bluuebunny/crossref_metadata_2025 huggingface dataset:
Die Biosynthese von 3‐Hydroxymethylpyridin bei Mycobacterium bovis Stamm BCG
Authors: GROSS D. , FEIGE A. , SCHÜTTE H. R. , ZURECK A.
Published: March 2005

Mycobacteria are able to produce nicotinic acid and 3‐hydroxymethylpyridine (pyridine‐3‐carbinole). Experiments using growing cells of Mycobacterium bovis strain BCG showed that applied 14C‐labelled nicotinic acid is utilized to form radioactive 3‐hydroxymethylpyridine. The resulting incorporation rate was comparatively high. On feeding resting cells with 3‐hydroxymethyl‐[4,6‐14C]pyridine, the reverse reaction was also noticed.

Isolated end‐products were degraded to estimate the isotope distribution. The radioactivity of applied [carboxy‐14C]nicotinic acid was almost entirely found in the carbinolic moiety of the isolated pyridine‐3‐carbinole, whereas the label of administered 3‐hydroxymethyl‐[4,6‐14C]‐pyridine was localized in the pyridine nucleus of the isolated nicotinic acid.

Results obtained from these feeding experiments showed a direct biogenetic relationship between nicotinic acid and pyridine‐3‐carbinole.
The Purification of Pig Brain Mitochondrial Monoamine Oxidase
Authors: TIPTON K. F.
Published: March 2005

A simple procedure for the extraction and purification of monoamine oxidase from pig brain mitochondria is described. The enzyme purified in this way appears to be homogeneous by cellulose acetate electrophoresis and the molecular weight was estimated to be approximately 102,000 by gel‐filtration. The purified enzyme is inhibited by iproniazid, chelating agents and a sulphydryl reagent. The Km value for tyramine has been determined as has its Ki value for high substrate inhibition.
Biochemical Studies on Lipopolysaccharides of Salmonella R Mutants
Authors: DRÖGE W. , RUSCHMANN E. , LÜDERITZ O. , WESTPHAL O.
Published: March 2005

Analyses performed on the lipopolysaccharides of several Salmonella R‐mutants as well as on their acid degradation products revealed differences in their phosphate content. Two groups of lipopolysaccharides (P+ and P−) could be distinguished, differing by approximately one phosphate group per two heptose units.

The additional phosphate group in P+ lipopolysaccharides is linked to the heptosyl‐heptose unit, since heptose phosphate esters could be isolated by the procedure of Slein and Schnell. When degraded P+ polysaccharides were subjected to methylation and methanolysis no peaks of methylated or partially methylated heptose were detected by gas chromatography.

Gel filtration on Sephadex revealed that P+ degraded polysaccharides contain heterogenous fractions of higher molecular weight, while the corresponding hydrolysates from P− mutants contain only oligosaccharides of lower molecular weight. Accordingly, P−“lipopolysaccharides” are termed “glycolipids”. It is concluded, that the additional phosphate groups function as diester bridges, thus causing the formation of larger molecules.

P+ lipopolysaccharides and P− glycolipids of corresponding chemotypes were found to be distinct serologically when tested by hemagglutination inhibition.
Rapidly Labelled High Molecular RNA from Rat Liver
Authors: SCHÜTZ G. , GALLWITZ D. , SEKERIS C. E.
Published: March 2005

The rapidly labelled RNA extracted from rat liver homogenate after a 4 min pulse sediments heterogeneously from 10 S to 80 S. After 30 min labelling time an accumulation of radioactivity between 30 S and 50 S is observed. A comparison of these sedimentation patterns of RNA derived from different labelling times indicates a greater metabolic instability of the rapidly labelled heterogeneously sedimenting RNA in relation to that of the 45 S r‐precursor.

The RNA sedimenting heavier then 45 S has a base composition which resembles that of DNA. The rapidly labelled RNA is resistant to DNase, pronase and EDTA treatment and thus aggregate formation with protein, DNA and bivalent cations is unlikely.

Experiments with actinomycin D and cold orotic acid show a shift of radioactivity to lower sedimentation values which also reflects the metabolic instability of the heavy RNA. Labelling of the ribosomal peaks after actinomycin D treatment could be achieved only after the 30 min pulse, and not after the 4 min pulse, suggesting the absence of an appreciable amount of r‐precursor in the shorter pulse.

The RNA extracted from nuclei prepared with Triton X‐100 remained in the upper fractions when analyzed on a sucrose gradient, reflecting the high susceptibility to degrading enzymes.

