bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Triamcinolone on Renal and Hepatic Phosphoenolpyruvate Carboxykinase in the Newborn Rat
Authors: MENCHER David , RESHEF Lea 
Published: March 2005

The effects of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase activity in the developing rat were investigated. The hormone induced increases in pre‐existing enzyme activity of both tissues in fetal and neonatal rats, yet did not cause the primary appearance of phosphoenolpyruvate carboxykinase activity in utero.Neonatal hepatic phosphoenolpyruvate carboxykinase activity was increased 2–3‐fold by triamcinolone from the 3rd to the 15th postnatal day. This was shown to be additive to the effect of Bt2cAMP on enzyme activity. The increases in phosphoenolpyruvate carboxykinase activity were demonstrated to be due to increased synthesis of the enzyme, which was accompanied by a proportionate increase in the amount of functional phosphoenolpyruvate carboxykinase mRNA, as measured by the polyribosomal and poly(A)‐containing RNA directed cell‐free synthesis of the enzyme.The demonstration of a triamcinolone effect on kidney and liver phosphoenolpyruvate carboxykinase activity in fetal and neonatal rats provides support for a possible role of glucocorticoids in the regulation of phosphoenolpyruvate carboxykinase activity during development.
Intrinsic Fluorescene of Cowpea‐Chlorotic‐Mottle‐Virus Protein
Authors: KRÜSE Jacob , VERDUIN Benedictus J. M. , VISSER Antonie J. W. G. 
Published: March 2005

Changes in the environment of the aromatic amino acid residues in the protein subunits of cowpea chlorotic mottle virus were studied with fluorescence techniques. The fluorescence properties of the protein in nucleoprotein particles, empty protein shells and dimers of the coat protein subunit were determined.According to fluorescence spectra (excitation at 295 nm, emission maxima at 342–346 nm depending on pH and ionic strength) the tryptophan residues are located in a polar environment. With excitation at 235 nm a strong shoulder at about 300 nm in the total emission spectrum can be observed. This emission must be attributed to the tyrosine residues in the protein and can also be observed as a fast component in the fluorescence decay curve. The lifetimes were compared with those of the other bromoviruses which are different in amino acid composition. From excitation spectra of cowpea chlorotic mottle virus it can be concluded that energy is transferred from tyrosine to tryptophan residues.Increase in the quantum yield of the tryptophan emission of the nucleoprotein particles upon raising the ionic strength and pH are attributed to a diminishing interaction between the protein and the RNA. The fluorescence properties of the protein hardly change when the empty protein shells are dissociated into protein dimers.Quenching studies with potassium iodide and acrylamide indicate that the tryptophan residues in the protein are attainable more easily at low ionic strength than at high ionic strength.
A Method for Designing Peptide Substrates for Proteases
Authors: POZSGAY Marianne , ELÖDI Pál , GÁSPÁR Rezsö , BAJUSZ Sándor 
Published: March 2005

The kinetic parameters of 25 peptidyl‐p‐nitroanilide substrates were investigated with subtilisin Carlsberg as model enzyme.
For a series of 12 substrates, the contribution of various side chains to the affinity constant was computed by regression analysis. From these contributions the sequence of a new and better substrate, N‐benzyloxycarbonyl‐arginyl‐norleucyl‐norleucyl‐p‐nitroanilide (Z‐Arg‐Nle‐Nle‐Nan) was predicted. The compound was synthesized and assayed. Its calculated 1/Km value, 43.5 mM−1, was in a good agreement with the value of 40.0 mM−1 that was determined experimentally.
On expanding the series to 19 substrates, it was found that the productivity of enzyme‐substrate binding is influenced primarily by those subsites which have a significantly greater contribution to the affinity constants than others.
The additivity principle applied reasonably well for the contribution of individual side chains to the kinetic parameters. This fact suggests that regression analysis can be used for the prediction of the amino acid sequence of better substrates than those already tested, probably not only for subtilisin but also for other proteolytic enzymes.
Glutathione S‐Conjugate Formation from 1‐Chloro‐2,4‐dinitrobenzene and Biliary S‐Conjugate Excretion in the Perfused Rat Liver
Authors: WAHLLÄNDER Axel , SIES Helmut 
Published: March 2005

