bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Electrophoretic Study of Native Myosin Isozymes and of Their Subunit Content
Authors: d'ALBIS Anne , PANTALONI Colette , BECHET Jean‐Jaques 
Published: March 2005

Myosin polymorphism in muscles has been studied by a variety of electrophoretic techniques, in non‐dissociating and in dissociating conditions. The analysis of myosin isozymes in the native state was achieved in pyrophosphate buffer and required only minute amounts of protein; identical results were obtained with purified or crudely extracted myosin. The determination of the subunit content of each isozyme was done in the presence of sodium dodecyl sulphate or urea for light chain, and in a phenol, acetic acid and urea system for heavy chain screening.Electrophoresis in non‐dissociating conditions has led to the separation of up to a dozen of myosin isozymes, differing in mobilities by as much as 30%. Muscle specificity of myosin was clearly established. Apart from a few exceptions, all the muscles tested were shown to contain more than one myosin species; fast‐twitch muscles for instance all contained the same three isozymes, but in variable ratios. Class specificity of myosin appeared related to the relative proportions of isozymes in a given muscle.A second electrophoresis in dissociating solvents of the myosin bands first resolved in pyrophosphate buffer has then allowed a further characterization of the various isozymes. The differences in mobilities observed in the native state were shown to come either from the light chains, or from the heavy chains, or from both. The first case was illustrated by the three species present in fast muscles, which were shown to correspond to three alkali light‐chain isozymes, the heterodimer representing in some instances up to 40% of the total. Next to light‐chain muscle type specificity, electrophoresis in the phenol, acetic acid, urea system has led to the detection of differences in the heavy chains of fast, slow and cardiac myosins.The application of these various electrophoretic techniques to the analysis of the modification of myosin isozymes during development or in pathology studies can be considered.
The Calcium‐Dependent Phosphatidylinositol‐Phosphodiesterase of Rat Brain
Authors: IRVINE Robin F. , HEMINGTON Norma , DAWSON Rex M. C. 
Published: March 2005

1. The activity of the soluble, calcium‐dependent phosphatidylinositol‐specific phosphodiesterase (EC 3.1.4.10) against [32P] phosphatidylinositol has been investigated.2. KCl (only at neutral pH), Mg2+, positively‐charged proteins such as histone, and phospholipids containing a choline headgroup are all inhibitory to the enzyme. Choline‐phospholipids cause a 90% inhibition at an equimolar ratio to phosphatidylinositol.3. Other phospholipids (phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid) are all potent stimulators of the enzyme: maximum stimulation being observed at a ratio of 1 mol activator/5–10 mol phosphatidylinositol.4. Unsaturated amphiphiles such as oleic and oleoyl alcohol also stimulate the activity, maximum stimulation being observed at about an equimolar ratio to phosphatidylinositol. Saturated amphiphiles (such as stearic acid and stearoyl alcohol) are less effective.5. The activation by acidic phospholipids and unsaturated amphiphiles appear to be independent as they are additive and, under certain conditions, synergistic.6. Both types of stimulator (independently or together) can reverse the inhibition caused by histone or phosphatidylcholine.7. Possible mechanisms of the suppression of the phosphatidylinositol phosphodiesterase in vivo, of its activation, and of the amplification of phosphatidylinositol breakdown are discussed.
The Insertion of Pig Microvillus Aminopeptidase into the Membrane as Probed by [125I]Iodonaphthylazide
Authors: NORÉN Ove , SJÖSTRÖM Hans 
Published: March 2005

The insertion of pig intestinal microvillus aminopeptidase (EC 3.4.11.2) into the membrane was studied by the hydrophobic photolabel [125I]iodonaphthylazide. The aminopeptidase was either labelled in the microvillus membrane and purified, or labelled after detergent solubilization and purification in a buffer containing Triton X‐100, and then isolated from the reaction mixture.Of the three subunits A (Mr 162000), B (Mr 123000) and C (Mr 62000) of the aminopeptidase, A and B, but not C contained radioactivity, indicating that both subunit A and B carry anchoring peptide. The radioactivity when released from the subunits by trypsin treatment was connected to low‐molecular‐weight material.
Affinity Labeling of the Escherichia coli Aspartate‐β‐Semialdehyde Dehydrogenase with an Alkylating Coenzyme Analogue
Authors: BIELLMANN Jean‐François , EID Pierre , HIRTH Christian 
Published: March 2005

