bluuebunny/crossref_metadata_2025_split huggingface dataset:
Carboxylesterases in Rat and Human Sera and Their Relationship to Serum Aryl Acylamidases and Cholinesterases
Authors: TSUJITA Takahiro , OKUDA Hiromichi 
Published: March 2005

The three enzyme activites, carboxylesterase, aryl acylamidase and cholinesterase activities, have been found in rat and human sera. Rat serum carboxylesterase associated with serum aryl acylamidase activity, but not with serum cholinesterase activity, was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE‐cellulose, blue Sepharose and QAE‐Sephadex, and then electrophoresis. Evidence for the identity of the two enzymes, carboxylesterase and aryl acylamidase, was their co‐elution profiles and co‐purification in the different steps, including electrophoresis, with constant ratios of specific activities and percentage recoveries.Human serum carboxylesterase associated with serum cholinesterase, purified earlier, was compared with the rat serum esterase. Human serum carboxylesterase and aryl acylamidase activities were inhibited by serotonin and neostigmine, whereas rat serum carboxylesterase and aryl acylamidase activities were not affected by these compounds. Tyramine activated human but not rat aryl acylamidase. Rat and human serum esterase activities were both strongly inhibited by the diisopropylfluorophosphate. Both esterases catalyzed the hydrolysis of short‐chain triacylglycerols, such as tributyrin, and medium‐chain monoacylglycerols, such as monocaprin, but not the hydrolysis of long‐chain triacylglycerols.
Localization of Ellipticine Derivatives Interacting with Membranes
Authors: TERCE François , TOCANNE Jean‐François , LANEELLE Gilbert 
Published: March 2005

The interaction with membranes of three anti‐cancer drugs of the ellipticine family was studied by fluo‐ rescence quenching of membrane probes. The fluorescence of three probes, located at different levels in mem‐ branes, was quenched by addition of two types of ellipticine derivatives, one amphiphilic drug (9‐methoxy‐ ellipticine) and two dipolar molecules (9‐aminoellipticine and 9‐hydroxyellipticine). By comparing the quenching curves obtained, the following can be proposed.a) 9‐Methoxyellipticine can penetrate deeper in the lipid layers than 9‐aminoellipticine and 9‐hydroxy‐ ellipticine can.b) The three drugs are able to penetrate at least between the first methylene groups of the acyl chains of lipids in liposomes.c) In an isolated bacterial membrane, only 9‐methoxyellipticine can be located in the region of the first methylene groups of lipids, the two dipolar drugs being adsorbed on the membrane surface.It was also shown that cholesterol hindered the penetration of 9‐methoxyellipticine in the bilayer of liposomes.
Detection and Regulation of the mRNA for the Inhibitor of Extracellular cAMP Phosphodiesterase of Dictyostellum discoideum
Authors: ROSSIER Claude , FRANKE Jak ob , MULLENS Isabel A. , KELLEY Kenneth J. , KESSIN Richard H. 
Published: March 2005

The inhibitor of the cAMP phosphodiesterase of Dictyostelium discoideum is a cysteine‐rich glycoprotein, which binds to the enzyme and inactivates it. When the inhibitor is removed, enzymatic activity is restored. Following translation in vitro of RNA from developing cells and immunoprecipitation with anti‐inhibitor serum, newly synthesized inhibitor can be detected by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography. The inhibitor can be labeled using [35S]cysteine but not [35S]methionine, in agreement with the previously determined amino acid composition, and can be detected after cell‐free translation only if it has been previously acetylated. Purified native inhibitor blocks immunoprecipitation of the inhibotor polypeptide synthesized in vitro.No inhibitor mRNA was detected in growing cells. Translatable mRNA was present 2 h after the beginning of starvation, reached a maximal level after 3 h, and decreased thereafter. Addition of 1 mM cAMP at the beginning of starvation delayed the appearance of translatable inhibitor mRNA. In the presence of 5 μM adenosine cyclic‐3′,5′‐phosphorothioate, a slowly hydrolyzed cAMP analogue, no translatable mRNA could be detected. Following removal of the analogue, the mRNA appeared within one hour and inhibitor was secreted after another hour.
Conformational Changes of Apigenin 7‐O‐(6‐O‐malonylglucoside), a Vacuolar Pigment from Parsley, with Solvent Composition and Proton Concentration
Authors: MATERN Ulrich , HELLER Werner , HIMMELSPACH Karl 
Published: March 2005

