bluuebunny/crossref_metadata_2025_split huggingface dataset:
Some Properties of NADPH‐Cytochrome c Reductase Reconstitutively Active in Fatty‐Acid ω‐Hydroxylation
Authors: Ichihara Kosuke , Kusunose Emi , Kusunose Masamichi 
Published: March 2005

A NADPH‐cytochrome c reductase (called Fraction II) has been solubilized with Triton X‐100 from porcine liver microsomes and purified.1. Fraction II can reconstitute with a cytochrome P‐450 fraction (called Fraction I) solubilized with Triton X‐100 from porcine kidney cortex microsomes to exhibit laurate ω‐hydroxylation activity.2. When the purified Fraction II is subjected to disc electrophoresis on polyacrylamide gel in the presence of 0.1% Triton X‐100, it shows a single band. In contrast, on electrophoresis in the absence of Triton X‐100, a polydisperse state is obtained. Fraction II is estimated to have a molecular weight of about 400000 by gel‐filtration with detergent, and it does not contain iron.3. Either gel filtration on a Sephadex G‐100 column or treatment with trypsin at 4 °C results in a conversion of Fraction II to a form, which migrates at a much faster rate on disc gel electrophoresis than dose Fraction II. This fast‐moving form (called Fraction IIf) is indistinguishable from either steapsin or trypsin‐solubilized NADPH‐cytochrome c reductase, judging from several criteria including electrophoretic and sedimentation behaviors, and molecular size.4. Fraction IIf is reconstitutively inactive in the hydroxylation, unless supplemented with spinach ferredoxin‐NADP reductase. Incubation of Fraction IIf with trypsin at 30 °C results in a remarkable enhancement in the reconstitution activity in the presence of ferredoxin‐NADP reductase. The results in this paper along with an earlier report [11] suggest that Fraction II may contain two different electron carrier components, possibly participating in the electron transfer from NADPH to cytochrome P‐450.
Isolation and Characterization of Chloroform‐Soluble Proteins from Rat‐Liver Mitochondria and Other Fractions
Authors: Hadváry Paul , Kadenbach Bernhard 
Published: March 2005

Chloroform‐soluble proteins have been isolated free from phosphatides from rat liver mitochondria, microsomes and cytosol by chromatography on Sephadex LH‐20 columns.
The amino acid composition of the chloroform‐soluble and insoluble proteins has been estimated. The chloroform‐soluble proteins from mitochondria and cytosol have a higher content of hydrophobic and a lower content of acidic amino acids compared to the corresponding chloroform‐insoluble proteins. No marked differences were found between chloroform‐soluble and insoluble proteins from microsomes. In contrast to the acidic chloroform‐insoluble proteins, all chloroform‐soluble proteins are basic.
From the amino acid composition the amount of chloroform‐soluble proteins has been estimated. Mitochondrial membranes contain 1.6%, mitochondrial matrix proteins less than 0.025%, microsomes 3.0% and the cytosol 0.15% from total proteins.
The molecular weight distribution of chloroform‐soluble proteins synthesized in mitochondria has been measured by gelelectrophoresis. The molecular weights range from about 7000 to 30000.
The turnover of chloroform‐soluble proteins from mitochondria and cytosol is higher than from the corresponding chloroform‐insoluble proteins. A part of mitochondrial chloroform‐soluble proteins (about 30%) is synthesized within the mitochondria.
Ethoxyformylation of Helix pomatia Haemocyanin
Authors: Engelborghs Yves , Lontie René 
Published: March 2005

Ethoxyformylation of Helix pomatia haemocyanin was studied to investigate the role of histidine residues in the binding of copper and in the dissociation properties of the protein. At neutral pH ethoxyformylation gives rise to a difference spectrum with a maximum at 240 nm, characteristic of ethoxyformylimidazole.Initial‐rate studies at 250 nm reveal the reaction to be bimolecular. The rate constant depends on pH and follows the titration of the histidine residues. The complete time course of the reaction is complicated by hydrolysis of the reagent and of the reaction products.Ethoxyformylation of α‐haemocyanin at pH 6.0 causes the protein to dissociate into halves, at pH 6.5 and higher the dissociation proceeds to tenths. β‐Haemocyanin does not dissociate at pH 6.0, at higher pH values it dissociates completely into tenths. An induction period is seen in the dissociation reaction, indicating that a certain number of groups have to be modified before dissociation starts. This number is lowered by raising the pH.The hydrolysis of the ethoxyformylimidazole groups can be enhanced with NH2OH · HCl. During hydrolysis the halves reassociate quantitatively into wholes, the tenths reassociate into large aggregates.Modification at pH 7.0 and above destroys the copper binding site at high reagent concentration. Ethoxyformylation of the apoprotein prevents reconstitution. In both cases a linear relationship was found between the copper band and the copper content. Parallel to the liberation of copper, tyrosine residues become accessible, as shown by the negative difference spectrum at 278 nm upon ethoxyformylation or by spectrophotometric tyrosine titrations.
Two Forms of the DNA Ligase of Human Cells
Authors: Pedrali Noy Guido C. F. , Spadari Silvio , Ciarrocchi Giovanni , Pedrini Antonia M. , Falaschi Arturo 
Published: March 2005