The major part of the rapidly labelled RNA remains associated with the nuclear fraction.
Molecular Weight and Quaternary Structure of Lactic Dehydrogenase
Authors: JAENICKE R. , KNOF S.
Published: March 2005

Isoenzymes of lactic dehydrogenase from different sources have been reported to show identical molecular weights between 115,000 and 154,000. As compared to the protomer weight of 36,000 this corresponds to association numbers between 3 and 4 suggesting either dissociation‐association equilibria between the native enzyme and its protomers or systematic errors in the methods applied.

To clarify this alternative the monodispersity of the homogeneous heart (H4) and muscle (M4) isoenzymes has been analysed comparing the mean values M̄ from sedimentation, light scattering and osmosis.

Considering all correction terms in the evaluations, the results in the total range of enzymic activity clearly establish the monodisperse tetramer as the native unit giving M̄s,D=M̄w=M̄n= 142,000 ± 3,600. No anomalous dependence of the molecular data on protein concentration and temperature have been observed on calculating this figure.

Dissociation into subunits requires high net charge or high ionic strength ás well as high concentrations of guanidine. HCI, or urea at high ionic strength. Under all conditions dissociation is accompanied by conformational changes thus restricting a more detailed insight into the native quaternary structure in solution. Regarding the interprotomer binding sites variation of the medium proves Coulombic attractions to prevail in the stabilization of the tetrameric state.
A Correlation Study between Myocardial Substrate Levels and the Blood Glucose Level in Normal, and in Acute and Chronic Alloxan‐Diabetic Rats in vivo
Authors: KRAUPP O. , ADLER‐KASTNER L. , NIESSNER H. , PLANK B.
Published: March 2005

A correlation study has been carried out on data obtained by Kraupp et al. [1] for the myocardial levels of citrate, malate, 2‐oxoglutarate, acetoacetate, 3‐hydroxybutyrate, intracellular glucose (estimated), glucose 6‐phosphate, fructose 6‐phosphate, fructose 1,6‐diphosphate, pyruvate, lactate, glycogen, ATP and free fatty acids (FFA) in relation to the corresponding blood glucose level in I‐normal, II‐acute alloxan‐diabetic, and III‐chronic alloxan‐diabetic rats in vivo. A variation in blood glucose level within each group had been achieved by starvation of about 50% of the animals.

In the normal rats there was a significant, negative correlation between the blood glucose level and the myocardial levels of citrate, malate and 2‐oxoglutarate, acetoacetate and 3‐hydroxybutyrate, and glycogen. A significant, positive correlation was obtained between the blood glucose level and the estimated intracellular glucose level and the fructose 1,6‐diphosphate/fructose 6‐phosphate ratio in the myocardium.

In the diabetic rats, a generally reverse pattern was obtained. The acute alloxan‐diabetic rats showed a significant, positive correlation of the blood glucose level to the myocardial levels of citrate and malate (not 2‐oxoglutarate), acetoacetate and 3‐hydroxybutyrate, estimated intracellular glucose, hexose monophosphates, lactate, and the lactate/pyruvate ratio. The fructose 1,6‐diphosphate/fructose 6‐phosphate ratio was negatively correlated with the blood glucose level.

In the chronic alloxan‐diabetic rats myocardial citrate, malate, estimated intracellular glucose, glucose 6‐phosphate and pyruvate were positively correlated with the blood glucose level. The negative correlation of the fructose 1,6‐diphosphate/fructose 6‐phosphate ratio to the blood glucose level became of even greater significance than in group II (acute diabetic) animals and a negative correlation in respect to the myocardial ATP level appeared. The myocardial ketone‐body levels were uniformly low and showed no correlation to the blood glucose level.

In no group was there any correlation between cardiac FFA levels and the blood glucose level.

In another series of experiments with fed and starved, acute alloxan‐diabetic rats, there was no correlation between the blood glucose level and the extracellular levels of FFA, ketone bodies, lactate or pyruvate, nor the 3‐hydroxybutyrate/acetoacetate and lactate/pyruvate ratios.

