bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Sequence around the Active‐Center Tyrosyl of Porcine Carboxypeptidase B
Authors: Roholt Oliver A. , Pressman David 
Published: March 2005

Iodination of porcine carboxypeptidase B causes a loss of both peptidase and esterase activity; the presence of the inhibitor β‐phenylpropionate during iodination prevents the loss.A residue in the active site of the enzyme was labeled by a procedure in which one portion of the enzyme was iodinated with 125I‐labeled iodine and a second portion with 131I‐labeled iodine in the presence of the inhibitor β‐phenylpropionate. The two preparations were mixed, digested with elastase and the iodopeptides separated. Two iodopeptides which appear to have come from the active site were isolated; both had high 125I/131I radioactivity ratios relative to the unfractionated digest. The labeled residue is tyrosyl in both iodopeptides. The same results were obtained when the inhibitor ɛ‐aminocaproate was used instead of β‐phenylpropionate. The yield of the two iodopeptides approached a total of about 1 mole/mole of enzyme in each of several experiments. The sequences of the two iodopeptides were found to be Thr‐Ile‐(monoiodo)Tyr‐Pro‐Ala and Ile‐(monoiodo)Tyr‐Pro‐Ala, and both appear to represent the same tyrosyl residue in the site of porcine carboxypeptidase B.
Excretion of Steroid Hormones in Adults
Authors: Eriksson Håkan , Gustafsson Jan‐Åke 
Published: March 2005

The steroids in faeces from three healthy persons, one woman and two men, were analyzed by gas‐liquid chromatography and gas chromatography‐mass spectrometry. Both C19O2, C19O3, C21O2 and C21O3 steroids were found in the free steroid and in the steroid monosulphate fractions.Incubation of various precursor steroids with intestinal contents recovered from the ileostoma of a colectomized patient demonstrated the presence of intestinal bacterial 16α‐ and 21‐dehydroxylating enzymes. Furthermore, it was shown that a Δ16‐C21 steroid was converted to a 17α‐pregnane derivative and that the side chain of 11β,21‐dihydroxy‐4‐pregnene‐3,20‐dione could be removed by the intestinal bacteria.
The Interaction of Fungal Laccase with Hydrogen Peroxide and the Removal of Fluoride from the Inhibited Enzyme
Authors: Brändén Rolf , Malmström Bo G. , Vänngård Tore 
Published: March 2005

Electron paramagnetic resonance analysis shows that H2O2 added to native fungal laccase binds to one specific Cu2+ of the four copper atoms in the enzyme (the so‐called Type 2 or “nonblue” Cu2+).
This binding of H2O2 is associated with a new absorption band in the visible and nearultraviolet spectrum of the enzyme with its maximum absorbance at 400 nm.
Fluoride bound to Type 2 Cu2+ of the enzyme, as commonly prepared, can easily be removed by dialysis of the reduced enzyme. Presence of H2O2 in this dialysis makes the removal of fluoride faster.
Isotope Discrimination in Steroid Hydroxylations
Authors: Bjökhem Ingemar 
Published: March 2005

The possible occurrence of isotope effects in steroid hydroxylations by rat and guinea‐pig liver was studied in vitro and in vivo. In vitro, the 7α‐hydroxylation of [24‐14C + 7α−3H]taurodexycholic acid was found to involve a moderate isotope effect, whereas 6β−hydroxylation of [4−14C + 6β−3H]taurochenodeoxycholic acid and 7α−hydroxylation of [4−14C + 6−3H, 7α−2H]cholesterol and [4−14C + 6−3H, 7α−2H]‐cholestanol did not involve significant isotope effects. An isotope effect was observed in the autoxidative 7α‐hydroxylation of [4−14C + 6−3H, 7α−2H]cholesterol. In vivo, [4−14C + 7α−3H]cholesterol was converted into chenodeoxycholic acid and cholic acid and [4−14C + 6β−3H]chenodeoxycholic acid into α‐ and β−muricholic acid without the occurrence of isotope effects, whereas [24−14+ 7α−3H]deoxycholic acid was converted into cholic acid with the occurrence of a moderate isotope effect.The results show that breaking of the C‐H bound is not rate limiting in the 7α−hydroxylation of cholesterol and cholestanol or in the 6β−hydroxylation of taurochenodeoxycholic acid but may be rate limiting in the 7α−hydroxylation of taurodeoxycholic acid.
On the Mechanism of Hemoglobin‐Haptoglobin Complex Formation
Authors: Pavlíček Zdeněk , Jaenicke Rainer 
Published: March 2005

