bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxygen‐Dependent Cleavage of the Vinyl‐Ether Linkage of Plasmalogens
Authors: Yavin Ephraim , Gatt Shimon
Published: March 2005

The low‐molecular‐weight component of rat brain supernatant, which catalyzed the oxygen‐dependent cleavage of the vinyl‐ether linkage of plasmalogen, was purified by chromatography on SE‐Sephadex and DEAE‐Sephadex. A compound was obtained with an absorption peak at 266 nm. It catalyzed the commensurate vinyl‐ether cleavage and oxygen reduction. Evidence is presented that this purified active component is identical with, or very similar to ascorbic acid. Commercial ascorbic acid mimicked the vinyl‐ether cleaving properties of the active component of rat brain supernatant.

Contrary to the rat brain supernatant, the purified active component was unstable and lost activity on heating or storage. Furthermore, vinyl‐ether cleavage and oxygen uptake ceased after 2–3 min. Addition of a protein of rat brain, which by itself was inactive, extended the reaction time for 15–20 min.

In the course of the reaction, 3.5–4 mol oxygen were consumed and 0.3 mol of ascorbate disappeared for each mole of vinyl‐ether‐linkage cleaved. Small quantities of compounds which reacted with thiobarbituric acid were also formed in the course of the reaction.

The products of the reaction were lysolecithin and several split products produced from the aldehydogenic moiety of the plasmalogen molecule; these reacted with p‐nitrophenylhydrazine. Using silicic acid chromatography, about 10–20% of the total carbonyl‐containing compounds were eluted with chloroform—methanol (95:5, by vol.), these were identified as pentadecanal in the presence of smaller quantities of heptadecanal.

The p‐nitrophenylhydrazones of the products were prepared and separated into 3–4 spots on thin‐layer silica gel plates. The fastest moving spot was identified by infrared and mass spectrometry as a hydroxy‐aldehyde, probably α‐hydroxyhexadecanal.

A reaction mechanism is proposed as follows: A ferrous‐ascorbate complex reduces and forms an “active” oxygen. This attacks the vinyl‐ether‐linkage of the plasmalogen forming a diol. Hydrolysis of this diol yields α‐hydroxy‐hexadecanal; oxidative cleavage of this bond yields pentadecanal.
Zur Kinetik der Glucose‐Aufnahme in Erythrocyten
Authors: Lacko Ladislav , Wittke Barbara , Kromphardt Hedwig
Published: March 2005

A method was developed which allowed the measurement of sugar uptake into red cells after only 4 sec.

The uptake of [14C]glucose from a saline‐glucose medium was followed at two temperatures (0° and 20°C) and at three different trans‐concentrations of non labelled glucose inside the cells: at equilibrium distribution with [Str] = [Scis], for non‐exchange with [Str] = zero at time zero, and for exchange with [Str] = 34 to 35 mM at time zero.

For these conditions, we found linear plots against time, which enabled us to determine the initial velocity for the uptake of label, i.e. the unidirectional influx.

Influx depended on the widely varying cis‐concentration of glucose in the medium in the manner of a simple saturation‐relationship. The parameters Jmax and Km, determined from a reciprocal plot, were different for the three chosen [Str]‐conditions. Some of these parameters were comparable to values of other papers, determined by measuring the efflux of glucose from red cells.

The results were compared with predictions derived from two models of a mobile carrier: the symmetrical model of Levine, Oxender and Stein [1] and the asymmetrical, but passive model of Geck [2]. The relationship between glucose uptake plotted against time and influx plotted against cis‐concentration corresponded to both models. Some, but not all, ratios between the measured parameters matched the predictions of the symmetrical model, whereas all ratios were compatible with the asymmetrical model.

From both models, the translocation resistance comes out to be much greater for the free carrier than for the glucose‐carrier complex: 10 to 30 fold at 20°C and circa 200 fold at 0°C. Hence, the mobility of the free carrier appears to be very much reduced in the cold.
Transcriptional Control of Tryptophanase Synthesis by Cyclic AMP in Escherichia coli
Authors: Ramírez Juan M. , Conde Francisco , DEL Campo Francisca F.
Published: March 2005

When Escherichia coli grows in a casamino acid‐supplemented medium, the removal of amino acids results in the inability to synthesize tryptophanase even in the presence of the specific inducer. However the transcription of the tryptophanase gene is still operative, since readdition of amino acids, even in the presence of an inhibitor of RNA synthesis, elicits enzyme production. Therefore, under these conditions tryptophanase is synthesized in two distinct steps, (a) accumulation of untranslated mRNA during amino acid deprivation, and (b) translation of the previously accumulated mRNA on readdition of amino acids plus an inhibitor of RNA synthesis. The first step is stimulated by cyclic AMP and inhibited by compounds which cause transient repression, while the second step is unaltered by either type of effector. In consequence it is concluded that cyclic AMP controls tryptophanase synthesis at the transcriptional level.
On the Kinetics of the Reaction of Hemoglobin with CO
Authors: Schmelzer Uwe , Steiner Rudolf , Mayer Adalbert , Nedetzka Teja , Fasold Hugo
Published: March 2005

Flash‐photolysis measurements were performed on normal and on chemically modified human adult hemoglobin solutions in the concentration range between 1 to 100 μM (for complete molecule) and in the pH range between pH 5 and pH 9. The energy of the irradiating flash was varied continuously from 20 to 400 Joules. It is shown that the fast reaction observed after complete or partial flash photolysis is pertinent both to dimeric hemoglobin molecules and to tetrameric hemoglobin molecules which are locked in the state of quaternary structure corresponding to completely liganded hemoglobin. The data suggest that an all‐or‐none transition of the quaternary structure of hemoglobin accompanies the third step of the ligation process.
The Study of the Active Center of Human Ceruloplasmin by Iodination
Authors: Vasiletz Ivan M. , Shavlovsky Michael M. , Neifakh Solomon A.
Published: March 2005