Malonylated apigenin 7‐O‐glucoside was prepared from malonyl‐CoA and apigenin 7‐O‐glucoside using a malonyltransferase from parsley cell cultures. The enzyme product, as well as the flavonoid substrate, was analyzed by high‐resolution nuclear magnetic resonance spectroscopy, confirming that malonic acid had been attached to the primary hydroxyl of the glucose moiety of apigenin 7‐O‐glucoside. During these studies in several solvents, it became apparent that, in particular, the chemical shift of H‐6” A and the coupling constants J5,“6”A and J5,“6”B were dependent on solvent composition and proton concentration. Similar changes of sugar proton resonance frequencies were also observed with methyl 6‐O‐malonyl‐β‐d‐glucopyranoside, but not with apigenin 7‐O‐[6‐O‐(4‐coumaroyl)glucoside]. The change of coupling constants is ascribed to a conformational modification of the sugar portion in the malonylglucosides.Malonylated flavonoid glycosides are exclusively located within the vacuoles of parsley cells. We propose that acylation of flavonoid glycosides which are synthesized in the cytoplasm facilitates transport of the substrates into the vacuole. The conformational modification which is induced by changes in proton concentration may provide a mechanism to trap flavonoids in situ.
Biosynthesis of the Major Glycoprotein Associated with Zymogen‐Granule Membranes in the Pancreas
Authors: HAVINGA Johan R. , STROUS Ger J. A. M. , POORT Cees 
Published: March 2005

The biosynthesis of GP‐2, the major glycoprotein associated with zymogen‐granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse‐chase experiments, using [35S]methionine, were performed and the processing of GP‐2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis.GP‐2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr= 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr= 78000–80000). Only the precursor form of GP‐2 is sensitive to digestion with the glycosidase endo‐β‐N‐acetylglucosaminidase H, indicating that the observed conversion reflects the processing of ‘high‐mannose’ oligosaccharides into complex type oligosaccharides.Acinar cells cultured in the presence of increasing concentrations of the N‐glycosylation inhibitor tunicamycin synthesize 5–6 distinct precursor GP‐2 species with apparent molecular weights decreasing from 73000–61000. We conclude that GP‐2 contains five or six N‐linked carbohydrate chains.From cell fractionation studies it was established that the precursor GP‐2 is present in a microsomal fraction with high density (> 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP‐2 is localized in low density microsomes (< 1.130 g/ml) probably Golgi vesicles. The GP‐2 in zymogen granule membranes is also in the mature form.
Revision of Enzyme Nomenclature
Authors: Dixon (Chairman) H. B. F. , Cornish‐Bowden (Secretary) A. 
Published: March 2005

A new edition of Enzyme Nomenclature is being prepared. During preparation it has become clear that some of the references in the present (1978) edition and its four supplements [Eur. J. Biochem. 104, 1–4 (1980); 116, 423–435 (1981); 125, 1–13 (1982); 131, 461–472 (1983)] are incorrect. Unfortunately, it is impracticable to check all 4000 references to ensure that each does indeed refer to the enzyme indicated. Nonetheless, most of the errors are probably known to biochemists interested in the particular enzymes in whose listing the errors occur, and it would also be relatively easy for individual biochemists to check whether the entries for the enzymes of which they have specialist knowledge are correct. II you know a any errors, please write to Professor Edwin C. Webb. Vice‐Chancellor, Macquarie University, North Ryde, New South Wales, Australia 2113.Although we are primarily concerned with actual errors, we would like to repeat the standing invitation for suggestions of new entries and improvements for old ones, including the citation of more appropriate references. We should be most grateful for all such help.
The Pyruvate Dehydrogenase Complex of Escherichia coli K12
Authors: STEPHENS Paul E. , DARLISON Mark G. , LEWIS Hilary M. , GUEST John R. 
Published: March 2005

The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined using the dideoxy chain‐termination method. The aceF gene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho‐independent terminator. The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (aceE) of the pyruvate dehydrogenase component (E1) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon.The amino terminus, carboxy‐terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein. The predicted molecular weight (Mr= 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78 000–89 000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration. The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains: the lipoyl domain and the subunit binding and catalytic domain. The lipoyl domain corresponds to the amino‐terminal segment of the protein. It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues. The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy‐terminal segment.
Kinetic Studies of Aminoglycoside Acetyltransferase and Phosphotransferase from Staphylococcus aureus RPAL
Authors: MARTEL Annie , MASSON Maryse , MOREAU Nicole , Le GOFFIC François 
Published: March 2005