In order to elucidate the mechanism of complex formation between human haptoglobin, Hp II, and bovine hemoglobin, Hb, measurements of the peroxidase activity and histidine determinations, as well as spectrophtometric and optical rotatroy dispersion titration experiments were performed.Following the raaction by slowly mixing the constituents of the complex in small portinos, the geometry of the complex turns out to depend on the sequence of mixing of the reactants. Adding Hb to Hp, a final complex Hb2→ Hp is formed. Teh inverse sequence of mixing leads to a complex of identical composition (Hp → Hb2), but different arrangement of the constituents, as shown by differences in the accessibility of histidine residues in both types of the complex. As suggested by the comparison of the histidine balance of both complexes, Hb2→ Hp may be described by the combination of αβ‐dimers of Hb with Hp (Hp (αβ)4), while Hp → Hb2 corresponds to the complex with two Hb tetramers (Hp(α2β2)2).Investigating the saturation process of complex formation in greater detail by spectrophtometric titration and optical rotatory dispersion, intermediary complexes are observed which prove the mechanism of complex formation to be a consecutive association process.Both series of complexes, Hp → Hb and Hb → Hp, seem to generate closely related configurations characterized by similar spectral and functional properties.
Zur Biosynthese der 2‐n‐Alkyl‐4‐hydroxychinolinderivate (Pseudane) bei Pseudomonas aeruginosa
Authors: Ritter Christiane , Luckner Martin 
Published: March 2005

Sixty strains of the bacterium Pseudomonas aeruginosa (Schroeter) Migula were proved to synthesis 2‐n‐alkyl‐4‐hydroxyquinolines (pseudans). The compounds were detexted in most of the strains, but only few of them showed high alkaloid production. The pseudans are located in the cells. Excretion into the medium after long cultivation seems to be due to autolytic processes.Thin‐layer chromatography of the purified alkaloid fraction and gas chromatography of the acids producd by oxydative degradation of the alkaloids hve shown that all strains form pseudan VII (2‐n‐heptyl‐4‐hydroxyquinoline) and pseudan IX (2‐n‐nonyl‐4‐hydroxyquinoline) only.Experiments on the physiology and biosynthese of the pseudans were carried out using strain 2977, which produces these compounds in good yields. The formation takes place during the idiophase (Fig. 1). After feeding anthranilic acid and acetate the production rate was considerably increased (Table 1). Strain 2977 degrades tryptophan by the “aromatic pathway” forming anthranilic acid as an interrmediate (Fig. 2). The production of higher amounts of this compound seems to be essential for a good rate of pseudan formation.To study the biosynthesis, isotope labelled preparations of anthranilic acid, tryptophan, acetate, malonate and β‐ketodecanoic acid methyl ester were fed (Table 2). For localization of the incoroprated radioactivity, procedures for the chemical degradation of the pseudan molecule were developed. They permit the serate estimation of the C‐atoms 4–8a and the nitrogen as anthranilic acid, C‐atoms 2 and 3 each as CO2, the two terminal C‐atoms of the side chain as CO2 and methylamine respectively and the side chain as a whole. The results obtained have shown that the pseudan molecule is formed from anthranilic acid and acetate. Feeding malonic acid, it was demonstrated that the side chain is built starting from the terminal C‐atoms (Fig. 4). The high sepcific incorporation of β‐keto[3H]decanoic acid methyl ester indicates that a 3‐keto acid is an intermidate. Thus the pathway shown in Fig.5 for the biosynthesis of the pseudans may be assumed to be valid. The incorporation of [2‐14C]acetate into the pseudans was twice that of [1‐14C]acetate. This effect may be due to an equilibrium between malonyl CoA and free malonic acid, which enables the formation of [1,3‐14C2]malonyl‐CoA from [1‐14C]malonyl‐CoA. During the formation of the β‐keto acid half of the radioactivity is lost from this compound.
Purification from Crude Extract by Affinity Chromatography of the Inhibitor of Deoxyribonuclease I
Authors: Lindberg Uno , Eriksson Sigbritt 
Published: March 2005

A one step procedure, consisting of affinity chromatgography, was worked out for the purification from crude extracts of calf thymus of the protein inhibitor specific for pancreatic deoxyribonuclease (DNAase I).For this purpose DNAase I was immobilized on agaros. This solid phase enzyme conjugate was shown to have the ability to adsorb from crude extracts one major protein, which could be eluted again with 3 M guanidine‐HCl in 1.0 M soudium acetate and 30% glycerol. This protein preparation had only a little DNAase inhibiting activity, but could be idnetified by gel electrophoresis and peptide mapping as the inhibitor protein described recently. The low recovery of inhibitor activity was due to poor stability of the inhibitor protein in the solvent system found necessary to effect its elution from the column material.
Amino Acid Sequence of Lima Bean Protease Inhibitor Component IV
Authors: Tan Celline G. L. , Stevens Frits C. 
Published: March 2005