To clarify the functional role of amino acid residues in copper binding and in the maintenance of ceruloplasmin catalytic activity, the protein was chemically modified by iodination. It is shown that addition of 20 mol iodine per 1 mol ceruloplasmin mainly produces iodination of tyrosine residues, while the protein modification by higher iodine concentrations (60 mol and more per 1 mol protein) leads also to iodination of histidine. Iodination of ceruloplasmin is followed by loss of copper and the oxidase activity. There is a correlation between copper loss and ceruloplasmin catalytic activity and iodinated histidine content at high concentrations of iodine in the medium. It appears that tyrosine residues are involved in maintaining the active conformational state of ceruloplasmin, while histidine residues directly bind copper in its catalytic center. Peptide maps for 131I‐labeled ceruloplasmin show that most tyrosine peptides of the protein iodinated at small iodine concentrations are localized in the region of acid and alkaline peptides. Histidine peptides, whose iodination in accompanied by decrease in the oxidase activity and copper content, are mainly localized in the neutral peptide region. One of the neutral peptides having a well‐defined Pauly reaction was isolated in a pure form. N‐ and C‐terminal amino acids of the peptide and its amino acid composition were determined.
Structure primaire de la caséine β bovine
Authors: Dumas Bruno RIBADEAU , Brignon Ghislaine , Grosclaude François , Mercier Jean‐Claude
Published: March 2005

This article is the sixth and last of a series devoted to the primary structure of bovine β‐casein [1–5]. In the preceding paper, we presented a partial sequence of this protein in which 156 out of the 209 amino‐acid residues were exactly positioned [5]. Only a few data were given on the tryptic peptides T2 and T3 [1] which corresponded to the unknown part of the molecule. In the present communication, we establish the amino‐acid sequence of these 2 peptides, and discuss the salient features of the β‐casein molecule.

Bovine β‐casein A2 is a phosphoprotein constituted of a single polypeptide chain containing 209 amino‐acid residues. Among them are five phosphoseryl residues. From sequence data, it can be written: Asp4, Asn5, Thr9, Ser11, SerP5, Glu17, Gln22, Pro35, Gly5, Ala5, Val19, Met6, Ile10, Leu22, Tyr4, Phe9, Trp, Lys11, His5, Arg4. Its exact molecular weight is 23982, a little higher than that of αs1‐casein, which is 23616 [6]. The total number of 27 amide groups (Asn + Gln) agrees well with previous data obtained by Pion et al. [7] and Peterson et al. [8]. The average hydrophobicity, calculated according to Bigelow [9], is 1330 and it places β‐casein among the most hydrophobic proteins.

The sequence obtained emphasizes the uneven charge distribution along the peptide chain. A high negative net charge is located in the first 50 residues. The high proline residue content (16.7%) and the rather uniform repartition of this residue exclude the possibility of a high α‐helix content. Furthermore, the complete absence of segments containing more than 2 adjacent prolyl residues seems to preclude the possibility of poly‐L‐proline structure that was expected from optical‐rotatory‐dispersion studies [10].

An interesting extension of the present work is the discovery by Gordon et al [11] that γ‐, TS‐, R‐ and S‐caseins are fragments of β‐casein likely originating from tryptic‐like cleavages of the peptide chain next to the carbonyl groups of some lysyl residues. The γ‐, TS · A2‐ and S‐, TS · B‐ and R‐caseins respectively represent residues 29 to 209, 106 to 209 and 108 to 209. These results explain the occurrence of only one phosphorus atom in the γ‐caseins, the absence of phosphorus in the TS‐, R‐ and S‐caseins [12]. Localization of the substitutions which differentiate the known variants of bovine β‐casein also explains the relationship between β‐, γ‐, R‐, S‐ and TS‐caseins [12,13].

The NH2‐terminal tryptic phosphopeptide, previously analyzed by Peterson et al. [14], has been sequenced independently by Manson and Annan [15]. Their results are identical to those reported in a preceding paper [4].
Conformation of Single‐Stranded Polynucleotides
Authors: Gruenwedel Dieter W.
Published: March 2005

Sedimenting the alternating copolymer, synthetic poly[d(A‐T)], through increasing concentrations of CH3HgOH at pH 6.8 in solutions of different ionic strength reveals that there exists a critical CH3HgOH concentration at which double‐stranded poly[d(A‐T)] becomes denatured. The denaturation reaction is accompanied first by a sudden increase in the rate of sedimentation followed by as sudden a decrease. The change occurs over a narrow concentration range of added CH3HgOH indicating that the transition is highly cooperative. At CH3HgOH concentrations insufficient to induce denaturation, the organomercurial does not interact with poly[d(A‐T)] as judged from the invariance of s20,w with increasing concentrations of CH3HgOH. By contrast, s20,w of denatured poly[d(A‐T)]is strongly dependent on CH3HgOH concentration.

It appears that in solvents of low ionic strength s20,w of single‐stranded poly[d(A‐T)] is determined largely by the increase in mass caused by the addition of the methylmercury group to the various binding sites of adenine and thymine. In more concentrated supporting electrolyte solutions, on the other hand, the variation of s20,w with organomercurial concentration can no longer be explained solely on the basis of a variation in molecular weight but presumably involves also changes in the frictional properties of the polymer.
Effects of Aldosterone on RNA and Protein Synthesis in the Toad Bladder
Authors: Rousseau Guy , Crabbé Jean
Published: March 2005

The hypothesis of a stimulatory effect of aldosterone on RNA, and consequently protein synthesis has been investigated. The influence of this hormone on the incorporation of precursors into RNA and protein was examined in the toad urinary bladder through which sodium transport is stimulated by aldosterone in vitro.