A working theory is proposed for the correlation between certain myocardial substrate levels and the blood glucose level in diabetic rats.
Influence of Gramicidin D on Permeability of Swine Erythrocytes to Ions
Authors: BIELAWSKI J.
Published: March 2005

Gramicidin 0.01 μg/ml is found to cause swelling and subsequent haemolysis of swine erythrocytes suspended in NaCI solutions of different concentrations (120 to 800 mM). The swelling can be explained by increased permeability of the erythrocyte membranes to Na+ which enters the cells according to the Gibbs‐Donnan equilibrium. The gramicidin induced permeability of the membranes is higher for K+ than Na+. Sucrose as well as proteins do not penetrate the membrane in the presence of gramicidin. The permeability of the membranes induced by gramicidin is reversible. Erythrocytes can thus be loaded with sodium or potassium ions in the presence of gramicidin and the impermeability to these ions can be restored by subsequent washing.
Identification des sites d'attachement spécifiques d'arginine VIII et de valine dans des RNA d'origines différentes
Authors: BELJANSKI M. , FISCHER‐FERRARO C. , BOURGAREL P.
Published: March 2005

The binding sites, to which [14C]arginine and [14C]valine are linked to a messenger‐like RNA of Alcaligenes faecalis in the presence of ATP and a purified “polypeptide synthetase” preparation from the same organism, have been identified. From a large scale incubation mixture, [14C]Arg‐RNA was isolated and degraded with ribonuclease. By electrophoretic and chromatographic methods two compounds were obtained, CpUp‐[14C]Arg, and CpUp‐[14C]Arg‐Cp. Evidence is presented indicating that the carboxylic group of the amino acid was bound in ester linkage to the hydroxyl in 2′‐position of the uridylic ribose. The same compound were obtained when an RNA fraction from Escherichia coli was used. Similar experiment with [14C]valine yielded CpCp‐[14C]Val and CpCp‐[14C]Val‐Cp with either RNA fraction. With TYMV‐RNA as acceptor, CpCp‐[14C]Val was identified. The possible significance of these results is discussed.
Early Effects of Phytohaemagglutinin on Lymphocyte RNA Synthesis
Authors: KAY J. E.
Published: March 2005

Phytohaemagglutinin was found to cause a rapid increase in the incorporation of [3H]‐uridine into lymphocyte RNA and an increase in the rate of incorporation of [14C]leucine into protein which began about 2 h after its addition.

During the first few hours after its addition phytohaemagglutinin stimulated the labelling of cytoplasmic RNA more than the labelling of nuclear or whole cell RNA.

The cytoplasmic RNA labelled during the first 2 h of incubation of lymphocytes with phytohaemagglutinin sedimented mainly at 4 S when analyzed by sucrose gradient centrifugation, but part of it could be distinguished from transfer RNA. Phytohaemagglutinin stimulated the labelling of both fractions of the labelled cytoplasmic RNA more than the labelling of whole cell RNA.

Labelled ribosomal RNA was first found in the cytoplasm after incubation of lymphocytes with [3H]uridine for 3 h. Incubation with phytohaemagglutinin markedly stimulated the labelling of this fraction of RNA.
Dosage des pyridine nucléotides oxydés et réduits dans le sang et les tissus animaux
Authors: CARTIER P. H.
Published: March 2005

A fluorometric method is described for the estimation of di‐ and triphosphopyridine nucleotides (oxidized and reduced) in animal tissues.

The continuous cycling reduction of DPN+ (or TPN+) in the presence of excess of ethanol and alcohol dehydrogenase (or glucose‐6‐phosphate and glucose‐6‐phosphate dehydrogenase) was coupled with non enzymatic reoxidation of pyridine nucleotides with phenazine methosulfate.

Phenazine methosulfate reduced the non fluorescent compound, resazurine to the highly fluorescent compound, resorufine. The rate of production of resorufine is proportionnal to the concentration of pyridine nucleotides.

The procedure is relatively simple and requires only two commercial enzyme preparations for complete determination. This method can be used for estimation of pyridine nucleotides in concentrations as low as 0.1 μM and quantities as small as 10 pmoles.

The acid and alkaline extraction procedure was carried out essentially according to the method of Lowry. Conditions affecting the specificity, accuracy and reliability have been studied. During extraction, interfering material is formed, inhibiting the rate of reduction. A calibration procedure is described which overcomes this difficulty. Values for the concentrations of oxidized and reduced pyridine nucleotides in rat liver and in rat and human blood are presented and are in fairly good agreement with those of Lowry.
A Simplified Method for the Determination of the Nucleotide Composition of Polyribonucleotides by Spectrophotometric Analysis
Authors: RICHARDS E. G.
Published: March 2005

A simple method for detemining the base composition of ribonucleic acid hydrolysates is described. The method depends on the measurement of a difference spectrum between an aliquot of the hydrolysate at pH 7 and one at pH 11.8. This difference spectrum depends only on the concentrations of UMP and GMP present. The quantities of CMP and AMP are obtained from a spectrum at pH 7 after allowance for the contribution of the UMP and GMP.