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6′‐N‐aminoglycoside acetyltransferase and a 2”‐O‐aminoglycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography.The kinetic mechanisms of each activity have been determinated from studies of initial velocities, as well as product and dead‐end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed.However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.
Improved Preparation of Lipoteichoic Acids
Authors: FISCHER Werner , KOCH H. Uwe , HAAS Renate 
Published: March 2005

A procedure is described for measuring the extraction of lipoteichoic acids from gram‐positive bacteria in absolute terms. Virtually complete extraction was achieved from various bacteria by hot phenol/water if the cells were disrupted. Extraction of whole and delipidated cells and of the membrane fraction gave considerably lower yields.Most of the nucleic acids co‐extracted from disrupted cells was removed by treatment with nucleases. Nuclease‐resistant nucleic acid, protein, polysaccharide, and teichoic acid were separated from lipoteichoic acid by anion‐exchange chromatography on DEAE‐Sephacel or hydrophobic interaction chromatography on octyl‐Sepharose. Purified preparations were essentially free of polymeric contaminants, retained their alanine ester substitution, and were in the sodium salt form.Hydrophobic interaction chromatography also made it possible to recognize contamination of lipoteichoic acid with its deacylated and Iyso‐form, and to discriminate molecular species containing two and three, or two and four acyl groups.
Characterization of Benextramine as an Irreversible α‐Adrenergic Blocker and as a Blocker of Potassium‐Activated Calcium Channels
Authors: PLOTEK Yael , ATLAS Daphne 
Published: March 2005

An irreversible α‐adrenergic blocker, benextramine [N,N′‐bis(o‐methoxybenzylamine‐n‐hexyl)‐cysteamine] was used as a probe to study the possible interrelationship between α‐adrenoceptors and the K+‐activated Ca2+‐channels.Benextramine, a tetraamine disulfide, acts irreversibly both on the α1‐adrenoceptor (t1/2= 3 min) and the α2‐adrenoceptors. These studies were carried out on rat brain synaptosomes, [3H]prazosin and [3H]clonidine binding. Benextramine blocked Ca2+ influx in rat brain synaptosomes under both depolarizing (75 mM KCl) and normal conditions (5 mM KCl). Its action at the channel is reversible with IC50= 10 ± 5 μM of the net Ca2+ influex. This makes benextramine a most potent Ca2+ blocker compared to verapamil or nicardipine (IC50= 200 μM and 170 μ, respectively).Pretreatment of rat brain slices with benextramine gave a synaptosomal preparation which was devoid of either α1‐adrenergic or α2‐adrenergic binding capacity due to the irreversible binding of benextramine, but with an undistrubed Ca2+ influx. Thus, these results suggest that the α‐adrenoceptors and the Ca2+‐channels are independent of each other, and that full occupancy of the α‐receptors does not affect the net calcium flux.
The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes
Authors: ANDUS Tilo , GROSS Volker , TRAN‐THI Thuy‐Anh , SCHREIBER Gerhard , NAGASHIMA Mariko , HEINRICH Peter C. 
Published: March 2005

The biosynthesis and secretion of α2‐macroglobulin, transferrin, α1‐acid glycoprotein and α1‐proteinase inhibitor were studied in rat hepatocyte primary cultures. After labelling with [35S]methionine, two forms, which can be separated electrophoretically differing by molecular weight, were found for each of the four glycoproteins. The following molecular weights were estimated for the intracellular precursors and the secreted forms: α2‐macroglobulin, 176 000 and 182 000; transferrin, 84 000 and 86 000; α1‐acid glycoprotein, 39 000 and 43 000–60 000; α1‐proteinase inhibitor, 49 000 and 54 000.Carbohydrate moieties could be removed from intracellular forms by treatment with endoglucosaminidase H indicating that their oligosaccharide chains were of the high‐mannose type. The extracellular forms were sensitive to sialidase. They incorporated [3H]galactose and [3H]fucose showing that their oligosaccharide chains were of the complex type.Pulse‐chase experiments revealed a precursor‐product relationship for the high‐mannose and the complex type glycoproteins. In the hepatocyte medium newly synthesized albumin was detected after 30 min and newly synthesized glycoproteins after 60 min.Unglycosylated α2‐macroglobulin (162 000), transferrin (79 000), α1‐acid glycoprotein (23 000), and α‐proteinase inhibitor (41 000) were found in the cells as well as in the medium, when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked reduction of the secretion of α2‐macroglobulin, α1‐acid glycoprotein and α1‐proteinase inhibitor, whereas the secretion of transferrin was less affected.
A Simple Model for Studies on the Regulation of Cholesterol Synthesis Using Freshly Isolated Hepatocytes
Authors: CIGHETTI Giuliana , GALLI Giovanni , KIENLE Marzia GALLI 
Published: March 2005