Aldosterone increases total nuclear RNA synthesis and also uridine uptake. Particular fractions of this RNA characterized by density gradient centrifugation might be specifically influenced. No effect of aldosterone on cytoplasmic RNA turn‐over rate or on mRNA synthesis could be demonstrated. Examination of the pattern of protein synthesis at the polysome level, of ribosome processing, and of protein turn‐over rate also failed to disclose any definite influence of aldosterone on these parameters.

The proposal that the effect of aldosterone on sodium transport is mediated by its action on transcription and translation in target tissues remains therefore founded on indirect evidence to date.
Nuclease Degradation and the Structure of Ribosomes
Authors: Pinder Jennifer C. , Gratzer Walter B.
Published: March 2005

Well‐defined patterns of fragments are produced when Escherichia coli ribosomes, both native, dissociated, unfolded, and partially denuded of proteins by treatment with caesium chloride, are digested with pancreatic ribonuclease, and the extracted RNA is fractionated by polyacryl‐amide gel electrophoresis. Procedures are given for determining the degree of congruence of a pair of digestion patterns, and analysing the results in terms of probability of matching. By this means it has been established that all the digestion patterns which have been obtained are closely related. The similarity between native 70‐S ribosomes and completely unfolded nucleo‐protein particles in EDTA indicates that the greater part of the RNA is accessible on the surface of the 70‐S ribosome. It has not been possible to show a close relationship between the patterns generated by the nucleoprotein particles in various states and these from free ribosomal RNA. We infer that a number of the labile points in the RNA are protected by bound proteins. The digestion proceeds much more slowly with increasing stabilisation of structure by magnesium ions, both in free RNA and ribonucleoprotein. It is shown that the labile points of the chain are segments of some length, and not isolated phosphodiester links.
Binding of Analogues of the Antibiotics Distamycin A and Netropsin to Native DNA
Authors: Zimmeer Christoph , Luck Gerhard , Thrum Heinz , Pitra Christian
Published: March 2005

Binding effects of DNA with two distamycin A analogues containing four and five 1‐methyl‐pyrrole residues (distamycin A contains three of these residues) and with a degradation product of netropsin have been investigated by ultraviolet absorbance, melting and circular dichroism measurements.

Increasing numbers of the methylpyrrole groups (3 to 5) enhance the thermal stability of the oligopeptide complex. On the other hand elimination of the basic groups of the netropsin molecule decreases the melting temperature of the oligopeptide · DNA complex enormously. The circular dichroism spectrum of DNA is also less affected by the degradation product compared to netropsin. Circular dichroism spectra of the complexes exhibit a pronounced band at the longer wavelength region and indicate an increasing chirality in the complex with increasing number of chromophores of the oligopeptides.

From the results it is concluded that electrostatic attraction of the oligopeptides by interaction of the propionamidino and guanidino groups with the negative phosphate sites of DNA is important for the formation of the complex. However, non‐ionic forces, which have been discussed earlier, seem to be involved and may be attributed to the chromophore residues oriented in the helical grooves.

The different binding tendency of the oligopeptides with DNA is discussed together with the biological activity of the oligopeptides.
The Pathway of Glutamate Oxidation in Rabbit Reticulocytes
Authors: Rost Johann , Rapoport Samuel M.
Published: March 2005

It is shown by means of experiments on intact cells with 14C‐labelled substrates that the first step in the oxidative metabolism of glutamate in rabbit‐reticulocytes is a transamination with oxaloacetate to form aspartate.

The aspartate is metabolized further in such a manner that CO2 is formed from all C‐atoms. With tracer amounts of glutamate no accumulation of aspartate occurs, in contrast to isolated mitochondria. The mechanism of the utilization of aspartate appears to be a decarboxylation of oxaloacetate to pyruvate, which is the main reaction of oxaloacetate under conditions of loading with glutamate.

On account of the minute contribution of the amino group of glutamate to the ammonia formation, which in itself is smaller than expected from the CO2 formation, an acceptor for the amino group of amino acids is postulated.
Aminoacylation of Fragment Combinations from Yeast tRNAPhe
Authors: Thiebe Rainer , Harbers Klaus , Zachau Hans G.
Published: March 2005

The aminoacylation of 15 fragments from yeast tRNAPhe and of various mixtures of these fragments was studied in detail. The aminoacylation conditions were systematically varied in order to obtain maximal incorporation of phenylalanine.

No fragment by itself accepted phenylalanine.

Fragment combinations in which parts of the anticodon loop or the dihydrouridine loop were missing, accepted phenylalanine to an extent of 20–75% compared to intact tRNAphe. Modifications in both regions together inactivated the molecule. With the anticodon stem and/or the minilooop missing, low but significant activity was found. Splits in the dihydrouridine loop and in the T‐Ψ‐C loop together did not abolish the phenylalanine acceptance. Combinations in which parts of the dihydrouridine stem or of the T‐Ψ‐C stem were missing could not be amino‐acylated.

Complete association of complementary fragments was observed in all cases. The results of charging and heating experiments, however, indicate the coexistence of different fragment combinations in a fragment mixture. The incomplete aminoacylation of fragment mixtures is explained by the presence of fragment combinations with an unsuitable three‐dimensional structure.