It is demonstrated that the results obtained by calculating the concentration of a component from measurements of absorbances at two wavelengths only are of comparable accuracy to those obtained from the more conventional technique of employing measurements at many wavelengths. It is concluded that the use of a digital computor is not required for the application of this method. A theoretical explanation is offered for this observation.

The accuracy obtained is of the order of 0.02 in the mole fraction of a component.
Short Term Kinetics of RNA Labelling in HeLa Cells
Authors: SZAPARY‐WINCKELMANS D. , BRUNFAUT M. , EERRERA M.
Published: March 2005

The short term kinetics of labelling RNA fractions of HeLa cells with [5‐3H]uridine have been studied, as well as the distribution of 32P among their nucleotides after 30 minutes incubation. The fractions were obtained from a methylated bovine albumin serum column after amyl alcohol‐chloroform isolation. Emphasis was given especially to a “DNA‐bound RNA” stable in caesium sulfate and to a “free RNA component”, both eluting with the DNA from the methylated bovine albumin serum column.

The results indicate that the “DNA‐bound RNA” is rich in [32P]uridylic plus adenylic acid, whilst the free component is rich in [32P]cytidylic plus guanilic acid; [3H]uridine appears in both these fractions before it can be detected in the other RNA and both become more rapidly saturated with the label. After very short incubation times, the “DNA‐bound RNA” is completely resistant to ribonuclease, but its susceptibility to the enzyme increases progressively at longer incubations.

These results are compatible with a possible precursor role of the “DNA‐bound RNA” for mRNA and perhaps for rRNAs and tRNAs as well.
Formation of Polysomes in T2‐Infected Escherichia coli during Inhibition of Protein Synthesis
Authors: HAUGE J. G.
Published: March 2005

A comparison has been carried out between RNA made in the presence and absence of methionine in a T2‐infected RCstringentEscherichia coli requiring methionine. RNA was labelled with H32PO4‐or [3H]uracil for 4 minutes during the early phase of the infection. After sucrose density gradient centrifugation, the radioactive RNA formed had highest specific activity in the polysome region, and this activity was essentially the same for the two experimental conditions. The radioactive RNA from the polysomes was demonstrated to be hybridizable with T2 DNA. The findings indicate the presence of a mechanism which allows polysome formation and RNA synthesis to proceed normally under circumstances where deprivation of a required amino acid greatly reduces protein synthesis.
Effect of Steric Factors on Digestibility of Peptides Containing Aromatic Amino Acids by Cathepsin D and Pepsin
Authors: KEILOVÁ H. , BLÁHA K. , KEIL B.
Published: March 2005

Of the oligomers of phenylalanine studied, tri‐ and tetra‐phenylalanine methyl esters were found to be cleaved by cathepsin D at the Phe‐Phe bond. Substitution of the amino group by the benzyloxycarbonyl group renders the substrate resistant to cleavage. By contrast pepsin readily cleaves di‐phenylalanine methyl ester and its action is not inhibited by the substitution of the amino group.

In the hexapeptide Gly‐Phe‐Leu‐Gly‐Phe‐Leu, bonds 2–3, 3–4, and 5–6 are cleaved by pepsin. The same substrate is cleaved by cathepsin D only at bond 2–3, the value of the Michaelis constant for this reaction being Km= 3.3×10−4 M.

A change in configuration of the amino acid involved in the sensitive bond completely inhibits the cleavage of the linear hexapeptide by both enzymes. Differences in the steric arrangement of diastereomeric substrates affect the action of cathepsin D considerably more than that of pepsin. d‐Isomers competitively inhibit the cleavage of the l‐isomer.

The cyclic hexapeptide Gly‐Phe‐Leu‐Gly‐Phe‐Leu is cleaved neither with pepsin nor cathepsin and it does not inhibit the cleavage of the linear hexapeptide.
On the Metabolism of Cholesterol in Rat Liver Homogenates
Authors: BJÖRKHEM I. , DANIELSSON H. , EINARSSON K.
Published: March 2005

The metabolism of endogenous cholesterol was studied in fractions of liver homogenates obtained from rats that had received a single injection of [1,2‐3H]cholesterol 48 hours before preparation of the homogenates. The pattern of labeled products formed in incubations of the 800×g supernatant fluid, of the mitochondrial fraction, and of the 20,000×g supernatant fluid was essentially the same as that described in earlier investigations, in which the labeled cholesterol had been added to the homogenates in acetone solution or as an emulsion. The results are consistent with the view that a major pathway for the conversion of cholesterol into 5β‐cholestane‐3α,7α,12α‐triol involves the intermediate formation of cholest‐5‐ene‐3β,7α‐diol, 7α‐hydroxycholest‐4‐en‐3‐one, and 7α,12α‐dihydroxycholest‐4‐en‐3‐one.
Initiation of DNA Synthesis in a System of Synchronized L‐Cells: Rhythmicity of Thymidine Kinase Activity
Authors: MITTERMAYER C. , BOSSELMANN R. , BREMERSKOV V.
Published: March 2005

