, since about 80% of the decarboxylase was oriented in the proteoliposomes with the substrate binding site facing to the interior. The orientation was determined from the increase of methylmalonyl‐CoA decarboxylase activity by destruction of the membrane permeability barrier. It was also deduced from the amount of enzyme which was not accessible from the outside to inactivation by avidin.With these reconstituted vesicles, a steady‐state internal Na+ concentration was established by methylmalonyl‐CoA decarboxylation which under optimized conditions was about 30‐fold higher than in the incubation medium. Sodium ion accumulation in the presence of the Na+ ‐carrying ionophores nigericin or monensin was practically nil. In the presence of valinomycin or carbonylcyanide‐p‐trifluoromethoxy phenylhydrazone the rate of Na+ transport and its steady‐state internal concentration were considerably higher than in the controls which is consistent with the function of methylmalonyl‐CoA decarboxylase as an electrogenic Na+ pump.
Age‐related growth and protein turnover in the thymus of normal and glucocorticoid‐treated rats
Authors: KELLY Frank James , GOLDSPINK David Frank 
Published: March 2005

Age‐related changes in the growth, nucleic acid content and protein turnover of the thymus have been studied in normal male rats.
A rapid and massive atrophy was found at each age 24 h after exposure to cortisone acetate or dexamethasone; the thymus of sexually mature rats being most severely affected.
The steroids decreased (70–90%) total protein synthesis in the thymus more than whole body synthesis (15%), indicating a more pronounced action on the thymus compared with other body tissues.
Initially protein breakdown increased in the steroid‐treated thymus but at longer times this change was reversed.
Isolation and characterization of a precursor form of M collagen from embryonic chicken cartilage
Authors: VON DER MARK Klaus , VAN MENXEL Marion , WIEDEMANN Hanna 
Published: March 2005

A disulfide‐cross‐linked collagen has been extracted with neutral salt solutions from organ cultures of embryonic chick sternal cartilage. This collagen, which we term pM collagen, is presumed to be the native extracellular precursor molecule to disulfide‐cross‐linked collagen fragments recently described. Cleavage of pM collagen under native conditions with pepsin gives rise to the collagen fragments M1 and M2, which had also been isolated from pepsin extracts of chick hyaline cartilage [K. von der Mark, M. van Menxel & H. Wiedemann (1982) Eur. J. Biochem. 124, 57–62]. Native pM collagen was purified by DEAE‐cellulose chromatography and agarose gel filtration. On agarose and following polyacrylamide gel electrophoresis, the unreduced molecule migrates with an apparent Mr of 300 000. Reduction of disulfide bridges produces two subunits with Mr 80000 (pMa) and 60 000 (pMb) when compared with collagen standards. Cyanogen bromide cleavage of pMa and pMb, excised from dodecyl sulfate gels, resulted in different peptide maps, indicating that both components are genetically distinct polypeptide chains. The occasional appearance of the unreduced pM collagen as a doublet band on dodecyl sulfate gels and the observation that pMa and pMb occur in non‐stoichiometric ratios suggests that pMa and pMb form separate native molecules, although their incorporation into a single pM molecule cannot be excluded. Native pM collagen was completely digested with bacterial collagenase, and contained hydroxyproline and proline in a ratio of 1.15:1, indicating the absence of significant non‐collagenous domains. Thus it represents, despite several pepsinlabile sites, more likely a largely triplehelical, processed form of collagen rather than a procollagen‐like molecule containing globular domains.Processing of pM collagen to M1 and M2 fragments or other intermediate forms was not observed in cartilage organ culture or in chondrocyte cell cultures within 18h.
The catalytic subunits of protein phosphatase‐1 and protein phosphatase 2A are distinct gene products
Authors: TUNG H. Y. Lim , RESINK Therese J. , HEMMINGS Brian A. , SHENOLIKAR Shirish , COHEN Philip 
Published: March 2005