3‐Chloroacetylpyridine‐adenine dinucleotide phosphate (clac3PdADP+, a NADP+ alkylating analogue, irreversibly inactivates aspartate‐β‐semialdehyde dehydrogenase with pseudo‐first‐order kinetics. NADP+ and NADPH, but not the substrate, protected the enzyme from inactivation. The pH dependence of the inactivation kinetics was determined. Incorporation of 1 mol cl[14C]‐ac3PdADP+/dimer totally inactivates the enzyme.Successive alkylation by the coenzyme analogue and by the substrate analogue, l‐2‐amino‐4‐oxo‐5‐chloropentanoic acid, was studied. After inactivation with the coenzyme analogue, no incorporation of the substrate analogue was detected. However, when the enzyme was first inactivated with the substrate analogue, the protein could subsequently be alkylated with the coenzyme analogue. The binding of NADP+ and NADPH to aspartate‐β‐semialdehyde dehydrogenase was determined by fluorescence.
Characterization of Rat‐Liver Microsomal Glutathione S‐Transferase Activity
Authors: MORGENSTERN Ralf , MEIJER Johan , DEPIERRE Joseph W. , ERNSTER Lars 
Published: March 2005

Rat liver microsomes were shown to catalyze the conjugation of 1‐chloro‐2,4‐dinitrobenzene with glutathione and this activity has been characterized. It cannot be removed from the microsomes by washing or other procedures which release loosely bound material from membranes. The microsomal glutathione S‐transferase can be activated up to eight fold by treatment with N‐ethylmaleimide. This activation also affects the apparent Km of the enzyme(s) for both glutathione and 1‐chloro‐2,4‐dinitrobenzene.Upon subcellular fractionation of the liver the N‐ethylmaleimide‐activateable glutathione S‐transferase distributes in the same manner as a marker for the endoplasmic reticulum and unlike markers for the other organelles and for the cytoplasm. Treatment of microsomes with proteases revealed that the enzyme is at least partially exposed on the cytoplasmic surface of the endoplasmic reticulum. Finally, three inducers of drug‐metabolizing systems–i.e. phenobarbital, methylcholanthrene, and trans‐stilbene oxide–all increase the activity of the cytoplasmic glutathione S‐transferases, but they do not affect the microsomal activity.These and other considerations indicate that the microsomal glutathione S‐transferase(s) is distinct from the cytoplasmic enzymes catalyzing similar reactions. The microsomal enzyme is likely to be involved in drug metabolism and the possibility of activating it through attack on a sulfhydryl group may represent an important physiological response to certain xenobiotics.
On the Mechanism of the Cytochrome c Oxidase Reaction. Bound Fe(III) Cytochrome c as an Intermediate in the Reductive Half Reaction
Authors: PETERSEN Lars Chr. , COX Raymond P. 
Published: March 2005

The reaction between cytochrome c and Cytochrome aa3 in the presence of reductant was studied using transient and steady‐state kinetic methods. To investigate the effect of complex formation the reaction was studied at both high and low ionic strength in potassium phosphate buffer (pH 7.4).
Steady‐state kinetic experiments were performed in which the oxidation level of cytochrome c was measured during the anaerobic‐aerobic transition of a system containing cytochrome aa3, cytochrome c and ascorbate. Such measurements confirm previous suggestions [Mochan and Nicholls (1972) Biochim, Biophys. Acta. 172, 37–51] that cytochrome c forms a complex with cytochrome oxidase which is not easily accessible to ascorbate reduction. The results suggest that one oxidised cytochrome c per oxidase is bound at low ionic strength.
The reduction of ‘oxygen‐pulsed’ oxidase in the presence of dithionite and various concentrations of cytochrome c was investigated by stopped flow spectrophotometry. Exposure of buffer containing cytochrome aa3, cytochrome c and dithionite to a brief pulse of oxygen caused an immediate oxidation of the oxidase and of up to one equivalent of cytochrome c per oxidase. Under conditions where complex formation is favoured the subsequent reduction of cytochrome c is many times slower than can be accounted for by the reaction between dithionite and free cytochrome c. This suggests that the bound oxidised cytochrome c is not readily accessible to dithionite reduction.
It is proposed that the complex between oxidised cytochrome c and the oxidase is an intermediate of the reductive half‐reaction and it is shown that the kinetic responses of the complex are rapid enough to be in agreement with this suggestion.
Kinetic and Thermodynamic Parameters of the Iron Spin‐State Transition in Human Aquomethemoglobin
Authors: OKONJO Kehinde O. 
Published: March 2005