The rate of aminoacylation was lower for fragment combinations than for tRNAPhe. The Michaelis constants of 3 fragment combinations were identical to the one of tRNAPhe. Two fragment mixtures inhibited the rate of charging of tRNAPhe.

Phenylalanine was the only amino acid which was accepted by the fragment mixtures with synthetases from yeast or Escherichia coli. No mischarging was observed.

The results together with those of related publications are discussed with respect to recognition sites of tRNAPhe for the cognate synthetase.
Studies on T4 Lysozyme
Authors: Jensen Harald B. , Kleppe Kjell
Published: March 2005

T4 lysozyme has been purified to apparent homogeneity from lysates of T4‐infected Escherichia coli B cells. In the purification procedure advantage was taken of the affinity of the enzyme for chitin. Binding of the enzyme to chitin under suitable conditions was found to be very efficient and complete, depending on the pH and ionic strength of the medium. The conditions for adsorption and desorption are different from those of hen egg‐white lysozyme. The enzyme could be desorbed from the chitin columns by increasing the ionic strength at a low pH.
A Model of Bacterial Flagella Based on Small‐Angle X‐Ray Scattering and Hydrodynamic Data which Indicate an Elongated Shape of the Flagellin Protomer
Authors: Bode Wolfram , Engel Jürgen , Winklmair Dieter
Published: March 2005

An arrangement of protomers in flagella is suggested which is based on the finding that flagellin molecules in solution are highly elongated. The model offers an explanation of the V‐shaped arrow tips and tail ends in contrast to earlier models constructed from spherical subunits. A number of protomers of 8 to 10 follows from the mass per length ratio of flagella obtained from smallangel X‐ray scattering. The latter is consistent with a hollow cylinder of outer diameter 15 nm and inner diameter 3 nm.
Caractérisation des variants génétiques des caséines αsl et β bovines
Authors: Grosclaude François , Mahé Marie‐Françoise , Mercier Jean‐Claude , Ribadeau‐Dumas Bruno
Published: March 2005

The three major casein species of cow's milk are known to show a genetic polymorphism: four variants of αsl‐casein (A, B, C, D), five of β‐casein (A1, A2, A3, B, C) and two of χ‐casein (A, B) have been discovered until the present time in the western breeds of cattle (Bos taurus) (see review by Aschaffenburg [1]). These variants, whose relative mobilities in gel electrophoresis at pH 8.6 and at pH 3.0 are shown in Fig.1, represent markers of the structural loci synthesizing the three casein species (αsl‐Cn, β‐Cn and χ‐Cn).

The salient feature of the genetics of cattle caseins lies in the very close linkage of these three loci, which are likely to be clustered on the chromosome [2–5]. As a consequence of this close linkage, associations of αsl‐, β‐and χ‐casein variants are transmitted en bloc, as “gene complexes”. Our interpretation of available data on the frequencies of the gene complexes in cattle populations had led us to propose a phylogeny of the genetic variants of αsl‐and β‐caseins, and also to suggest that the mutation sites corresponding to the more common ones are particularly close together on the chromosome [6]. These hypotheses prompted us to identify and to position the alterations specific for each of the variants of αsl‐ and β‐caseins.

We have already, in recent publications, characterized the αslC, αslA and βA3 variants [7–9].

The present work, which in the case of β‐casein was guided by indications on amino‐acid substitutions given by Pion et al. [10], Peterson et al. [11], Groves and Gordon [12], and which refers to the sequences of αsl B and βA2 caseins recently established by Mercier et al. [13] and Ribadeau‐Dumas et al. [14], deals with the four other variants, αslD, βA1, βB, βC.

The variant αsl D differs from αsl B in that the alanyl residue in position 53 of the polypeptide chain is replaced by a phosphothreonyl residue (53 Ala → ThrP). There is no serine → proline substitution as previously proposed [15].

Compared to βA2, the three variants βA1, βB and βC all differ by a 67 Pro → His substitution.

In βB, in addition to the 67 Pro → His substitution, the seryl residue in position 122 is replaced by an arginyl residue, as previously mentioned [16].

In βC, in addition to the 67 Pro → His substitution, the glutamyl residue in position 37 is replaced by a lysyl residue, and the seryl residue in position 35 is no more phosphorylated.

Table 3 summarizes our results on all the genetic variants of αsl‐ and β‐caseins. These results make it possible to deduce phylogenic relationships (Fig. 6) that support and expand our former hypotheses [6]. The concept that the initiator end of β‐Cn locus is contiguous to the terminal end of αsl‐Cn locus accounts for the rare occurrence of recombinant types between αsl‐Cnc and β‐CnA1, β‐CnB, β‐CnC, since the distances between the corresponding mutation sites would be only 73, 128 and 43 codons, respectively.
Role of the Redox State in Ethanol‐Induced Suppression of Citrate‐Cycle Flux in the Perfused Liver of Normal, Hyper‐ and Hypothyroid Rats
Authors: Lindros Kai O.
Published: March 2005

The mechanism of the ethanol‐induced suppression of the citric acid cycle has been studied by single‐passage perfusion of livers from normal, triiodothyronine‐treated and propylthiouraciltreated rats.

Ethanol oxidation increased the lactate/pyruvate ratio in the perfusion medium to about 50 and depressed the 14CO2 formation from [1‐14C]acetate and [1‐14C]hexanoato (taken to reflect citrate cycle flux) by 67% and 60%, respectively, in normal livers. In the livers of the hyperthyroid animals ethanol suppressed [1‐14C]acetate oxidation by 22% and [1‐14C]hexanoate oxidation by 34%, and the lactate/pyruvate ratio was only increased to about 20. In the hypothyroid group ethanol enhanced the lactate/pyruvate ratio slightly more than in the controls and suppressed the oxidation of acetate and hexanoate by 79 and 62%, respectively.