Three forms of protein phosphatase‐1 were isolated from rabbit skeletal muscle that had Mr values of 37000, 34000 and 33000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the β‐subunit of phosphorylase kinase very much faster than the α‐subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase‐3 and Mg‐ATP for activity by incubation with inhibitor‐2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr‐37000 and Mr‐34000 species were converted to the Mr‐33000 form by incubation with chymotrypsin. It is concluded that the Mr‐33000 and Mr‐34000 forms are derived from the Mr‐37000 component by limited proteolysis. Conversion of the Mr‐37000 to the Mr‐33000 form was accompanied by a two‐fold increase in activity, indicating that an Mr‐4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity.The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr‐37000 (15–20 kU/mg) or Mr‐33/34000 (40–50 kU/mg) forms of protein phosphatase‐1. It dephosphorylated the α‐subunit of phosphorylase kinase 4–5‐fold faster than the β‐subunit, was unaffected by inhibitor‐1 or inhibitor‐2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase‐3 and Mg‐ATP‐dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr‐37000 form of protein phosphatase‐1 to the Mr‐33000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observerd with protein phosphatase 1. Antibody to protein phosphatase‐2A raised in sheep did not cross‐react with any of the forms of protein phosphatase‐1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase‐1 and protein phosphatase‐2A are distinct gene products.
Evidence for the presence of calmodulin in fish mucus
Authors: FLIK Gert , VAN RIJS Jeanne H. , WENDELAAR BONGA Sjoerd E. 
Published: March 2005

Partly purified mucus collected from the skin of three species offish contains a protein that, on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, comigrates with bovine brain calmodulin and shows the same calcium‐dependent shift in electrophoretic mobility as calmodulin.
Fish mucus contains a heat‐stable activator of cyclic nucleotide phosphodiesterase; activation is concentration dependent and sensitive to the specific calmodulin inhibitor calmidazolium (R 24571).
The presence of calmodulin in fish mucus is further indicated by means of a specific radioimmunoassay.
A drop in the calcium concentration of the water induces an increase in the immunoassayable calmodulin concentration of mucus, which indicates that the function of calmodulin in mucus is related to control of permeability of the skin epithelium to water and ions.
Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N‐terminal region
Authors: van EIJK Jan H. , VERHEIJ Hubertus M. , DE HAAS Gerard H. 
Published: March 2005

In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the ɛ‐amidinated derivatives without reaction of the α‐amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity.Starting from AMPA, chemical modification reactions were carried out at the α‐amino function. This group was blocked with a tert‐butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N‐terminal asparagine, resulting in the formation of des‐Asn1‐AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine8 residue.Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N‐terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N‐terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid‐bound enzyme.
Primary structure of flavocytochrome b2 from baker's yeast
Authors: GHRIR Rachid , BECAM Anne‐Marie , LEDERER Florence 
Published: March 2005

Reverse‐phase high‐pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment α. Acetonitrile gradients at acid and/or neutral pH using μBondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, αCB1 and αCB2, could only be separated from each other by trifluoroacetic acid/1‐propanol gradients on μBondapak‐CN columns. The various systems tested are presented and compared.The elucidation of the amino acid sequence of αCB2 (95 residues), αCB3 (67 residues) and αCB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment αCB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin‐layer finger‐printing and/or high‐pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator.The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305–316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120 279–287], the present results enable us to present the complete sequence of fragment α (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alternation of α helices and β structure. The possibility that the non‐heme‐binding portion of fragment α could constitute a flavin‐binding domain is discussed.
Solubilization of mannosyltransferase activities for the biosynthesis of mammary glycoproteins
Authors: PRAKASH Chandra , KATIAL Albine , KANG Mohinder S. , VIJAY Inder K. 
Published: March 2005