Commmerical lima bean inhibitor was fractionated into four apparently homogeneous components as previously described by Jones, Moore and Stein in 1963.Reduced and alkylated component IV was hydrolyzed with tryspin and the resulting peptides were separated by ion exchange charomatography on Dowexs 50 X2 or gel filtration on Bio‐Gel P‐6. Where necessary, further purification of the peptides was carried out by paper chromatography, paper electrophoresis or a combination of both.Seven peptides were obtained in pure form and their compositions are reported here. The amino acid sequenceof six of these peptides was determined, using classical methods. When allowance is made for the occurence of two homologous peptides, presumably resulting from heterogeneity in the original protein preparation, the tryptic peptides isolated, account for the complete composition of the protein.In an attempt to obtain overlapping sequences the tryptic digest was also performed on the reduced, alkylated and guanidinated protein. Five tryptic peptides, including one containing homoarginine as the carboxy‐terminal residue, were isolated in pure form from the digest; their amino acid compositions are reported.
X‐Ray Small‐Angle Scattering of Yeast Glyceraldehyde‐3‐phosphate Dehydrogenase as a Function of Saturation with Nicotinamide‐Adenine‐Dinucleotide
Authors: Durchschlag Helmut , Puchwein Gerd , Kratky Otto , Schuster Inge , Kirschner Kasper 
Published: March 2005

Structural changes of yeast glyceraldehyde‐3‐phosphate dehydrogenase as a function of saturation with NAD have been measured with the X‐ray small‐angle scattering technique. Samples of yeast glyceraldehyde‐3‐phosphate dehydrogenase were investigated at pH 8.5 and 40° and five different degrees of saturation with the coenzyme NAD (degree of saturation Y = 0, 0.23, 0.46, 0.72, 0.99). Because of the temperature‐sensitivity of the sample, the use of a special flow device became necessary to keep the time of exposure of the samples to 40° and to X‐ray irradiation below 1 h.Apo‐enzyme revealed a contraction in volume depending upon the degree of saturation. Fully saturated the contraction amounts to 7%. As the observed degree of contraction is not linearly proportional to the degree of saturation, the simple sequential mechanism cannot explain the observations. To the extent that they coincide with the quantitative predictions of the allosteric model for this particular enzyme, based on calculations involving the equilibrium constants obtained from chemical relaxation experiments under identical conditions, they strongly support the latter mechanism.Comparison of the experimental scattering curves with theoretical scattering curves of compact full cylinders did not bring about satisfactory agreement in the range of the first submaximum. Hollow circular cylinders with h:2r1= 0.6:1 (apo‐enzyme) or h:2r1= 0.58:1 (holo‐enzyme) may be regarded as sufficiently equivalent in scattering as far as the form of the main maximum is concerned as well as the position and relative intensity of the submaximum. Even if the molecule consists of four subunits they consequently must be arranged in such a way that the entire particle is very similar to a hollow cylinder.The experimental determination of the radius of gyration (R= 32.1 Å in the case of the apo‐enzyme, or 31.7 Å in the case of the holo‐enzyme) leads to the calculation of the absolute particle dimensions: Apo‐enzyme: h= 48.5 Å, r1= 40.4 Å, r2= 6.1 Å, Holo‐enzyme: h= 46.4 Å, r1= 40.2 Å, r2= 6.0 Å.The nature of the structural changes occuring in the allosteric transition can now be described in terms of an increase of the inherent anistropy and of a considerable decrease in the solvent included in the tetrameric assembly.Exposure of the enzyme to 40° over a longer period of time leads to significant alterations in the scattering curves. These can be interpreted on the basis of growing aggregates in polydisperse solutions. This process is accompanied by a considerable decrease of enzyme activity.
Effects of Hydroxyurea and 1‐β‐D‐Arabinofuranosyl‐cytosine on Deoxyribonucleotide Pools in Mouse Embryo Cells
Authors: Skoog Lambert , NordenskjÖLD Bo 
Published: March 2005