Hepatic ethanol elimination was slightly slower in the hyper‐ and hypothyroid groups than in the controls. Hexanoate (1 mM) stimulated oxygen uptake in all groups but inhibited ethanol oxidation by 45% in the hyperthyroid group and only by 18% in the hypothyroid group. Pyruvate, on the other hand, stimulated ethanol elimination only slightly in the hyperthyroid group, by about 50% in the controls and by about 70% in the hypothyroid group.

Oxidation of acetate and hexanoate was also depressed by acetaldehyde. This effect was potentiated by pyrazole, which blocked the reduction of acetaldehyde and caused an increased shift in the lactate/pyruvate ratio, thus indicating that acetaldehyde also affects the citric acid cycle by causing a shift in the redox state.

A highly significant correlation was observed between the effect of ethanol on the output of 14CO2 from the labelled substrates and the shift in the lactate/pyruvate ratio. Experiments with different input levels of lactate and pyruvate demonstrated that the citric acid cycle is susceptible to the fall in pyruvate resulting from the shift in the redox state during ethanol oxidation.
Ribosomal Subunits from Rat Liver
Authors: Reboud Anne‐Marie , Arpin Monique , Reboud Jean‐Paul
Published: March 2005

Ribosomal subunits from rat liver were prepared with a mild method which consisted of centrifuging ribosomes through a sucrose layer containing 0.3 M KCl. Isolated 40‐S subunits were obtained completely pure and 60‐S subunits were contaminated by less than 5% of 40‐S. In the presence of Mg2+ subunits reassociated into monosomes, which had high activity in polyphenylalanine synthesis. 40‐S, but not 60‐S, subunits could bind [3H]poly(U) and [14C]phenylalanyl‐tRNA.

Both subunits could be lyophilized and then kept at room temperature without detectable change in activity nor in sedimentation profile.

Determination of the chemical composition of both subunits showed that 40‐S had a higher protein content than 60‐S subunits. However concentrated‐salt washings of the 40‐S resulted in step‐wise extraction of proteins without inactivation of the subunits, whereas extraction with salt of a small amount of proteins from the 60‐S subunits completely inactivated them.

Study of thermal inactivation showed that 40‐S had a significantly higher heat stability than 60‐S subunits.
Ribosomal Subunits from Rat Liver
Authors: Reboud Anne‐Marie , Buisson Monique , Reboud Jean‐Paul
Published: March 2005

Large subunits from rat liver ribosomes, dialyzed against monovalent cation‐free buffer, were completely inactive in polyphenlyalanine synthesis and failed to reassociate with standard 40‐S subunits. Small subunits dialyzed under exactly the same conditions kept about 2/3 of their‐activity in poly(U)‐directed [14C]phenylalanyl‐tRNA binding or protein synthesis and could reassociate with standard 60‐S subunits. The nature of the monovalent cation present in low concentrations in the dialysis buffer was important in determining the activity of the 60‐S, but not of the 40‐S subunits: K+, Rb+ and NH+4 were more effective while Cs+, Li+ and Na+ were less effective.

The various monovalent cations could be used at higher concentrations to prepare active subunits, except for Cs+ and Li+ which selectively inactivated the 60‐S subunits while modifying their conformation.

Experiments with bivalent cations also showed the cationic content of the medium to be more critical for maintaining the functional form of 60‐S than of 40‐S subunits.
A General Strategy for Parameter Estimation from Isosteric and Allosteric‐Kinetic Data and Binding Measurements
Authors: Reich Jens Georg , Wangermann Gert , Falck Margrit , Rohde Klaus
Published: March 2005

A strategy suitable for computer programming is presented which permits the estimation of kinetic parameters and of their range of statistical variation from initial‐rate measurements or from binding data. It can be used for steady‐state rate equations, rapid‐equilibrium mechanisms, binding kinetics, descriptive models such as Adair's or Hill's equations, and for the study of more complicated allosteric models. The desired kinetic equation is addressed from a model catalogue.

The strategy consists of four main steps: (1) formulation of a goodness‐of‐fit criterion using the concept of the distance between observed and predicted behaviour of the model, avoiding assumptions about the statistical structure; (2) localization of the best fit, i.e. of the parameter point of minimum distance from the data by a search algorithm derived from Marquardt's method; (3) exclusion of discrepancies between model and data by testing the randomness of the vector of residual deviations and the structure of the information matrix; (4) exploration of the parameter variability based on a linear approximation of the fit criterion.

Input to the programme are ligand concentrations and measured‐response data, a code number of the desired model, and a guessed starting point of the parameters which, in contrast to older methods, need not be precise. The output is either a set of parameter values with a respective region of variation, supplemented by tables and graphs of the fit obtained, or the rejection of the data with comments on the reason of failure. The possible reasons include statistical fluctuation or systematic trends in the data, lack of required information (poor experimental design), and inappropriate model (non‐unique or inconsistent kinetic model).

Tests on isosteric and allosteric kinetic data lead to the conclusion that the convergence of the strategy is very good when the fit surface is well defined (good data and correct model).
Single‐Chain Structure of Human Ceruloplasmin
Authors: Rydén Lars
Published: March 2005

In many reports human ceruloplasmin has been shown to contain several polypeptide chains. Their molecular weights have been estimated to 17000 and 59000. These findings formed the basis for proposed models of ceruloplasmin quarternary structure. Recently it was however observed, that the low molecular weight peptide chains found in commercial samples of human ceruloplasmin are absent if the protein is prepared directly from fresh serum, and evidence was presented that the earlier observed subunits were the result of limited proteolysis.