The microsomal preparation from the lactating bovine mammary tissue was solubilized by treatment with nonionic detergent, NP‐40, at a protein/detergent ratio of 1.5:1 and a detergent concentration of 0.5%. Following centrifugation at 147000 ×g for 120 min, the supernatant fraction was incubated with labeled sugar nucleotides, GDP‐Man and UDP‐GlcNAc. It was found to synthesize a series of lipid‐linked saccharides up to (Man)5‐ (GlcNAc)2. The solubilized glycosyltransferases retained up to about 60% of the activity after two weeks of storage at 4°C. The biosynthesis of glycolipids was stimulated by a mixture of lipids obtained by extracting the mammary microsomes with CHCl3/CH3OH (2:1).A labeled lipid‐linked tetrasaccharide of the structure Manα1 → 3Manβ→ GlcNAcβ→ GlcNAc was isolated by labeling baby hamster kidney cells with [2‐3H]mannose under conditions of glucose starvation followed by extraction of the cells with CHCl3/CH3OH (2:1) and separation of the lipids by high‐performance liquid chromatography. When this lipid‐linked tetrasaccharide was incubated with the solubilized bovine mammary microsomes and GDP‐Man, it was elongated to a lipid‐linked heptasaccharide having the structure Manα1 → 2Manα1 → 2Manα1 → 3(Manα1 → 6)Manβ→ GlcNAcβ→ GlcNAc. The kinetics of the elongation reaction also revealed the intermediary formation of smaller amounts of lipid‐linked pentasaccharide and hexasaccharide. The elongation reaction did not require any divalent metal ion and had a broad pH optimum between 6.8 and 7.6. The lack of inhibition of the elongation reaction by EDTA or amphomycin support earlier studies that GDP‐Man rather than mannosylphosphoryldolichol, is the direct donor of mannosyl residues for the biosynthesis of glycolipids up to (Man)5(GlcNAc)2. Mannosylphosphorylretinol was ineffective as mannosyl donor for the elongation reaction.
Short‐term control of mitochondrial adenine nucleotide translocator by thyroid hormone
Authors: MOWBRAY John , CORRIGALL Jane 
Published: March 2005

1. An improved simple technique for measuring adenine nucleotide translocator activity at low medium substrate concentrations is described.2. Confirming previous reports, thyroidectomy was shown to lead to lowered translocator activity in rat liver mitochondria. The rapidly exchangeable portion of the matrix nucleotide also decreased in hypothyroid preparations even though the total nucleotides increased substantially.3. The apparent Km of translocator for ADP increased from 2.8 to 6.2 μM in hypothyroid preparations: Mg2+ ions raised this to about 20μM.4. All of these changes in adenine nucleotide translocation were entirely reversed by 15 min after a single intravenous near‐physiological dose of triiodothyronine.
Adenine nucleotides affect the binding of 3‐phosphoglycerate to pig muscle 3‐phosphoglycerate kinase
Authors: VAS Mária , BATKE József 
Published: March 2005

Pig muscle 3‐phosphoglycerate kinase was complexed with 1‐anilino‐8‐naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme‐dye interaction did not influence the enzymic activity under the experimental conditions used.By measuring the substrate‐dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd≤ 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20°C. The dissociation constants obtained for the separate bindings of 3‐phosphoglycerate, MgATP, 1,3‐bisphosphoglycerate and MgADP were 0.03 ± 0.01 mM. 0.15 ± 0.1 0mM, 0.00005 ± 0.00001 mM and 0.15 ± 0.10mM respectively.Binding of 3‐phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3‐phosphoglycerate in this ternary complex (0.25 ± 0.08 mM) is comparable to its Km value (0.38 ± 0.10mM). The same weakening can be observed in the non‐productive ternary complexes where MgATP is replaced by MgADP (Kd= 0.20 ± 0.10 mM) or AMP (Kd= 0.12 ± 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3‐hosphoglycerate.In contrast to 3‐phosphoglycerate, the binding of the substrate analogue, glycerol 3‐phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 ± 0.10 mM) is comparable to its inhibitory constant (0.70 ± 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3‐phosphate binding (0.40 ± 0.10mM) and the Km value of 3‐phosphoglycerate (0.38 ± 0.10mM) suggest that, during the enzymic reaction, binding of 3‐phosphoglycerate occurs probably without involvement of the carboxyl group.
Inactivation of pig muscle 3‐phosphoglycerate kinase by thiol modification depends on reagent size
Authors: DÉKÁNY Katalin , VAS Mária 
Published: March 2005