The rate constants for the iron spin‐state transition in human aquomethemoglobin, as well as the corresponding thermodynamic parameters, have be determined at pH 6. klh, the rate constant for the low‐to‐high spin transition, varies between 242.6 s−1, and 480.6 s−1 in the temperature range 13–27°C; khl, the rate constant for the high‐to‐low spin transition, ‐varies between 57.4 s−1 and 69.4 s−2 over the same temperature range. The activation energies are 34.59 kJ mol−1 and 9.78 kJ mol−1 respectively, while the entropy change associated with the high‐to‐low spin transition is −99.7 J mol−1 K−1.The simple scheme postulated for formate binding to aquomethemoglobin [Okonjo, K. O. & Ilgenfritz, G. (1978) Arch. Biochem. Biophys. 189. 499–507] is shown to be inadequate. It is demonstrated that a scheme that includes a fast spin transition of the iron atoms, preceding formate binding, adequately accounts for formate binding to aquomethemoglobin in the T and R quaternary states.Evidence is presented to show that, in a given pure solvent, the spin‐state transition appears to be insensitive to differences in solute composition. By contrast, the spin transition appears to be quite sensitive to differences in solvent composition of ethylene glycol/water mixtures.In the presence of inositol hexakisphosphate. methemoglobin formate exhibits three relaxation times; the two with high amplitudes are affected by changes in inositol hexakisphosphate concentration. This indicates that inositol hexakisphosphate perturbs both the a and ß chains of methemoglobin and may be evidence to support the proposal that inositol hexakisphosphate, at acid pH. causes a quaternary structure transition in aquomethemoglobin.
Isolation and Immunochemical Characterization of the Euonymus europaeus Lectin Receptor from the Major Sialoglycoprotein of Human O Erythrocytes
Authors: PETRYNIAK Jerzy , PETRYNIAK Bronistawa , WASNIOWSKA Kazimiera , KROTKIEWSKI Hubert 
Published: March 2005

Purified and desialized blood‐group‐M glycoprotein from human O erythrocytes precipitated with a lectin isolated from Euonymus europaeus seeds. The precipitation was inhibited completely by lacto‐N‐fucopentaose I and lactose.
The H2 N‐terminal tryptic fragment of glycoprotein M (MT), containing most of carbohydrate and all fucose of glycoprotein M [Lisowska and Jeanloz (1973) Carbohydr. Res. 29, 181 ‐ 191], did not precipitate with the lectin but did inhibit the precipitation.
Reduced alkali‐labile oligosaccharides and glycopeptide containing alkali‐stable oligosaccha‐ride were isolated from the desialized MT glycopeptide by alkaline borohydride degradation and gel nitration. Only glycopeptide with the alkali‐stable oligosaccharide inhibited the precipitation and on a molar basis it was as active as glycopeptide MT‐ Both gave 50% inhibition at about 0.76 mM.
Two desialized oligosaccharides obtained as hydrazinolysis products of glycoprotein M, were also as active as glycopeptide MT in the inhibition test. This shows that both oligosaccharides as well as alkali‐stable oligosaccharide linked to glycopeptide MT have determinants of the same structure.
The chemical composition of alkali‐stable oligosaccharides and their pattern of inhibition of precipitation strongly suggest that l‐fucose α1‐linked at the nonreducing end is an immuno‐dominant group and that grouping l‐Fucα1 → DGal →(GlcNAc, Gal, Man) is present in their molecules.
The inhibition of the precipitation by glycopeptide MT is in agreement with the presence of only one alkali‐stable oligosaccharide chain on the glycoprotein M molecule [Tomita, Furthmayr and Marchesi (1978) Biochemistry, 17, 4756 ‐ 4770]. These results as well as the restriction of lectin receptor site to alkali‐stable oligosaccharide, show that glycopeptide MT is monovalent in the reaction with the lectin.
Fluorescence of Cowpea‐Chlorotic‐Mottle Virus Modified with Pyridoxal 5′‐Phosphate
Authors: KRÜS Jacob , VERDUIN Benedictus J. M. , VISSER Antonie J. W. G. 
Published: March 2005

Cowpea chlorotic mottle virus (CCMV), which is stable at pH 5.0, has been modified at this pH with 0.5‐0.7 pyridoxal 5′‐phosphate molecules per protein subunit. The fluorescence properties of the labelled CCMV protein in different aggregation states of the virus provide information about the labelled part of the protein and the changes induced in its environment, when the nucleoprotein particles are swollen or dissociated. Fluorescence excitation and emission spectra indicate the presence of radiationless energy transfer from the aromatic amino acid residues to the label. Comparison of the fluorescence lifetimes of the labelled and the unlabelled protein confirms the existence of energy transfer.The mobility of the labelled part, which can be estimated from the fluorescence polarization of pyridoxal phosphate chromophore, is higher than expected from the dimensions of the virus and the protein subunits. Polarization values and the fluorescence lifetimes depend on the presence of small amounts of NaCl or MgCl2 in the buffer solution at pH 7.5. This is due to structural changes in the vicinity of the pyridoxal phosphate label of the RNA and of the protein part.
Isolation and Characterization of Profilactin and Profilin from Calf Thymus and Brain
Authors: BLIKSTAD Ingrid , SUNDKVIST Ingegerd , ERIKSSON Sigbritt 
Published: March 2005