The subunit structure of human ceruloplasmin has now been reconsidered using the new preparation. The reduced and cyanoethylated protein has been studied in solutions of 6 M guanidine hydrochloride. Its intrinsic viscosity and sedimentation coefficient, as well as data from gel filtration on calibrated columns of agarose gels are consistent with the presence of a single continuous polypeptide chain of about 1050 amino acid residues with a molecular weight around 120000. Together with the carbohydrate this accounts for the molecular weight of the native protein as determined by sedimentation equilibrium.
The Amount of Ribosomal RNA Genes in Tetrahymena pyriformis in Different Physiological States
Authors: Engberg Jan , Pearlman Ronald E.
Published: March 2005

DNA‐RNA hybridization studies have been performed to determine the amount of rRNA genes present in purified nuclear DNA of Tetrahymena pyriformis cultured under various conditions of growth. It was found that 0.32% of the nuclear DNA isolated from exponentially growing cells hybridizes with an equimolar mixture of 17‐S and 25‐S rRNA. Nuclear DNA isolated from starved cells and from cells in the stationary growth phase hybridizes 30–40% less with rRNA than the nuclear DNA of the exponentially growing cells. This variation of the amount of rRNA genes to the total amount of nuclear DNA suggests that the replication of the rRNA genes and the rest of the nuclear DNA are under separate controls. With the possible exception of a minor homology between 17‐S and 25‐S rRNA, the saturation and competition experiments indicate that there are separate genes for the two classes of rRNA.
The Self‐Association of Rabbit‐Muscle Phosphofructokinase
Authors: Leonard Kevin R. , Walker Ian O.
Published: March 2005

The sedimentation pattern of phosphofructokinase indicates that the enzyme undergoes a self‐association reaction. Alkaline pH and high ionic strength prevented the reaction. At pH 10.5 fructose 1, 6‐bisphosphate stabilised the “monomer” of the self‐association, which had s020,w= 11.9 ± 0.4 S, and D020,w= 3.22 ± 0.33 × 10−7 cm/sec giving a molecular weight of 340000. Sedimentation equilibrium gave a molecular weight of 330000 ± 12000.

The Z‐average molecular weight, measured at pH 8 by sedimentation equilibrium, increased in a sigmoid fashion with concentration from 340000 at 0.5 mg/ml to 2000000 at 7 mg/ml. By comparing the experimental curve for the variation of molecular weight versus concentration with calculated curves for open and closed polymerisations, it is suggested that the monomer associates to give a closed hexamer probably via an intermediate dimer or trimer which is present in very small amounts. The apparent association constant at pH 8.0 and 20°C was 6 × 1023 M−1 for the monomer‐hexamer reaction.

The minimum molecular weight obtained in 6 M guanidine hydrochloride, 0.1 M 2‐mercaptoethanol was 85000 ± 10000 measured by a sedimentation velocity method and 76000 ± 5000 measured by sedimentation equilibrium. This implies that the “monomer” is composed of four polypeptide chains of approximately equal molecular weight.
Kinetic Properties of a Soluble Catechol O‐Methyltransferase of Human Liver
Authors: Ball Peter , Knuppen Rudolf , Haupt Margitta , Breuer Heinz
Published: March 2005

When 2‐hydroxyoestradiol‐17β was incubated in the presence of S‐adenosylmethionine with a 380‐fold purified catechol O‐methyltransferase from human liver, 2‐methoxyoestradiol‐17β and 2‐hydroxyoestradiol‐17β 3‐methyl ether were identified as the only metabolites. No dimethylation, transmethylation or demethylation was observed.

The purified enzyme was active only in the presence of cysteine and divalent cations; magnesium was found to be the most effective cation. The temperature optimum for the methylation of 2‐hydroxyoestradiol‐17β was 42°C; the activation energy amounted to 20.9 kcal/mol. The enzyme activity had two pH optima; the pH optimum between 6.8 and 8.4 was due to maximal formation of 2‐methoxyoestradiol‐17β, whereas the pH optimum at 9.2 was due to the formation of both the 2‐ and 3‐monomethyl ether.

The formation of the two isomeric monomethyl ethers increased to a maximum at a substrate concentration of 50 μM (Km value 14 μM) and then decreased with an inflection point between 75 and 100 μM (K1 value under standard assay conditions 95 μM). Whereas both monomethyl ethers were formed at the same rate up to 50 μM substrate concentration, the decrease of formation of 2‐hydroxyoestradiol‐17β 3‐methyl ether was more pronounced than that of 2‐methoxyoestradiol‐17β. Both substrate inhibition and the ratio of methylation depended on the concentrations of S‐adenosylmethionine and MgCl2. Product inhibition was also demonstrated with K1 values of 24 μM for 2‐methoxyoestradiol‐17β, 80 μM for 2‐hydroxyoestradiol‐17β 3‐methyl ether and 39 μM for S‐adenosylhomocysteine. In contrast, increasing amounts of S‐adenosylmethionine did not produce inhibition or changes of the ratio of methylation (Km value 8.5 μM).
Cell Envelope and Shape of Escherichia coli K12 Properties of a Temperature‐Sensitive rod Mutant
Authors: Henning Ulf , Rehn Kurt , Höhn Barbara , Braun Volkmar , Sschwar Uli
Published: March 2005

A mutant of Escherichia coli K12 is described which, depending on temperature and Mg2+ concentration, can grow as a rod or sphere. The mutational defect was traced to an alteration in the cytoplasmic membrane. Rod‐shaped cells ànd spherical cells did not differ measurably in the chemistry of their murein.
The Reversible Inactivation of Rat‐Liver Arginase at Low pH
Authors: Hosoyama Yoshiyuki
Published: March 2005

Rat liver arginase (l‐arginine amidinohydrolase) is inactivated by treatment with acid at 0 °C.