Two thiol groups (out of the total seven) of pig muscle 3‐phosphoglycerate kinase can be alkylated in 0.1 M Tris/HCl buffer, pH 7.5, at 20°C either with methyl iodide or with iodoacetamide in a second‐order reaction with rate constants 0.05 ± 0.02 M−1 s−1 and 0.23 ± 0.05 M−1 s−1 respectively. The slow reaction of the remaining five thiols with Ellman's reagent (Nbs2), which requires the unfolding of the protein, is not affected by the nature of the alkylating reagent.While methylation of the two reactive thiols does not affect either the specific activity of the enzyme or the Km values of the substrates, carboxamidomethylation is accompanied by the loss of enzymic activity.Both the methylated and the carboxamidomethylated enzymes bind 3‐phosphoglycerate or MgATP practically with the same binding constants as the native enzyme, as detected by fluorimetric titration of enzymes complexed with 1‐anilinonaphthalenesulfonate (ANS). Thus, inactivation during carboxamidomethylation cannot be due to changes in the substrate‐binding ability of the enzyme.The substrate‐caused changes in the fluorescence emission spectrum of ANS bound to the carboxamidomethylated enzyme are different from the changes observed with the native enzyme. The fluorescence properties of the methylated enzyme do not differ from those of the native enzyme. These differences may reflect the different mode of substrate binding to the carboxamidomethylated enzyme as compared to the native or the methylated one. Thus, the special steric requirements of the enzymic reaction are possibly not fulfilled after carboxamidomethylation.The presence of the equilibrium mixture of substrates lessens the reactivity of the fast‐reacting thiols towards both alkylating agents, a finding that indicates the effects of substrates on the local conformation around these groups.
Specific changes in lactate levels, lactate dehydrogenase patterns and cytochrome b559 in Dictyostelium discoideum caused by queuine
Authors: SCHACHNER Elfriede , ASCHHOFF Hans‐Jürgen , KERSTEN Helga 
Published: March 2005

Higher eukaryotes contain tRNA transglycosylases that incorporate the guanine derivative queuine from the nutritional environment into specific tRNAs by exchange with guanine at position 34. Alterations in the queuosine content of specific tRNAs are suggested to be involved in regulatory mechanisms of major routes of metabolism during differentiation. Dictyostelium discoideum has been applied as a model to investigate the function of queuine or queuine‐containing tRNAs. Axenic strains are supplied with queuine by peptone, but they grow equally well in a defined queuine‐free medium. Queuine‐lacking amoebae, starved in suspension culture for 24 h, lose their ability to differentiate into stalk cells and spores, whereas amoebae sufficiently supplied with queuine will overcome this metabolic stress and undergo further development when plated on agar.The results presented here show that d(–)‐lactate occurs in the slime mould in millimolar amounts and that its level is remarkably decreased in queuine‐lacking cells after 24 h of starvation in suspension culture. On isoelectric‐focusing polyacrylamide gels, nine different forms of NAD‐dependent d(–)‐lactate dehydrogenase can be separated from extracts of vegetative cells, and six forms from extracts of the starved cells. Under queuine limitation, one form is missing in the starved cells. Low amounts of l(+)‐lactate are usually found in vegetative amoebae but significantly less in queuine‐lacking cells. Five forms of NAD‐dependent l(+)‐lactate dehydrogenase are detectable in extracts from vegetative, queuine‐treated cells, and slight alterations occur in queuine‐deficient amoebae. In the starved cells only one form of l(+)‐lactate dehydrogenase is found, irrespective of the supply of queuine to the cells. A cytochrome of type b with an absorption maximum at 559 nm accumulates during starvation only in queuine‐lacking cells; it might be a component of an NAD‐independent lactic acid oxidoreductase as is cytochrome b557 in yeast and be responsible for the reduced level of lactate in cells lacking queuine in tRNA.