Mouse embryo cells incubated in medium with 0.5% serum contain only few cells which synthesize DNA. When the serum concentration was raised, DNA synthesis was induced and the pools of the deoxyribonucleoside triphosphates expanded. In such cultures the pool of dGTP was the smallest of the deoxyribonucleoside triphosphate pools and its size corresponded to only approximately 30 sec of DNA synthesis. The dCTP pool was 10–20 times larger than the dGTP pool.Addition of hydroxyurea resulted in an almost instantaneous loss of the pool of dGTP and also in a decrease in the size of the dATP pool. In contrast the pools of dCTP and dTTP increased. However, the turnover of the dTTP pool was greatly diminished.Removal of hydroxyurea from the medium resulted in a rapid normalization of the triphosphate pools but only in a more gradual restoration of DNA synthesis.During incubation in 1‐β‐D‐arabinofuranosyl‐cytosine (arabinosyl‐cytosine), the pools of dATP, dGTP and dTTP expanded, while the pool of dCTP showed an initial decrease. The turnover of the dTTP pool was not markedly affected in spite of the fact that DNA synthesis was greatly reduced. When arabinosyl‐cytosine was removed the pools were normalized and DNA synthesis was slowly restored.Our results thus show that the inhibition of DNA synthesis by hydroxyurea is correlated with diminishing pools of dGTP and dATP and a decreased turnover of dTTP, indicating a generally decreased deoxyribonucleotide formation presumably due to the inhibition of the enzyme ribonucleoside‐diphosphate reductase. The inhibition of DNA synthesis by arabinosyl‐cytosine is not correlated with a similar depletion of deoxyribonucleoside triphosphate pools.
Propriétés des ribosomes et du RNA synthétisés par Escherichia coli cultivé en présence d'éthionine
Authors: Beaud Georges , Hayes Donal H. 
Published: March 2005

Escherichia coli EA1 (met−, bio−, RCstr) grown in the absence of methionine but in the presence of ethionine synthesises ribosomes containing ethionine in their proteins, and whose RNA is sub‐methylated. When the sedimentation behaviour of such ribosomes is examined in the presence of low concentrations (100 μM) of Mg++ they are found to consist mainly of 50 S and 30 S particles which resemble normal ribosomes, but to contain some particles which sediment between 30 S and 50 S. Furthermore, the ratio of 30 S to 50 S particles in the ethionine ribosomes is higher than that observed for normal ribosomes. These modifications, in the synthesis of ribosomes seem to result more from the slowing of bacterial growth in the presence of ethionine than from a specific inhibition of the formation of 50 S particles. 50 S and 30 S subunits formed in the presence of ethionine are defective in the capacity to associate at high Mg++ concentration (10 mM) to form 70 S particles. Reconstruction experiments suggest that this defect is inherent in the structure of ribosomes synthesised in the presence of ethionine. When bacteria, cultured in the presence of ethionine, are sub‐cultured in the presence of methionine, or in the presence of methionine and chloroamphenicol their defective ethionine ribosomes regain normal association properties without loss of incorporated ethionine. These experiments led us to study the level of methylability of the RNA of these ribosomes.Using an in vitro system, with an S 100 as a source of methylases, we have shown that the RNA, isolated from ethionine ribosomes, accepts methyl groups from S‐adenosyl‐[Me‐14C]‐methionine while normal ribosomal RNA or RNA isolated from bacteria grown successively in the presence of ethionine and methionine does not. Moreover, we have shown that ethionine ribosomes can be methylated in vitro to a level corresponding to 5–6 methyl groups per 70 S particle. When in vitro methylated ethionine ribosomes are analysed in sucrose gradients the methyl groups are found to be associated both with particles which sediment approximately at 30 S and 50 S and with particles which sediment between 30 S and 50 S. These results suggest that the defect in the association properties of ethionine ribosomes results from the submethylation of their RNA moieties. Preparations of ethionine ribosomes have been found to contain methylases since methylation of these particles occurs when external methylases (S 100) are omitted.
Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria
Authors: Wober Wolfgang , AlaupoviĆ Petar 
Published: March 2005