The inactivated or denatured enzyme can readily have its catalytic activity restored by incubation at 37 °C in neutral buffer. The amount of reactivation that occurrs depends on the Mn2+ ion concentration in the renaturation medium. The rate of renaturation also depends on Mn2.

Disc gel electrophoresis shows that dissociation into subunits occurrs by acid denaturation, the subunits having molecular weights of about 64000 and 32000, at pH 4, and 2 respectively.

The renaturation process is temperature dependent and shows first‐order kinetics. ΔH and ΔS for renaturation are 6.0 kcal/mol and – 29.8 cal/K, respectively, at 37 °C.

Some annealing process is important for renaturation and it may be a rate‐limiting process which makes the renaturation process an apparently first‐order reaction.

The renatured enzyme has the same optimum pH and Km as the native enzyme but loses its original activity in the neutral pH region.
Some Comparative Aspects of Carbamylation of Human Blood and of Hemoglobin with Cyanate and Carbamyl Phosphate
Authors: Carreras‐Barnes José , Diederich Dennis A. , Grisolia Santiago
Published: March 2005

Carbamylation of hemoglobin in the intact erythrocyte proceeds more readily with cyanate than with carbamyl phosphate. Carbamylation of hemoglobins A and S with carbamyl phosphate is lower at pH 6 than at pH 7 and even more so in the presence of azide. Carbamylation of insulin and histone also proceeds more readily with cyanate than with carbamyl phosphate. Mg2+, which diminishes carbamyl phosphate decomposition to cyanate, decreases also histone carbamylation with carbamyl phosphate. At pH 3.9 and in the presence of azide no histone carbamylation with carbamyl phosphate could be detected. On the bases of these experiments it is concluded that carbamylation of hemoglobin and other proteins with carbamyl phosphate proceed through cyanate. Rapid incorporation of potassium [14C]cyanate and [14C]carbamyl phosphate into erythrocyte ghosts was obtained. A discussion of the above results in relation to the suggested use of hemoglobin in sickle cell disease is presented.
Regulation of Ribosome Formation in Regenerating Rat Liver
Authors: Rizzo Arthur J. , Webb Thomas E.
Published: March 2005

The rate of formation of the 45‐S ribosomal precursor RNA in regenerating liver is approximately 50% higher at 19–26 h post‐operative, when the rate of ribosome formation is high, than at 36–42 h post‐operative, when the rate is returning to pre‐operative levels. Although a 1‐h treatment with cycloheximide or puromycin had no effect on the synthesis of the 45‐S precursor, the processing to nuclear 18‐S and 28‐S RNA was reduced approximately 20% in both cases, indicating that a protein(s) involved in the processing (possibly) ribosomal proteins becomes limiting in the 42‐h and even more so in the 19‐h regenerating liver. Cycloheximide, which preserves the polyribosome structure, had no effect on the transport of ribosomal sub‐units to the cytoplasm in the 42‐h regenerating liver. In contrast, puromycin, which causes the conversion of these functional components to inactive monomers, inhibited transport by over 75%. The results indicate that ribosome formation in regenerating liver is subject to control at both the transcriptional and post‐transcriptional levels.
Comparative Electron‐Microscope Studies on the Collagens Extracted from Cartilage, Bone, and Skin
Authors: Stark Marlies , Miller Edward J. , Kühn Klaus
Published: March 2005

Long spacing segment crystallites, prepared from soluble chick bone and skin collagen with the chain composition [α1(I)]2α2 and soluble chick cartilage collagen with a chain composition [α1(II)]3, were compared in electron microscope studies after staining with phosphotungstic acid and uranyl acetate. Although comparison of bone and skin collagen long‐spacing segment preparations with similar preparations of cartilage collagen revealed several differences in intensities of cross‐striation bands, no differences in the positions of the cross‐striation bands could be observed in the three preparations, indicating that the distribution of polar and apolar regions along the cartilage collagen molecule is markedly similar to that of molecules derived from bone and skin collagen.

Comparison of segment long‐spacing crystallites prepared from the various collagens also showed that the cartilage collagen is longer than the bone or skin collagen molecule by about 10–12 nm. The additional length of the cartilage collagen molecule is clearly discerned as an extra band located in the COOH‐terminal region, and is removed by treatment of the collagen with pepsin prior to precipitation as segment long‐spacing crystallites, indicating that this region contains one or more sequences in which the collagen helix cannot form.

In studies on fibrils formed by the three collagen preparations, it was noted that bone and skin collagen could be readily precipitated as broad, well‐structured fibrils in which a banding pattern characteristic of native type fibrils could be easily observed. On the other hand, cartilage collagen, precipitated under identical conditions, formed slightly smaller fibrils in which a banding pattern commonly associated with native type collagen fibrils was only faintly visible in certain segments of the fibrils. These results suggest that the relatively large number of bulky carbohydrate prosthetic groups covalently attached to hydroxylysine residues in the α1(II) chains of cartilage collagen sterically hinder the deposition of staining materials and thus prevent the clear manifestation of the banding pattern in fibrils of cartilage collagen.
N6‐Monobutyryladenosine 3′ : 5′‐Monophosphate as the Biologically Active Derivative of Dibutyryladenosine 3′ : 5′‐Monophosphate in HeLa S3 Cells
Authors: Kaukel Eckhard , Mundhenk Klaus , Hilz Helmuth
Published: March 2005

N
6,O2′‐dibutyryl cyclic AMP and O2′‐monobutyryl cyclic AMP had practically no affinity to Gilman's cyclic‐AMP‐binding kinase from muscle, while the N6‐monobutyryl derivative was bound to the protein to nearly the same degree as cyclic AMP.