Profilactin and profilin have been purified from calf thymus and calf brain. Thymus profilactin could be crystallized with a similar technique as described earlier for the spleen protein.
Preparations of profilactin from the three calf tissues spleen, thymus and brain contain a mixture of β actin and γ actin. The ratio β/γ differs between the tissues, but is constant throughout the purification steps.
The properties of profilin isolated from three sources (spleen, thymus and brain) indicate that it is a highly conserved protein.
Yeast Mannosyl Transferases Requiring Dolichyl Phosphate and Dolichyl Phosphate Mannose as Substrate
Authors: BABCZINSKI Peter , HASELBECK Anton , TANNER Widmar 
Published: March 2005

The first mannosyl unit of manno‐oligosaccharides of fungal mannoproteins is transferred in a dolichyl‐phosphate‐dependent reaction sequence to serine/threonine residues of the protein. The two membrane‐bound enzymes catalyzing this transfer in the yeast Saccharomyces cerevisiae have been solubilized by detergents. The enzyme transferring mannose from guanosine diphosphate mannose to dolichyl phosphate has been purified 18‐fold when based on membrane protein and 140‐fold when based on total cell protein. The enzyme transferring mannose from dolichyl phosphate mannose to protein has been purified 48‐fold and 380‐fold, respectively. A HCl‐treated cell‐wall mannoprotein from yeast served as acceptor protein for the second enzyme.The solubilized enzyme catalyzing the formation of dolichyl diphosphate mannose has a Km for guanosine diphosphate mannose of 7 × 10−6 M and is saturated with about 0.15 mM yeast dolichyl phosphate. The metal requirement, pH‐optima, and the detergent concentration necessary for optimal activity have been determined for both solubilized enzymes.
Specificity of Solubilized Yeast Glycosyl Transferases for Polyprenyl Derivatives
Authors: PALAMARCZYK Grazyna , LEHLE Ludwig , MANKOWSKI Tadeusz , CHOJNACKI Tadeusz , TANNER Widmar 
Published: March 2005

Four glycosyl transferases solubilized by detergents from Saccharomyces cerevisiae membranes have been tested for their specificity towards various polyprenols differing in chain length and saturation.

The formation of dolichyl diphosphate‐(N‐acetylglucosamine)1‐2 from uridine diphosphate N‐acetylglucosamine and dolichyl monophosphate showed an obligatory requirement for α‐saturated polyprenols. The apparent affinity for α‐saturated polyprenyl phosphates improves with increasing chain length; those shorter than seven isoprene units were inactive.
The rate of transfer of the chitobiosyl unit from dolichyl diphosphate chitobiose to the hexapeptide Tyr‐Asn‐Leu‐Thr‐Ser‐Val followed the sequence: α‐dihydropolyprenyl (C55) >(C80 > (C100).
The enzyme transferring the mannosyl group from guanosine diphosphate mannose to dolichyl monophosphate also showed a clear dependence on α‐saturation and chain length. In this case the chain length affects mainly the maximal velocity of the reaction. The highest value was obtained with C55α‐dihydropolyprenyl phosphate (100 nmol × h−1× mg protein−1), whereas the value with C35α‐dihydropolyprenyl phosphate was only 8 nmol × h−1× mg protein.−1.
The mannosyl transfer from dolichyl monophosphate mannose to dinitrophenylated tetrapeptide Asn‐Ala‐Thr‐Vol to form on O‐glycosidic bond strongly depended on α‐saturation and chain length of the polyprenyl phosphate. The rate of transfer correlated directly with the length of the polyprenyl residue. With C80α‐dihydropolyprenol less than half the rate of the C100 compound was observed, whereas C55 and C35α‐dihydropolyprenol are only 20 and 10% as active, respectively.
Structural Studies on the Glucose‐Heptose Region of the Proteus mirabilis R Core
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , FEIGE Ulrich , JENSEN Michael , KOTELKO Krystyna , FRIEBOLIN Horst , MAYER Hubert 
Published: March 2005