We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350‐fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4‐phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form.The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular‐weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme.
Studies on the Metabolism of Steroids in the Foetus
Authors: Plasse Jean‐Claude , Lisboa Belisário Penna 
Published: March 2005

Biosynthesis and site of formation of epitestosterone in the human foetus was investigated by incubation of androstenedione with subcellular fractions of mid‐gestational foetal liver. Testosterone and epitestosterone were purified by thin‐layer chromatography and resolved by gas‐liquid chromatography.Epitestosterone (and testosterone) were identified by gas chromatography‐mass spectrometry as silyl ether and methoximesilyl ether derivatives. The ratio testosterone: epitestosterone found in the incubations was about 20:1.Both 17β‐ and 17α‐hydroxysteroid oxidoreductase activities were found in the 105000 X g microsome fraction as well as in the cytosol fraction.It may be concluded that the human foetal liver is one of the sources of production of epitestosterone, and that androstenedione is one of its precursors. The possibility that androstenedione and epitestosterone are precursors of maternal urinary 17α‐hydroxyphenolic steroids during pregnancy is discussed.
The Association of Ribosomal Subunits of Escherichia coli
Authors: van Diggelen Otto P. , Bosch Leendert 
Published: March 2005

Association between ribosomal subunits of Escherichia coli can lead to the formation of two classes of 30‐S · 50‐S couples. The first class sedimenting between 50 S and 70 S, arises when native subunits associate, the second sedimenting at 70 S upon coupling of so‐called derived subunits. From cross experiments it is concluded that it is the 50‐S subunit which determines the nature of the association product, whereas the 30‐S subunit determines whether an association product can be formed. Native and derived 50‐S subunits are indistinguishable by sucrose gradient centrifugation and do not differ qualitatively in their ability to form polypeptides under the directions of bacteriophage RNA. Native 30‐S subunits lose a factor during isolation, probably IF‐3. They are therefore able to associate at 0° and 37 °C. On the other hand derived 30‐S subunits are unable to associate at 0 °C but they do so at 37 °C in the presence of 10 mM Mg2+.A number of trivial factors have been excluded to explain the difference between the two types of 50‐S subunits. The preparation method is not responsible for the difference. Nor do the derived subunits contain peptidyl‐tRNA, known to influence subunit association. The content of tRNA and aminoacyl‐tRNA has been determined quantitatively by polyacrylamide‐gel electrophoresis. None of the subunits nor the 70‐S ribosomes from which the derived subunits are prepared contain a tRNA species.
The Association of Ribosomal Subunits of Escherichia coli
Authors: van Diggelen Otto P. , Oostrom Henk , Bosch Leendert 
Published: March 2005

The two types of 30‐S · 50‐S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real s value being 70 S. The latter couples can be stabilized against pressure‐induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation.As the factor determining this intriguing difference in pressure sensitivity lies in the 50‐S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50‐S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low‐molecular‐weight components are responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ‐cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50‐S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50‐S differ in a ribosomal protein.
Quaternary Structure of Polynucleotide Phosphorylase from Escherichia coli
Authors: Portier Claude , Thang Minh Nguy , Grunberg‐Manago Marianne , van Rapenbusch Roland 
Published: March 2005

Purification of polynucleotide phosphorylase from Escherichia coli by a modified technique yeilds a protein that appears to be homogeneous when analyzed by disc‐gel electrophoresis. Two species of polypeptide chains are obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate or by filtration on Sephadex G‐200 column in the presence of guanidinium chloride; their molecular weights are 92000 and 48000.Renaturation experiments show that reassociation of the purified heavy chains yields an active enzyme endowed of both polymerization and phosphorolysis activities; accordingly, the active site(s) must reside on the heavy component.Reassociated renatured 92000‐Mr chains have a behavior very similar to that of the native enzyme on sucrose gradient and in disc gel electrophoresis, strongly suggesting a structure of the α2 type for polynucleotide phosphorylase. The origin and possible role of the 48000‐Mr component is discussed.
Studies on the Specificity of an Acid Deoxyribonuclease from Helix aspersa (Müll.)
Authors: Thiery Jean‐Paul , Bernardi Giorgio , Laval Jacques , Paoletti Claude , Ehrlich Stanislav D. 
Published: March 2005