Rat hepatocytes isolated by the procedure described here showed 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity in the range of that reported for rat liver at the maximum of the circadian cycle, even if they were taken from rats at the time of the minimum.The enzyme was present in cells as both its active dephosphorylated (20 ± 8%) and the inactive phosphorylated forms. The enzyme activity and the ratio between the two forms were unaltered during 3 h of cell incubation.25‐Hydroxycholesterol (50 μM) induced about 50% inhibition of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity during 1 h incubation but the relative amount of the two forms was not modified by the sterol.Cells isolated by the described procedure may therefore be a useful tool in studies on the regulation of cholesterol neogensis, both through the synthesis of the enzyme, which can be shown by measuring the activity after complete dephosphorylation of the enzyme, and via the rapid reversible shift of the inactive to the active form, resulting from the ratio between the two enzyme forms. The latter mechanism for the modulation of cholesterol synthesis cannot be tested in cell cultures because full activation of the enzyme occurs during hepatocyte plating.
Some Kinetic Properties of 4‐Hydroxyphenylpyruvate Dioxygenase from Pseudomonas sp. Strain P.J.874
Authors: RUNDGREN Marianne 
Published: March 2005

Steady‐state kinetic properties of purified 4‐hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P.J. 874 were examined by a 14CO2 method at pH 7.5 and 377deg;C. The results conform to a mono‐iso‐ordered bi‐bi mechanism with binding of 4‐hydroxyphenylpyruvate before O2 and release of CO2 before homogentisate. A Theorell‐Chance mechanism can not be excluded.The apparent DV and DV/Km ratios were about 1.1 and 1.2, respectively, for 4‐hydroxy[2,6‐2H2]phenylpyruvate when the initial O2 consumption was measured. The stoichiometry between the consumption of O2 and the formation of homogentisate and CO2 was 1:1:1: Loss of hydrogen at C2 or C6 of the ring of the substrate is thus not the rate‐limiting step during the reaction. Instead, this appears to be the conversion of an isomerized enzyme form.
Hysteretic Behaviour of Citrate Synthase. Alternating Sites during the Catalytic Cycle
Authors: LÖHLEIN‐WERHAHN Gerlinde , BAYER Ernst , BAUER Barbara , EGGERER Hermann 
Published: March 2005

Chemically and stereochemically pure (3S)‐citryl‐CoA was prepared enzymically and used as a substrate for citrate synthase to investigate the previously determined unexpectedly low rate of hydrolysis of the (3 RS)‐substrate. The unnatural R‐diastereomer of this mixture is not inhibitory.
At low enzyme concentrations the rate of citryl‐CoA hydrolysis was linear until the reaction went near to completion; the hydrolysis approached Michaelis‐Menten kinetics at high enzyme concentrations. In between these concentration extremes a biphasic rate dependence was detectable, where a fast initial phase lasting a few seconds was followed by a slow steady‐state phase.
Citrate synthase was characterized as a hysteretic enzyme existing in two interconvertible forms, which were designated according to their functions as hydrolase E and ligase E'.
The hysteretic behaviour originates in the cleavage of citryl‐CoA to acetyl‐CoA and oxaloacetate. This reaction occurs on the ligase form E', which represents a trap for enzyme form E, the hydrolase.
The conclusions given above are strengthened by the ordinary hydrolysis kinetics of (2S)‐malyl‐CoA, a substrate that is not subject to cleavage of the C‐C bond on the synthase.
The results satisfy the kinetic criterion for citryl‐CoA being an intermediate of the physiological synthase reaction and, therefore, establish the oscillation of the synthase between hydrolase and ligase states during the catalytic cycle.
A disorganization of these oscillations can be achieved by limited tryptic proteolysis of the synthase.
Porphyran Primary Structure
Authors: MORRICE Lora M. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B. 
Published: March 2005