Methylation analysis of the core oligosaccharide of the Proteus mirabilis mutant R4 (derived from serotype 028 was carried out in order to obtain information on the internal (glucose‐heptose) region of the P. mirabilis R core.The isolated core oligosaccharide was composed of glucose, l‐glycero‐d‐manno‐heptose, 3‐deoxy‐d‐manno‐octulosonic acid (dOclA) and phosphorus in a molar ratio of about 1:2:1:1.4. It was methylated either directly or after dephosphorylation. To localize the position of the phosphate substituents, the permethylated product was dephosphorylated with hydrogen fluoride and the oligosaccharide obtained was remethylated using C2H3I. Location of phosphate at C‐7 of the terminal heptose was shown by isolation of the sugar phosphate from partial hydrolysates and gas‐liquid chromatography/mass spectrometry of the permethylated product.Combining the data of the methylation analysis with the data of an NMR study allows one to formulate the structure of the core oligosaccharide as follows: DGlcp(β1→¾)LZD Hepp(1 →?)dOclA. ↑(1‐ 4/3) LαD Hepp‐7‐p
Acquisition of an Insulin‐Sensitive Activity of Adenosine‐3′,5′‐Monophosphate Phosphodiesterase during Adipose Conversion of 3T3‐L2 Cells
Authors: MURRAY Thomas , RUSSELL Thomas R. 
Published: March 2005

Mouse 3T3‐L2 fibroblasts differentiate when cultured and express a new phenotype which is characteristic of adipose cells. One aspect of this differentiation is the acquisition of hormone sensitivities typical of adipocytes. The sensitivity of adenosine‐3′,5′‐monophosphate (cAMP) phosphodiesterase to brief insulin treatment in 3T3‐L2 cells at various stages of adipose conversion was examined. The capacity to increase the activity of a low‐Km form of the enzyme in response to acute insulin exposure is a property which is acquired as the cells begin to express the adipose phenotype. Enzyme activity in preadipocytes is not affected by insulin, nor is there an effect in the non‐differentiating 3T3‐C2 line under any of the conditions tested. Since insulin receptors and cAMP phosphodiesterase are both present in the preadipocytes, one result of this cytodifferentiation is to effectively couple receptors to the enzyme activity.Insulin‐sensitive cAMP phosphodiesterase in 3T3‐L2 adipocytes is characterized by a 40–50% increase in enzyme activity during insulin treatment when determined at 0.1 μM cAMP. This activation results from an increase in the apparent V of the enzyme and does not involve a change in enzyme‐substrate affinity. Maximal stimulation is seen within 2–5 min and is sustained for at least 45 min when high levels of insulin (850 nM) are used. Lower concentrations of insulin (1.7 nM) also bring about rapid activation, although the activation is not completely sustained during longer incubations. Stimulated activity falls off to about 60% of peak values by 30 min. Re‐addition of insulin after 30 min raises enzyme activity back to the maximal level. Further, the insulin response is completely reversible in that the insulin‐sensitive cAMP phosphodiesterase activity disappears within 10 min after removal of insulin from the cultures.
Pancreatic Ribonucleases of Mammals with Ruminant‐Like Digestion
Authors: HAVINGA Johan , BEINTEMA Jaap J. 
Published: March 2005

High levels of pancreatic ribonucleases are found in ruminants, species that have a ruminant‐like digestion and several species with coecal digestion. Pancreatic ribonucleases from several independently evolved species with ruminant‐like digestion were investigated to test a hypothesis that glycosylation of ribonucleases may have some function in species with coecal digestion and that glycosylation of the enzyme may not be advantageous for ruminants. Ribonucleases from the hippopotamus, two‐toed sloth and three‐toed sloth were isolated by extraction with sulfuric acid and affinity chromatography. Complete amino acid sequences were determined for the ribonucleases from the hippopotamus and two‐toed sloth and a partial sequence for the enzyme from the three‐toed sloth. The amino acids 75–78 of hippopotamus ribonuclease were positioned by homology with other artiodactyl ribonucleases.In hippopotamus ribonuclease a heterogeneity was found at position 37, half of the molecules containing glutamine and the other half lysine. Hippopotamus ribonuclease differs less from pig and bovine ribonuclease than these differ from each other, because more ancestral characteristics have been retained. Although hippopotamus ribonuclease contains all four Asn‐X‐Ser/Thr sequences previously found to be glycosylation sites in one or more pancreatic ribonucleases, only the sequence Asn‐Met‐Thr (34–36) is glycosylated in the variant with glutamine at position 37, while the variant with lysine at this position is carbohydrate‐free.Both sloth ribonucleases are completely glycosylated at the sequence Asn‐Met‐Thr (34–36) with a simple type of carbohydrate chain. The amino acid sequence of two‐toed sloth ribonuclease shows some interesting coupled replacements.