The so‐called simple proteins isolated from the endotoxin of Serratia marcescens 08 and whole cells Escherichia coli 0 141 :K85(B) by aqueous phenol treatment were characterized by the determination of hydrodynamic properties, electrophoretic behavior, immunochemical specificity and chemical analysis. The chemical composition of both proteins revealed the presence of small amount of lipid A constituents such as glucosamine, phosphorus and fatty acids; the β‐hydroxymyristic acid regarded as the characteristic marker for lipid A was present in all protein preparations. Since the treatment of intact endotoxin or bacterial cells with aqueous phenol results in the formation of two fragments (a lipopolysaccharide and a lipid A‐protein), both of which are characterized by the presence of lipid A constituents, this dissociation seems to be caused by cleavage of a phenol‐sensitive linkage within the molecule of lipid A rather than at its point of attachment to the protein moiety. Thus, the “simple” protein preparations consist of the protein moiety and a small segment of lipid A. Contrary to previous views, such protein preparations are not simple proteins if this designation implies absence of constituents other than amino acids. However, for a historical reason we suggest that the term simple protein be retained for protein fragments isolated by aqueous phenol treatment of intact endotoxins.The simple protein from Serratia marcescens 08 was treated successively with trypsin and pronase. The resulting trypsin and pronase cores contained increasing amounts of lipid A constituents firmly bound to the residual protein moiety. Since lipid A was separated as an entity from the protein moiety only after acid hydrolysis of the pronase core, it is proposed that in intact endotoxin lipid A is covalently linked to the protein moiety. The absence of any detectable glucosamine and fatty acids in the mixture of peptides and amino acids released by trypsin and pronase, indicated a single point attachment between the lipid A and protein moieties.The protein moieties from Serratia marcescens and Escherichia coli were immunogenic and possessed a common antigenic determinant. Both trypsin and pronase cores retained the immunogenicity and revealed the presence of a common antigen with the parent simple protein. This second set of antigenic determinant(s) different from the specific O‐antigens seems to be located in the protein moiety close to lipid A.
The Synthesis of the Alternating Copolymer Poly [r(A‐s4U)] by RNA Polymerase of Escherichia coli
Authors: Cramer Friedrich , Gottschalk Ellen M. , Matzura Hans , Scheit Karl‐Heinz , Sternbach Hans 
Published: March 2005

The transcription of poly [d(A‐T)] by RNA polymerase from Escherichia coli in the presence of ATP and 4‐thiouridine triphosphate (s4UTP) lead to the synthesis of poly[r(A‐s4U)]. The investigation of the polymerisation kinetics furnished the kinetic parameter Km (ATP‐s4UTP) = 0.33 mM and Vmax (ATP‐s4UTP) = 204 nmoles AMP × mg enzyme−1× min−1.The structure of poly[r(A‐s4U)] was proved by nearest neighbour analysis and calculation of the AMP/s4UMP ratio from the absorption spectrum of poly[r(A‐s4U)]. As shown by sedimentation velocity analysis poly[r(A‐s4U)] exhibited a mean s20, w value of 12 S. Enzymatic hydrolysis and thermal denaturation of poly[r(A‐s4U)] revealed the existence of a large hyperchromicity in the absorption spectrum. The absorption‐temperature profile of poly [r(A‐s4U)] measured at 0.08 M Na+ showe a sharp cooperative transition with a Tm of 52°C.
Delayed Fluorescence and the High‐Energy State of Chloroplasts
Authors: Wraight Colin A. , Crofts Antony R. 
Published: March 2005

The relation between the “high energy state” of photophosphorylation and delayed fluorescence has been studied in spinach chloroplasts.
In the presence of an electron acceptor or cofactors, the kinetics of the rise in intensity of light emitted 1 millisecond after illumination showed two distinct phases,‐a rapid phase complete in less than 0.1 s, and a sigmoidal slow phase with a halfrise time of about 0.3 s.
The maximal intensity and time course of emission were found to vary with electron flow and the degree of coupling. With nigericin or with NH4CI, the slow phase of the intensity rise was abolished but the rapid phase was unaffected. With valinomycin the rapid phase was substantially inhibited, but the extent of the slow phase was increased to give the same maximal intensity as observed in the absence of ionophore. With carbony cyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP), or a combination of valinomycin and nigericin, both phases were substantially inhibited. This spectrum of sensitivities has been interpreted as showing that the slow phase depends on the pH component, and a part of the rapid phase on the electrical component, of the electro‐chemical H+‐gradient. Addition of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethyl‐urea always inhibited emission.
A mechanism relating delayed fluorescence to the “high energy state” is suggested. The photochemical act of System II is envisaged as occurring between acceptor and donor sites on opposite sides of the thylakoid membrane and in equilibrium with pools the mid point potentials of which are dependent on the pH of the phase. It is shown that in such a system the energy conserved in the oxido reductive poise and in the electrochemical H+ gradient are additive and that both contribute to a decreased activation energy for delayed fluorescence. By dissipating the H+ gradient, the activation energy requirement is increased so that the intensity of emission falls.
It is concluded that other mechanisms for emission also operate, in particular one dependent on electron flow as such.