bluuebunny/crossref_metadata_2025_split huggingface dataset:
Co‐fractionation of an Endonuclease Activity during the Purification of DNA Polymerase‐α from Regenerating Rat Liver
Authors: MECHALI Marcel , DE RECONDO Anne‐Marie 
Published: March 2005

The presence of endonuclease activity associated with DNA polymerase was detected during the purification of high‐molecular‐weight DNA polymerase‐α from regenerating rat liver by the use of a highly sensitive test. This endonuclease activity co‐fractionated with the DNA polymerase in a great variety of purification procedures involving ion‐exchange chromatographies or molecular weight fractionation, but was further completely separated from DNA polymerase activity by using affinity chromatography on DNA‐cellulose.The endonuclease acted on native or denatured DNA by introducing single‐strand nicks in the DNA molecules; its enzymatic properties indicate that it could act in polymerisation conditions in vitro.
Glucosylation of Galactosylhydroxylysyl Residues in Collagen in vitro by Collagen Glucosyltransferase
Authors: MYLLYLÄ Raili , RISTELI Leila , KIVIRIKKO Kari I. 
Published: March 2005

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500‐fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate‐soluble collagens from normal or lathyritic rats and isolated α1 and α2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values.A marked difference was found between native and heat‐denatured citrate‐soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 °C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 °C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 °C, similar to that observable in the melting curve of collagen. The data suggest that triple‐helical conformation of collagen prevents the glucosylation of galactosylhydroxylysyl residues.
Mitochondria and Peroxisomes from the Cellular Slime Mould Dictyostelium discoideum
Authors: PARISH Roger W. 
Published: March 2005

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter‐Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients.An isolation method was developed using Triton X‐100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub‐optimal Triton X‐100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane.The mitochondria and peroxisomes could not be clearly separated using conventional sucrose‐Ficoll density gradients.Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2‐p‐iodophenyl‐3‐p‐nitrophenyl‐5‐phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes (“heavy‐labelling”). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact.The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen‐peroxide‐producing oxidases were detected.The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 μM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.
Interactions of Colipase with Bile Salt Micelles
Authors: CHARLES Maurice , ASTIER Martine , SAUVE Paul , DESNUELLE Pierre 
Published: March 2005

A detailed investigation by ultracentrifugation of the colipase‐taurodeoxycholate system showed the formation of well‐defined mixed associations with a sedimentation coefficient of about 2.2 S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor.Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.
The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii
Authors: BRESTERS Tjarda W. , DE KOK Arie , VEEGER Cees 
Published: March 2005

The presence of activators (AMP and sulphate) or inhibitors (acetyl‐CoA) has no influence on the Hill coefficient of the S‐shaped [pyruvate]–velocity curve of either the pyruvate‐NAD+ overall reaction (h= 2.5) or that of the pyruvate‐K3Fe(CN)6 activity of the first enzyme (h= 1.3). pH studies indicated that the Hill coefficient is dependent on subunit ionization within the pyruvate‐containing complex and not on those in the free complex.It is concluded that pyruvate conversion rather than pyruvate binding is responsible for the allosteric pattern. The activity is, due to absence of a protein kinase, mainly regulated at the acetyl‐CoA/CoA, and NADH/NAD+ levels and by the value of the energy charge.
Amino‐Acid Incorporation into tRNA Fragments and into Heterologous Combinations of Fragments
Authors: WÜBBELER Wolfgang , LOSSOW Christine , FITTLER Friedrich , ZACHAU Hans G. 
Published: March 2005

When the CCA‐halves of tRNAAla1 (yeast) and tRNAVal1 (Escherichia coli) were incubated with the pG‐halves of tRNAVal1 (E. coli) and tRNAAla1 (yeast), respectively, heterologous complexes were detected. When a 10‐fold excess of one half was applied, up to 50% of the other half could be com‐plexed. 5–12% alanine and valine incorporation was observed into the heterologous combinations in which the pG‐halves were derived from tRNAAla1 (yeast) and tRNAVal1 (E.coli), respectively. Although the values are small they appear to be significant considering the results of a number of control experiments.The CCA‐half of tRNASer1.2 (yeast) and another fragment of this tRNA which extends from the dihydrouridine region to the CCA‐terminus were inactive in the aminoacylation assay but they could be converted into a form which accepted serine under standard conditions even in the absence of a complementary fragment. One activation procedure involved the addition of MgCl2 to Mg2+ ‐free fragment solutions, the other consisted in a brief heating‐cooling cycle of the fragment solutions at low Mg2+ concentrations. With the two procedures up to 20% or up to 40%, respectively, of the maximal serine incorporation were achieved. At 37 °C the active conformation of the fragments persisted only for a few minutes. Analogously, the CCA‐halves of tRNAphe (yeast), tRNAAla1 (yeast), and tRNAVal1 (E. coli) could be activated although here the extent of aminoacylation varied greatly from one experiment to the other. Mischarging of the activated CCA‐halves of tRNASer1,2 (yeast) and tRNAPhe (yeast) with phenylalanine and serine, respectively, was not observed.The results obtained with the hererologous fragment combinations and with the CCA‐halves alone, which at first sight seem to contradict each other, are discussed with respect to the confor‐mational requirements of synthetase‐tRNA recognition.
Temperature‐Sensitive Mutants of the Yeast Fatty‐Acid‐Synthetase Complex
Authors: SCHWEIZER Eckhart , KNOBLING Ansgar 
Published: March 2005