N
6‐monobutyryl cyclic AMP was the only cyclic nucleotide accumulating inside HeLa cells when cultures were incubated with N6,O2′‐dibutyryl cyclic AMP. N6‐monobutyryl cyclic AMP was also accumulated when cell homogenates acted on N6,O2′‐dibutyryl cyclic AMP, while cyclic AMP and O2′‐monobutyryl cyclic AMP were rapidly degraded.

The results indicate that N6‐monobutyryl cyclic AMP is the true intracellular imitator of cyclic AMP when N6,O2′‐dibutyryl cylic AMP is added to tissues.
The Lysines in Liver Alcohol Dehydrogenase
Authors: Mckinley‐McKee John S. , Morris David L.
Published: March 2005

Liver alcohol dehydrogenase is reversibly inactivated by pyridoxal 5′‐phosphate. When the enzyme is saturated with pyridoxal‐P the residual activity is about 20%. The enzyme can be completely inactivated by successive covalent labelling with pyridoxal‐P, using sodium borohydride reduction. A maximum of about 11 pyridoxal‐P groups per subunit were covalently incorporated into the enzyme.

The binding to the enzyme of several known inhibitors was investigated by studying their effect on the reversible inactivation of the enzyme by pyridoxal‐P at pH 8.0. The effect of these ligands on the activation of the enzyme by methyl picolinimidate was also studied. AMP protected competitively against inactivation by pyridoxal‐P and also protected strongly against activation by methyl picolinimidate. After picolinimidylation of the enzyme the binding of AMP at pH 10 was 8‐fold weaker.

AMP and NADH both protected one lysine residue per subunit from reaction with pyridoxal‐P. It is concluded that there is one lysine residue at the active centre of the enzyme and that this lysine plays an important part in binding the coenzyme by attracting the phosphate group of the AMP moiety of the coenzyme. Coenzyme binding to the picolinimidylated enzyme is weakened by steric interference between the picolinomidylated active‐centre lysine and the AMP moiety of the coenzyme. Since effects of neutral zinc‐binding ligands cannot be explained by steric or electrostatic effects, it is suggested that they produce conformational changes in the enzyme, perhaps by binding at a site other than the active‐centre zinc atom.
Kinetic Studies of the Interaction of Pigeon‐Liver NAD Kinase with Adenine Nucleotides and Divalent Cations
Authors: Apps David K. , Marsh Adrian
Published: March 2005

NAD kinase is inhibited by free Mg2+, free ATP and by a number of other adenine nucleotides.

Inhibition by ATP, ADPMg−, ADP‐ribose2− and AMP2− is competitive with respect to both ATPMg2− and NAD+.

ATP appears to bind equally well to the free enzyme and to binary enzyme · substrate complexes.

The other adenine nucleotides studied have markedly less affinity for the free enzyme than for binary complexes.

Mg2+ probably inhibits by forming a complex with NAD+, which binds to the enzyme but is inactive in the reaction.

Low concentrations of Mg2+ reduce KmNAD+ in an unexplained manner.

ATPCa2− is a substrate for NAD kinase, but the rate‐limiting step of the reaction may be different when other metals activate the enzyme.
Structural Studies on the Coat Protein of Alfalfa‐Mosaic Virus
Authors: Kraal Barend , DE Graaf J. Martien , Bakker Ton A. , VAN Beynum Gerard M. A. , Goedhart Menno , Bosch Leendert
Published: March 2005

The investigation described in this paper deals with various aspects of the primary structure of the coat protein of the plant virus alfalfa‐mosaic virus (AMV 425). The N‐terminus of this protein has been identified as N‐acetylserine, the C‐terminal structure is ‐Arg‐His · COOH. Cyanogen bromide cleavage of the reduced and carboxymethylated protein yields four major peptide fragments which is in accordance with the presence of three methionine residues in the intact protein chain. These fragments have been separated and characterized by amino acid analysis, electrophoretic behaviour on polyacrylamide gel, end‐group determination and molecular weight. Together they account for all the amino acid residues in the entire protein chain. The alignment of the peptide fragments along the chain is presented. The data lend strong support to the molecular size of this protein reported previously from this laboratory.
Comparison of Polypeptide‐Chain Structure of Four Mammalian Ceruloplasmins by Gel Filtration in Guanidine Hydrochloride Solutions
Authors: RydÉN Lars
Published: March 2005

In a recent paper in this journal it was shown that human ceruloplasmin contains a single continuous polypeptide chain composed of about 1050 amino acid residues. Together with the carbohydrate this accounts for the molecular weight of the native protein, 132000. In contrast to this porcine ceruloplasmin has been reported to contain two subunits of molecular weight 70000–80000. This was the reason for performing a direct comparison of polypeptide‐chains from ceruloplasmin from different mammalian sera.

Ceruloplasmin was prepared from sera of man, pig, horse and rabbit. The reduced and alkylated proteins were gel filtered on a calibrated column of 6% agarose in 6 M guanidine hydrochloride. The four proteins all contained a single main component eluting in the same position within errors of measurements. It is concluded that the four proteins investigated all contain a single polypeptide chain of very similar size.