The kinetics of degradation of calf thymus DNA by the DNAase from Helix aspersa has been investigated in its middle and terminal phase by determining both hyperchromic shift and average chain length of the digest. The results obtained are practically identical with those previously reported for acid DNAase from hog spleen. In contrast, the distribution of the isostichs in snail DNAase digests is significantly different from those found for spleen acid DNAase digests of similar sizes.The 3′‐phosphate and 5′‐hydroxy terminal nucleotides were found not to vary in their compositions in the average size range 34 to 10 and 70 to 12, respectively. In the 3′‐phosphate terminal position, deoxyadenosine is by far predominant (78%) and deoxycytidine almost absent (less than 1%); deoxyguanosine and thymidine form 6% and 16%, respectively, of the terminals. In the 5′‐hydroxy terminal position deoxyguanosine is predominant (45%), followed by deoxycytidine (31%), thymidine (14%) and deoxyadenosine (10%); in the 5′‐hydroxy penultimate position thymidine forms 38% of the nucleotides, deoxyguanosine 24%, deoxyadenosine 21% and deoxycytidine 17%.
Studies on Tyrosine Environments of Chicken Ovomucoid
Authors: Krysteva Milka A. , Mancheva Ivanka N. , Dobrev Ivan D. 
Published: March 2005

The particular environments of tyrosine side chains were suggested to be responsible for the non‐denaturation acidic difference spectra of ovomucoid as well as of abnormal ionization of its phenolic groups. The micro environment of the most exposed tyrosine in ovomucoid was studied. The tyrosine was first labelled by means of tetranitromethane. Nitrotyrosine peptide was isolated. The study of the nine steps of nitrotyrosine peptide degradation results in the following amino acid sequence: Ala‐Gly‐Ala‐Lys‐Thr‐Gln‐NO2Tyr‐Gly‐Gly. The analysis of neutral sugars as well as glycosamine gave negative results. The presence of sialic acid however was detected.On the basis of evidence obtained, the existence of a glycosidic bond formed from the hydroxyl group of tyrosine and the glycosidic hydroxyl group of sialic acid was suggested. The spectral behaviour of the tyrosine studied was discussed in the light of above assumption.
The Binding of Indole Derivatives by Borohydride‐Reduced β‐Cyclopiazonate Oxidocyclase
Authors: Steenkamp Daniël J. , Schabort C. 
Published: March 2005

Initial velocity studies on β‐cyclopiazonate oxidocyclase in the presence of the competitive inhibitor, 3‐acetyl‐5‐scatyl‐tetramic acid, indicates that this enzyme catalyzes the oxidocyclization of β‐cyclopiazonic acid by a Ping‐Pong‐Bi‐Bi mechanism.
It was possible to reduce the flavin coenzyme to the 3,4‐dihydroflavin by consecutive cycles of reduction with BH4‐ and reoxidation by atmospheric oxygen.
The BH4‐‐reduced enzyme shows a similar affinity for indole derivatives as does the native flavoquinone enzyme, but is inactive. β‐Cyclopiazonic acid binds to the enzyme in a simple equilibrium, and the reduction of the 3,4‐dihydroflavin to the 1,3,4,5‐tetrahydroflavin or a conversion to 1,5‐dihydroflavin does not proceed.
Partial reactivation of the enzyme by the photocatalyzed oxidation of the 3,4‐dihydroflavin to the flavoquinone form was observed
Studies of Sugar Transport in Acholeplasma laidlawii
Authors: Tarshis Mark A. , Migoushina Valentina L. , Panchenko Leonid F. , Fedotov Nikolay S. , Bourd Heinrich I. 
Published: March 2005

Different mechanisms of carbohydrate transport into Acholeplasma laidlawii cells have been studied. The following properties have been found.

The glucose, maltose and fructose uptake is dependent on the time of incubation, the substrate concentration in the external medium, as well as on the pH and temperature, thus pointing to the enzymatic nature of carriers.
The carbohydrate uptake is inhibited by a number of energy inhibitors and uncouplers, thus pointing to the active nature of this proces.
The carbohydrate uptake is highly sensitive to SH‐inhibitors, characterising carriers as proteins.
The noncompetitive inhibition suggests that glucose and maltose are transported by single carrier involving two active centres.
A New Immobilized NAD+ Analogue, Its Application in Affinity Chromatography and as a Functioning Coenzyme
Authors: Lindberg Margareta , Larsson Per‐Olof , Mosbach Klaus 
Published: March 2005

Alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement gave N6‐carboxymethyl‐NAD+. Condensation of this analogue with 1,6‐diaminohexane gave the analogue NAD+‐ N6‐[N‐(6‐aminohexyl)‐acetamide]. The coenzymic activity of the two derivatives was tested with malate dehydrogenase, alcohol dehydrogenase and lactate dehydrogenase. The efficiency relative to unsubstituted NAD+ was in the range of 50–100%.NAD+‐N6‐[N‐(6‐aminohexyl)‐acetamide] was attached to Sepharose 4B by the cyanogen bromide method. The immobilized NAD+ analogue thus obtained, exhibited cofactor activity when tested in a recycling three‐enzyme system (malate dehydrogenease‐citrate synthase‐lactate dehydrogenase). The immobilized NAD+ analogue proved to be and effective ligand in affinity chromatography. Thus a mixture of albumin, alcohol dehydrogenase and lactate dehydrogenase was resolved with good recovery. The enzymes were eluted with NAD+ and NADH, respectively.
Thymine Utilization in Escherichia coli K12 on the Role of Deoxyribose 1‐Phosphate and Thymidine Phosphorylase
Authors: Jensen Kaj Frank , Leer Johan Chr. , Nygaard Per 
Published: March 2005

Exogenously supplied thymine is only poorly utilized by wild‐type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine.Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1‐phosphate and/or the level of thymidine phosphorylase.Thymine incorporation in wild‐type cells could be observed if the cells possessed an increased deoxyribose 1‐phosphate pool.The kinetics of thymine uptake in wild‐type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration.The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1‐phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine phosphorylase showed that thymidine is synthesized from deoxyribose 1‐phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside.
A Study of the Interaction of Calcium Ions with Bovine αs1‐Casein Using Fluorescence Spectroscopy
Authors: Dalgleish Douglas G. 
Published: March 2005

The interaction of αs1‐casein with calcium ions at pH 7.0 has been studied, and it is shown that there is a stepwise increase in the fluorescence of the tryptophan residues of the protein as increasing amounts of Ca2+ are added, leading to eventual precipitation of the calcium‐caseinate complex.The first of these steps appears to result from a conformational change induced by Ca2+, without which the protein cannot precipitate. Further steps arise from interaction between protein molecules to produce particles of about 120 nm in diameter, then final aggregation of these particles to give a precipitate. All of these changes result in changes in the fluorescence emission spectra of one or both tryptophan residues in the casein, although most of the change in fluorescence behaviour in the first step of the interaction probably arises from tryptophan‐164 in the protein:
ω‐Oxidation of Stearic Acid in the Normal, Starved and Diabetic Rat Liver
Authors: Björkhem Ingemar 
Published: March 2005

ω‐Oxidation of stearate, catalyzed by 20000 ×g supernatant fluid of rat‐liver homogenate in the presence of an NADPH‐generating system, was stimulated three‐ to seven‐fold by starvation or diabetes. The soluble fraction of liver homogenate was responsible for the stimulation. Thus, addition of 100000 ×g supernatant fluid of liver homogenate from starved rats stimulated microsomal ω1‐oxidation of stearate two‐ to three‐fold whereas addition of 100000 ×g supernatant fluid of liver homogenate from untreated rats had no effect or an inhibitory effect. Evidence was obtained to indicate that the stimulatory effect of the 100000 ×g supernatant fluid from liver of starved rats was due to the presence in this fraction of liver alcohol dehydrogenase and NAD. The role of alcohol dehydrogenase might be to protect the ω1‐hydroxylase from product inhibition by oxidation of the 18‐hydroxystearic acid formed. The effect of 100000 ×g supernatant fluid from liver of untreated rats was shown to be due to an inhibition of the ω1‐hydroxylase reaction and not of the alcohol dehydrogenase reaction. The inhibitory effect of the 100000 ×g supernatant fluid was retained after boiling for 5 min but was partially destroyed after preincubation at 37°C or after repeated freezing and thawing. The inhibition was shown to be at least not mainly due to interference with formation of stearoyl‐CoA from stearic acid. Stearoyl‐CoA was ω1‐hydroxylated by the microsomal fraction or by the 20000 ×g supernatant fluid at a rate about half that of free stearic acid.The possibility is discussed that the low extent of ω1‐oxidation of long‐chain fatty acids observed under normal conditions might be partially due to the presence of the inhibitor(s) in the soluble part of the cell and that disappearance of this inhibition under conditions of starvation and diabetes might be of physiological importance.
Effect of Adenosine on Methionyl‐tRNA Synthetase
Authors: Lawrence Françoise 
Published: March 2005

The methionyl‐tRNA synthetase activities were shown to be inhibited by adenosine. In the ATP⇋PPi exchange, and in the hydroxamate reactions, the inhibition was competitive with respect to ATP and uncompetitive with respect to L‐methionine. In the methionine transfer reaction, inhibition patterns are more complicated, however the inhibition tends to be competitive with respect to ATP at high methionine concentration.The inhibition by adenosine of the ATP⇋PPi exchange reaction has been observed for several aminoacyl‐tRNA synthetases (seryl‐, cysteyl‐, isoleucyl‐, valyl‐, phenylalanyl‐, methionyl‐ and trypsin‐modified methionyl‐tRNA synthetases). However under standard conditions the degree of inhibition varies from one enzyme to the other.