By genetic complementation analysis, 88 independently isolated temperature‐sensitive fatty acid synthetase mutants have been assigned to the six different fas‐complementation groups II (fas 1), III (fas 1), Vb (fas 1), VI (fas 2), VIII (fas 2) and IX (fas 2). The complementation groups Va, Vc, Vd,IV and VII observed among nonconditional fas‐mutants have not been found among the temperature‐sensitive strains studied. From the failure to detect pantetheine‐deficient conditional fas‐mutants it is concluded that the yeast acyl‐carrier protein has an exceptionally stable tertiary structure. Furthermore, the lack of temperature‐sensitive mutants of complementation group IV possibly indicates that this group specifically represents only nonsense and frameshift mutations.Almost half of the temperature‐sensitive fas1 and fas2 mutants studied exhibited non‐complementing characteristics. These results confirm the existence of non‐complementing fas1 and fas2 missense mutations. From this it is concluded that both fatty acid synthetase loci encode multifunctional polypeptide chains rather than several monofunctional component enzymes. The possible existence of an independent acyl‐carrier protein, as suggested by the genetic data reported in this study, is discussed.With 10 different temperature‐sensitive fas1 and fas2 mutants the dependence of cellular growth rates on growth temperature and fatty acid supplementation was determined. With all mutants studied fatty‐acid‐independent growth was completely suppressed at non‐permissive temperatures (34–37 °C). In fatty‐acid‐supplemented media, however, these mutants exhibited the same growth characteristics as wild‐type yeast cells. In contrast to this, wild‐type yeast growth was found to be fatty‐acid‐independent at all temperatures studied. Other than in vivo, the purified fatty acid synthetase isolated from five different temperature‐sensitive fas1 and fas2 mutants exhibited in vitro no increased thermolability compared to the wild‐type enzyme. From this it is concluded that the specific conformation of fatty acid synthetase subunits either forms only at the ribosomal level during translation, or that this conformation is stabilized by the assembly of subunits into the multienzyme complex structure.
A New Method of Isolation and Some Properties of the Heavy Chain of Human Plasmin
Authors: RICKLI Egon E. , OTAVSKY Wendy I. 
Published: March 2005

A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase‐activated plasminogen with 2‐mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6‐aminohexanoic acid (yield 60–65%).Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate‐polyacrylamide electro‐phoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH2‐terminal amino acid was predominantly threonine.6‐Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor‐induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6‐aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6‐aminohexanoic acid.
Further Studies on Lipid‐Peroxide Formation in Isolated Hepatocytes
Authors: HÖGBERG Johan , ORRENIUS Sten , O'BRIEN Peter J. 
Published: March 2005

Lipid peroxide formation was initiated by the addition of either ADP‐complexed Fe3+or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30–60 min, and the response was dose‐related with iron concentrations ranging from 19 to 187 μM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The additionin vitroofN,N,N′;N′‐tetramethyl‐p‐phenylenediamine, menadione orp‐benzoquinone inhibited the iron‐induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (100 μM).The attenuation of the iron‐induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells.Cumene hydroperoxide‐induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 μM to the liver cell incubate. With concentrations below 150 μM, a lag phase was present which seemed to be glutathione‐dependent.It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPHviathe NADPH–cytochromeP‐450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron‐apoferritin complex formation.
d‐Glucose‐6‐Phosphate Dehydrogenase (Entner‐Doudoroff Enzyme) from Pseudomonas fluorescens
Authors: LESSMANN Dieter , SCHIMZ Karl‐Ludwig , KURZ Gerhart 
Published: March 2005

The existence of two different d‐glucose‐6‐phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner‐Doudoroff pathway and the other to the hexose monophosphate pathway.
A procedure is described for the isolation of that d‐glucose‐6‐phosphate dehydrogenase which forms part of the Entner‐Doudoroff pathway (Entner‐Doudoroff enzyme). A 950‐fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 μmol NADH formed per min per mg protein, was shown to be homogeneous.
The molecular weight of the Entner‐Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000.
The pI of the Entner‐Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner‐Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9.
The Entner‐Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for d‐glucose 6‐phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory.
The enzyme catalyzes the transfer of the axial hydrogen at carbon‐1 of β‐d‐glucopyranose 6‐phosphate to the si face of carbon‐4 of the nicotinamide ring and must be classified as B‐side stereo‐specific dehydrogenase.
RNA‐Dependent DNA Polymerase from a Cell Line Derived from the Bone Marrow of a Patient with Polycythemia vera
Authors: WEIMANN Bernd Jürgen , SCHMIDT Jörg , KLUGE Norbert , OSTERTAG Wolfram , WOLFRUM Dirk Ingo 
Published: March 2005