The isolated hemoglobin‐free perfused rat liver was used as a model to study the formation of a glutathione S‐conjugate and its disposition into the biliary as well as other extracellular (caval perfusate) compartments. 1‐Chloro‐2,4‐dinitrobenzene was chosen as substrate because it reacts with the glutathione‐S‐transferases at high activity to give rise to an S‐conjugate, S‐(2,4‐di‐nitrophenyl)‐glutathione, a product readily discernible from the parent compound.
S‐(2,4‐Dinitrophenyl)‐glutathione formation rates were equal to the infusion rates of 1‐chloro‐2,4‐dinitrobenzene up to 0.3 μmol × min−1× g liver−1. Steady‐state rates of S‐conjugate release were maintained until the glutathione content of the liver had decreased to about 40% of the control value.
Biliary excretion of the S‐conjugate was practically quantitative at low rates of S‐conjugate formation, up to about 30 nmol × min−1× g liver−1. and release of S‐conjugate into the caval perfusate gradually increased with the rate of formation. Maximal biliary S‐conjugate concentration was 36 mM.
Choleresis associated with biliary S‐conjugate excretion amounted to 18.6 pi of extra bile per μmol of net S‐conjugate excreted. This would correspond to a limiting concentration of 54 mM S‐conjugate in the water compartment associated with biliary transport, e.g. exocytotic vesicle in the context of an exocytosis‐like mechanism of bile formation.
The system described here is suited for further investigation of properties of bile formation, as is illustrated by the sensitive detection of changes in the concentration of S‐conjugate in the perfusate upon Ca2+‐depletion.
Thermal Responsiveness of 3‐Hydroxy‐3‐methylglutaryl‐Coenzyme‐A Reductase and Acetyl‐Coenzyme‐A Carboxylase in Neoplastic Guinea Pig Lymphocytes (L2C)
Authors: PHILIPPOT Jean R. , WALLACH Donald F. H. 
Published: March 2005

Hydroxymethylglutaryl‐CoA reductase and acetyl‐CoA carboxylase, rate‐limiting enzymes of cholesterol and fatty acid biosynthesis, respectively, do not show equivalent thermal responsiveness in leukemic L2C guinea‐pig lymphocytes. Both enzymes exhibit Arrhenius plots characteristic of membrane‐association but hydroxymethylglutaryl‐CoA reductase yields a single slope, compared with three slopes, crossing at 24°C and 12°C for acetyl‐CoA carboxylase. Preincubation with phosphatidylcholine liposomes before enzyme assay does not modify the thermal behavior of acetyl‐CoA carboxylase but introduces a discontinuity at 24 C in the Arrhenius diagramin of hydroxymethylglutaryl‐CoA reductase, conferring to this enzyme an activation energy close to that of normal cells.The Arrhenius plot for fatty acid biosynthesis in L2C cells parallels the thermotropism of the acetyl‐CoA carboxylase, with two discontinuities at 25 and 15 °C. However, cholesterol biosynthesis shows a discontinuity at 24.6 °C, whereas hydroxymethylglutaryl‐CoA reductase activity does not.The different thermal behavior of hydroxymethylglutaryl‐CoA reductase and acetyl‐CoA carboxylase is discussed in terms of lipid heterogeneities in membrane enzyme environments, i.e. enzyme localization in dissimilar lipid domains. We propose several hypotheses to account for the lack of correlation between the thermotropic responses of hydroxymethylglutaryl‐CoA reductase activity and of cholesterol biosynthesis in L2C cells.
Anticooperative Binding of l‐Tryptophan to Tryptophanyl‐tRNA Synthetase from Beef Pancreas
Authors: GRAVES Pierre‐Vincent , MAZAT Jean‐Pierre , JUGUELIN Héléne , LABOUESSE Julie , LABOUESSE Bernard 
Published: March 2005