Porphyran, a highly substituted agarose from Porphyra umbilicalis was degraded by highly purified β‐agarase I from Pseudomonas atlantica. This enzyme cleaved at the reducing side of units of β‐neoagarobiose (3,6‐anhydro‐α‐l‐galactopyranosyl‐(1 → 3)‐β‐d‐galactopyranose).The oligosaccharides were divided into fractions of low and high molecular weight by dialysis. The permeate (23% of total starting carbohydrate) was separated by ion‐exchange into neutral and anionic fractions. Gel filtration of the neutral fraction (19%) resolved two major oligosaccharides. These were shown by 13C‐NMR spectroscopy to be 63‐O‐methyl‐neoagarotetraose and 63,65‐di‐O‐methyl‐neoagarohexaose. Gel filtration of the anionic oligosaccharides (3.3%) revealed two novel monosulphated tetrasaccharides, 6‐O‐sulphato‐α‐l‐galactopyranosyl‐(1 → 3)‐β‐d‐galactopyranosyl‐(1 → 4)‐3,6‐anhydro‐α‐l‐galactopyranosyl‐(1 → 3)‐d‐galactopyranose and its 63‐O‐methylated derivative.The 13C‐NMR data from the sulphated tetrasaccharides provided a novel reference which was used to characterise higher, partially sulphated fragments in the dialysis permeate. The fraction retained on dialysis (77%) had an average degree of polymerisation of 40 and was homologous with the high‐molecular‐weight anionic permeate. From 13C‐NMR spectroscopy porphyran was found to comprise 49% sulphated disaccharide units and these were calculated to occur in stretches averaging 2.0–2.5 contiguous units.
Characterization of Cytochrome P2‐450 (20‐S) mRNA
Authors: IKEDA Toshihiko , ALTIERI Mario , CHEN Yuan‐Tsong , NAKAMURA Michitoshi , TUKEY Robert H. , NEBERT Daniel W. , NEGISHI Masahiko 
Published: March 2005

Mouse liver cytochrome P2‐450 is defined as the major isosafrole‐inducible form of P‐450 which is most specific for isosafrole metabolism. λAhP‐1 represents a 15.5 × 103‐base‐pair segment of mouse genomic DNA having the cytochrome P1‐450 gene (∼ 4600 base pairs) located in the middle portion. Using various subclones as probes, we investigated the differential expression of P1‐450 mRNA and P2‐450 mRNA induction as a function of association with the Ah locus, 3‐methylcholanthrene or isosafrole dosage, tissue specificity, and developmental age.Both P1‐450 (23‐S) mRNA and P2‐450 (20‐S) mRNA induction processes are regulated by the Ah receptor. P2‐450 mRNA is about 10‐fold more sensitive than P1‐450 mRNA to induction by either 3‐methylcholanthrene or isosafrole. Phenobarbital pretreatment has no effect at all on either P1‐450 mRNA or P2‐450 mRNA. Whereas both P1‐450 mRNA and P2‐450 mRNA are induced by 3‐methycholanthrene in C57BL/6N liver, P1‐450 (23‐S) mRNA but not P2‐450 (20‐S) mRNA is induced by 3‐methylcholanthrene in C57BL/6N kidney. P1‐450 mRNA induction by 3‐methylcholanthrene is measurable in C57BL/6N liver at day 15 of gestation, and the expression becomes enhanced with increasing age. P2‐450 mRNA induction by 3‐methylcolanthrene in C57BL/6N liver appears about 7 days later during development than 3‐methylcholanthrene‐inducible P1‐450 mRNA. Both 3‐methylcholanthrene‐induced P1‐450 mRNA and P2‐450 mRNA are detectable in DBA/2N liver; their appearance is later in development, however, and at lower concentrations than that seen with C57BL/6N liver. P1‐450 (23‐S) mRNA and P2‐450 (20‐S) mRNA appear to hybridize to a common 5′ fragment of the P1‐450 gene.