A RNA‐dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)‐cellulose, and glycerol gradient centrifugation.The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) · oligo(dG) and its molecular size of about 70 000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co‐purified. Upon dodecyl‐sulfate‐polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68 000 and 66 000 and three minor bands were detected.The enzyme preferentially used poly(rA) · oligo(dT) as template‐primer compared with poly‐(dA) · oligo(dT). It incorporated dGMP into polymer on poly(rC) · oligo(dG).
Purification and Properties of Potato 1,4‐α‐d‐Glucan: 1,4‐α‐d‐Glucan 6‐α‐(1,4‐α‐Glucano)‐Transferase
Authors: BOROVSKY Dov , SMITH Eric E. , WHELAN William J. 
Published: March 2005

Q‐Enzyme, the enzyme that synthesizes the 1,6‐α‐glucosidic branch linkages of amylopectin, has been purified from potato to near homogeneity.The molecular weight of the enzyme is 85000. The active enzyme is a monomer, with a molar activity at pH 7.0 and 24 °C of 15. The energy of activation is 25 kJ/mol below 15 °C, changing sharply to 63 kJ/mol above that temperature. Enzyme activity is not affected by Mg2+ or ATP. There are about 11 readily titratable sulfhydryl groups per molecule.The evidence that the enzyme is a single protein entity, without hydrolytic activity towards amylose, contrasts with an earlier report that Q‐enzyme consists of two components, a hydrolase with molecular weight 70000, and a transferase with molecular weight 20000.Q‐Enzyme acts on native and synthetic amyloses to give products resembling amylopectin in terms of average unit chain length, degree of β‐amylolysis and iodine stain. The profiles of the unit chains of these synthetic products are, however, different from that of native amylopectin. Additional branch linkages are introduced by Q‐enzyme into potato amylopectin, but the product bears no resemblance to phytoglycogen.
Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides
Authors: PETKOV Dimiter , CHRISTOVA Evdokia , POJARLIEFF Ivan , STAMBOLIEVA Nikolina 
Published: March 2005

The absence of both nonproductive binding and substrate activation and also the good solubility of the substrates make the urokinase‐catalysed hydrolysis of specific anilides a very suitable reaction for substrate structure–enzyme activity studies.Derivatives of α‐N‐acetyl‐l‐lysine anilide with high σ‐value substituents in the aniline ring were synthesized. Rate constants kcat. and apparent Michaelis‐Menten constants Km(app.) are presented. From the substituent dependence of kcat. and from the fact that kcat. is 13 to 37 times smaller than the deacylation rate constant it is concluded that the rate‐limiting step proceeds prior to deacylation.The catalytic rate constant kcat. obeys a linear free‐energy relationship of the Hammett type with ϱ=+ 0.72. Two different mechanisms implied by the results obtained from the model reaction (specific base and general acid‐base catalysed hydrolysis of N‐acetylglycine anilides under extreme conditions) are proposed in order to account for this positive and low ϱ‐value. In the first mechanism the breakdown of an enzyme tetrahedral intermediate is rate‐limiting, while in the second one its formation controls the overall rate. The discrimination between the two mechanisms, however, could not be found.
Premature Induction of Glucokinase in the Neonatal Rat by Thyroid Hormone
Authors: PARTRIDGE Nicola C. , HOH Chee Hoong , WEAVER Patricia K. , OLIVER Ivan T. 
Published: March 2005

It was shown that the development of liver glucokinase in the rat coincided with a peak in the levels of circulating thyroid hormone at about the 16th postnatal day.
Administration of thyroid inhibitors blocked the development of the enzyme and administration of thyroid hormone restored activity to normal levels.
Glucokinase could be induced prematurely as early as the 2nd postnatal day by the administration of thyroid hormone followed by daily injection of glucose (10 mg/g body weight).
Glucocorticoids and corticotropin failed to induce glucokinase activity prematurely.
The postnatal increase in circulating thyroid hormone levels together with increased intake of carbohydrate at weaning may be the normal physiological stimulus for induction of this enzyme.
Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: POLGÁR László 
Published: March 2005

The reactivity and the mode of activation of the essential – SH group (Cys‐149) of d‐glyceral‐dehyde‐3‐phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187–190 (1974)], capable of detecting the dissociated form of the thiol group in proteins.Alkylations of Cys‐149 of NAD‐free d‐glyceraldehyde‐3‐phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys‐149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK1= 5.5, pK2= 8.2).It is concluded that there are two reactive forms of the –SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion‐pair composed of a mercaptide ion and some base, possibly the imidazolium group of His‐176.The ion‐pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion.The two reactive forms are transformed into each other with pK2= 8.2. The ion‐pair decomposes to a nondissociated thiol group and a protonated base with pK1= 5.5.In the presence of NAD, only the pH‐rate profile of alkylation of d‐glyceraldehyde‐3‐phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion‐pair exists in a somewhat wider pH‐range in the enzyme‐coenzyme complex.An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.