Equilibrium dialysis and gel filtration studies show that tryptophanyl‐tRNA synthetase from beef pancreas binds two molecules of l‐tryptophan per dimer in an anticooperative way.The binding of tryptophan ellicits a series of spectroscopic changes in the protein as seen by absorbance, fluorescence and circular dichroism. The molar absorption change of the protein‐tryp‐ tophan system upon formation of the complex is Δɛ292= 10400 ± 1000 M−1 cm−1 per dimer. Taking an initial symmetrical dimeric protein the two dissociation constants for tryptophan at pH 8, 25 C are respectively K1= 2.0 ± 0.5 μM and K2= 10 ± 4 μM. They are respectively K1= 1 ± 0.25 μM and K2= 20 ± 8 μM if one considers a sequenced binding of the two tryptophan molecules.The dichroic band at 290 nm of the free protein disappears when tryptophan is bound. All observed changes are characteristic of tryptophan perturbation and none of tyrosine perturbation. They all exceed the effect that can be expected from the change in environment of the bound tryptophan molecules and modifications of the tertiary structure of the protein have to be taken into account to explain the observed spectroscopic data.
Deazaflavins as Cofactors for Flavocytochrome b2 from Baker's Yeast
Authors: POMPON Denis , LEDERER Florence 
Published: March 2005

Flavin‐free cytochrome b2 can be reconstituted with 5‐deaza‐5‐carba‐flavin mononucleotide (5‐deaza‐FMN). The resultant enzyme is reduced by lactate and reoxidized by pyruvate, but electrons are not transferred to the heme and no ferricyanide reductase activity can be measured. Tritium transfer experiments from labeled substrate to analog have yielded an isotope discrimination factor of 12. Back transfer (from enzymatically labeled analog to pyruvate) is essentially complete. The affinity of 5‐deaza‐FMN for flavin‐free cytochrome b2 has been found to be similar to that of FMN. Its redox potential is −230 mV. Sulfite forms a reversible covalent adduct with the analog (Kd= 2.8 μM) as it does with the normal cofactor. The reactivity of 2‐hydroxy‐3‐butynoic and 2‐oxo‐3‐butynoic acids toward bound 5‐deaza‐FMN has also been examined; no evidence could be obtained for covalent adduct formation. Difference spectra showed the appearance of a band centered at 423 nm upon binding of oxidized 5‐deaza‐FMN; this band was attributed to an interaction between heme and the analog; it is not observed with the natural cofactor.In contrast, no evidence was obtained for binding of 1‐deaza‐FMN to flavin‐free cytochrome b2.A comparison is presented between known data concerning flavocytochrome b2 and lactate oxidase from Mycobactevium smegmatis; they point to a highly similar environment around N‐5, C‐4a and O‐4 of the isoalloxazine ring in the two proteins. It is suggested that the difference between flavin‐dependent dehydrogenases/electron transferases and dehydrogenases/oxidases might be the interaction between flavin and protein in the N‐1‐O‐2 area.
Purification and Characterization of Proinsulin mRNA from Rat B‐Cell Tumor
Authors: ITOH Nobuyuki , NOSE Kiyoshi , OKAMOTO Hiroshi 
Published: March 2005

Proinsulin mRNA was purified from rat B‐cell tumor induced by streptozotocin and nicotinamide. The purification was achieved through the use of oligo(dT)‐cellulose chromatography and sucrose gradient centrifugation. As judged by translational activity in a cell‐free protein‐synthesizing system of wheat germ, proinsulin mRNA was purified 362‐fold from total nucleic acid of the B‐cell tumor. Purified proinsulin mRNA migrated as a single symmetrical peak on sucrose gradient centrifugation and as a single band on polyacrylamide gel electrophoresis in the presence of formamide. The translation product of purified proinsulin mRNA in the cell‐free protein‐synthesizing system was found to be only preproinsulin, as analyzed by dodecylsulfate/polyacrylamide gel electrophoresis and immunoprecipitation.The molecular weight of proinsulin mRNA, determined by polyacrylamide gel electrophoresis in the presence of formamide, was approximately 200000 (equivalent to approximately 570 nucleotides). Almost all of the proinsulin mRNA was bound to oligo(dT)‐cellulose, and the translation of proinsulin mRNA in a cell‐free protein‐synthesizing system of wheat germ was completely inhibited by 7‐methylguanosine 5′‐phosphate. These results indicated that the nucleotide sequences in proinsulin mRNA consisted of 58% translatable sequences and 42 % untranslatable sequences, presumably containing a capped structure, a 7‐methylguanosine residue, at the 5′ terminus and a poly(A) sequence at the 3′ terminus.The translation product of purified proinsulin mRNA, preproinsulin, was found to consist of two types of preproinsulin I (57%) and preproinsulin II (43%). This result indicated that the isolated proinsulin mRNA consisted of two types of proinsulin I mRNA and proinsulin II mRNA in approximately equal amounts.Complementary DNA was synthesized with proinsulin mRNA as a template. From the experiment of hybridization kinetics under conditions of excess RNA, the r0t1/2, of proinsulin mRNA was found to be 2.1 × 10−4 mol 1−1 s. This result also confirmed that proinsulin mRNA had been isolated in a high degree of purity.
1H‐Nuclear‐Magnetic‐Resonance Studies on Glycophorin and Its Carbohydrate‐Containing Tryptic Peptides
Authors: EGMOND Maarten R. , WILLIAMS Robert J. P. , WELSH E. Jane , REES David A. 
Published: March 2005

The proton nuclear magnetic resonance (1H‐NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of α‐helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met‐81 residue in glycophorin.Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C‐3 to C‐11 methylene resonances. The bound dodecyl‐sulphate probably increases the mobilities of the hydrophobic residues in the protein as these resonance intensities are increased by the binding. The carbohydrate chains in glycophorin were conformationally mobile; no evidence was found for tight carbohydrate‐protein interactions. The relevance of flexible carbohydrate chains in membrane glycoproteins is discussed in relation to cell surface chemistry.
Purification and Some Properties of a Hitherto‐Unknown Enzyme Reducing the Carbon‐Carbon Double Bond of α,β‐Unsaturated Carboxylate Anions
Authors: TISCHER Wilhelm , BADER Johann , SIMON Helmut 
Published: March 2005

2‐Enoate‐reductase, a previously unknown soluble enzyme is present in Clostridium kluyveri and another Clostridium species growing on (E)‐2‐butenoate. From the latter the reductase was purified 88‐fold with an overall yield up to 74%. The enzyme was pure as judged by polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate as well as by isoelectric focusing.The purification of the enzyme was performed in the presence of (E)‐2‐methyl‐2‐butenoate as substrate to keep the enzyme in the oxidized state and under anaerobic conditions. The purification procedure included an ammonium sulphate precipitation, chromatography on DEAE‐Sepharose CL‐6B, hydroxylapatite and Sepharose CL‐6B.The enzyme reduces different α,β‐unsaturated carboxylate anions such as (E)‐2‐butenoate, (E)‐2‐methyl‐2‐butenoate, (E)‐cinnamate and probably many others in a NADH‐dependent reaction to the saturated carboxylate anions. Fumarate, 3‐phenyl‐2‐propinate, 2‐enoyl‐methyl and CoA esters proved not to be substrates for the purified reductase. NADPH does not act as an electron donor.The enzyme was shown to have a molecular weight of about 450000 by gel chromatography. It consists of subunits with a molecular weight of 78000. Per subunit about 1 FAD, 3.5–3.8 atoms of iron and 4.0 labile sulphur atoms have been found indicating a conjugated iron‐sulphur flavoprotein. Copper could not be detected. The isoelectric point was 8.4.As shown by absorption spectroscopy the enzyme can be reduced by NADH and reoxidized with dichloroindophenol, hexacyanoferrate III, oxygen and substrates.Addition of 8 mol p‐hydroxymercuribenzoate to 1 mol subunit completely destroyed the activity of the reductase.So far no physiological role of the enzyme is known.
External and Internal Forms of Yeast Aminopeptidase II
Authors: FREY Jürgen , RÖHM Klaus‐Heinrich 
Published: March 2005

Intact cells of Saccharomyces cerevisiae catalyze the hydrolysis of various aminopeptidase substrates. This activity is not due to permeation of substrates and products but exerted by an external enzyme.
From its substrate specificity and the effects of pH and inhibitors the enzyme was identified as aminopeptidase II.
About 40% of total aminopeptidase II activity is detectable with untreated exponentially growing cells. Up to two thirds of the external enzyme is released into the medium during enzymic digestion of the cell wall, while little enzyme is liberated by osmotic shock. Membrane preparations contained only small amounts of aminopeptidase II; thus, the localization of the external enzyme appears to be similar to that of the so‐called ‘periplasmic’ yeast hydrolases.
By cytochemical methods the presence of aminopeptidase II in the cell envelope was visualized.
In contrast to aminopeptidase II, yeast dipeptidase is an entirely intracellular enzyme.
Effect of Sulphated Glycopeptides on Kinetics of 3‐OMethyl Glucose and 2‐Deoxyglucose Transport by Epithelial Cells Isolated from Rabbit Small Intestine
Authors: MIAN Nasi , ANDERSON Caroline E. , KENT Paul W. 
Published: March 2005

Suspensions of cells isolated from rabbit small intestine were prepared which showed a considerable rate of respiration after 4 h of their isolation. Rabbit intestinal cells accumulated 3‐O‐methyl‐glucose by an active transport mechanism which was Na+‐dependent whereas 2‐deoxyglucose was transported by a saturable pathway. Analysis of the initial transport data indicated that the uptake of both sugars by cells followed Michaelis‐Menten‐type kinetics. These cells also showed a considerable degree of 2‐deoxyglucose phosphorylation and the transport of the sugar into the cells appeared to be a rate‐limiting factor for its phosphorylation.Sulphated glycopeptide had no effect on the respiration rate of these cells but it increased the transport of 3‐O‐methyl‐glucose and 2‐deoxyglucose into cells by many fold compared with untreated cells. Kinetic analysis of the data indicated that the increase in the transport was due to increased V while the Km remained unchanged. Presence of sulphated glycopeptide had no effect on the kinetics of phosphorylation of 2‐deoxyglucose. These effects of sulphated glycopeptide on the transport activity of the cells have been discussed as a result of an increase in the number of sugar sites in the cell membrane due to unmasking or activation of preexisting sites owing to the interaction between the cell membrane components and highly polyanionic sulphated glycopeptides.
N‐Acetylaminoacyl‐p‐Nitranilidase from Human Placenta
Authors: UNGER Thomas , NAGELSCHMIDT Manfred , STRUCK Hansjürgen 
Published: March 2005

An enzyme hydrolyzing N‐acetylaminoacyl‐p‐nitroanilides has been isolated from mature human placentae by a six‐step procedure comprising extraction from a placenta homogenate, ammonium sulfate fractionation, treatment with isopentyl alcohol, chromatography on CM‐Sephadex, protamine sulfate precipitation and gel filtration on an Ultrogel AcA 34 column. About 2500‐fold enrichment was achieved from placenta homogenate. The purified enzyme preparation showed a single band on polyacrylamide disc electrophoresis. The molecular weight was estimated to be 380000 by gel filtration. Placental extracts contain two main isoenzymes of pI 3.9 and 4.5 respectively.Activity was strongly inhibited by chloromercuribenzoate, slightly inhibited by Ca2+ and cysteine; no activation could be detected. The enzyme exhibits an exopeptidase‐like activity towards acetyl‐dipeptides with a certain specifity towards N‐acetylalanyl‐alanine; N‐acetylalanine‐p‐nitroanilide, however, is hydrolyzed four times faster.With N‐acetylalanine‐p‐nitroanilide as substrate the pH optimum was 8.0–8.2; Km was 2.13 mmol/l. N‐Acetylleucine‐p‐nitroanilide and N‐acetyltyrosine‐p‐nitroanilide were split slowly; N‐acetylalanyl‐alanyl‐alanine‐p‐nitroanilide, N‐butyloxycarbonyl‐alanyl‐alanine‐p‐nitroanilide, un‐substituted analogous aminoacyl‐p‐nitroanilides and several protein substrates were not hydrolyzed.The functions of the enzyme are still unknown.
Ionized and Bound Calcium Inside Isolated Sarcoplasmic Reticulum of Skeletal Muscle and Its Significance in Phosphorylation of Adenosine Triphosphatase by Orthophosphate
Authors: PRAGER Rudolf , PUNZENGRUBER Christian , KOLASSA Norbert , WINKLER Franz , SUKO Josef 
Published: March 2005

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5–15 mM) on ice gives calcium loads of 50–60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis‐(2‐aminoethyl)‐N, N, N′, N′‐tetraacetic acid], but almost completely released by ionophore X‐537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non‐saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half‐saturation constant of 5.8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 μl/mg protein respectively.Calcium‐dependent phosphorylation of sarcoplasmic reticulum calcium‐transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium‐dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively.It is further suggested that the saturation of the low‐affinity calcium‐binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium‐dependent phosphorylation than in calcium‐independent phosphorylation from orthophosphate. Maximum calcium‐dependent phosphoprotein formation at 20°C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.