In mechanically synchronized L‐cells, the activity of thymidine kinase and dCMP deaminase shows a distinct periodicity during the mitotic cycle. The rise in enzyme activities occurs at about the onset of DNA synthesis, 6 hours after mitosis, and reaches a maximum 16 hours after mitosis. The maximum rate of thymidine incorporation into DNA is found at 10 hours. The increase of enzyme activity is 6 fold for thymidine kinase and 5 fold for dCMP deaminase. A rapid fall of both enzyme activities is observed between 18 and 22 hours, coinciding with the second synchronous mitosis. The increase in enzyme activities is probably due to de novo synthesis, as judged by the effect of actinomycin D and puromycin. It is suggested that during the presynthetic phase of the cell cycle, messenger RNAs are synthesized for these enzymes. The enzymes in turn are required for the following DNA synthesis.
Model Substrates for the Rapid Assay of Specific Ribonuclease Activity
Authors: MOLEMANS F. , FIERS W. , van MONTAGU M.
Published: March 2005

A new procedure is proposed for the assay of specific ribonuclease activity. The substrates are [14C]‐labeled benzyl esters of 3′‐ribonucleotides. Methods are described for preparing pure 3′‐uridylic acid, 3′‐cytidylie acid, 3′‐adenylie acid and 3′‐guanylic acid, and for the synthesis of [14C]phenldiazomethane. By reaction of the latter with the former, the four 3′‐ribonucleotide [14C]benzyl esters are readily obtained.

After enzymatic reaction of a ribonuclease preparation with one of the model substrates, the released [14C]benzyl alcohol is simply extracted by mixing with toluene. The radioactivity of the organic phase is then measured by liquid scintillation counting. The method has been used to detect contaminating nuclease activity in pancreatic ribonuclease preparations (specific for 3′‐uridylic acid and 3′‐cyidylic acid esters), and in Aspergillus T1 ribonuclease preparations (specific for 3′‐guanylic acid esters).
The Purification and Properties of Guanosine Diphosphate Glucose Pyrophosphorylase of Pea Seedlings
Authors: PÉAUD‐LENOËL C. , AXELOS M.
Published: March 2005

400 fold purification of GDP glucose pyrophosphorylase from pea seedlings concomitant with stabilization of enzyme activity has been accomplished by (NH4)2SO4 fractionation, followed by chromatography on Sephadex G200 and DEAE‐celluose columns. In this last step the enzyme is eluted with 0.20 M NaCl.

The enzyme is specifically activated by Mn2+. The apparent Km value for glucose‐1‐phosphate (37°, pH 7.5) is about 3 × 10−4 M. The GDP glucose'biosynthesis is found to be reversible, with the equilibrium in favour of pyrophosphorolysis.

The purified enzyme is not active with GTP plus mannose‐1‐phosphate or galactose‐1‐phosphate. No formation of nucleotide sugar is observed in the presence of glucose‐1‐phosphate plus ATP, ITP, dTTP, or CTP. A small residual UDPG pyrophosphorylase activity is activated by Mg2+.
Steroids in Germfree and Conventional Rats
Authors: GUSTAFSSON B. E. , GUSTAFSSON J.‐Å. , SJÖVALL J.
Published: March 2005

Steroid conjugates having the chromatographic properties of steroid sulphates were isolated from faeces of germfree rats. After solvolysis and purification of the steroids by silicic acid chromatography the trimethylsilyl ethers and O‐methyl oxime‐trimethylsilyl ether derivatives were analyzed by combined gas chromatography‐mass spectrometry. 3α,16α‐Dihydroxy‐5α‐pregnan‐20‐one was identified as the predominant C21O3 steroid. Small amounts of 3β,16α‐dihydroxy‐pregn‐5‐en‐20‐one and another 16‐hydroxylated pregnanolone, tentatively identified as 3β,16α‐dihydroxy‐5α‐pregnan‐20‐one, were also found.
Steroids in Germfree and Conventional Rats
Authors: GUSTAFSSON B. E. , GUSTAFSSON J.‐Å. , SJÖVALL J.
Published: March 2005

A fraction of solvolyzable sterol conjugates having the chromatographic properties of steryl sulphates was isolated from faeces of germfree and conventional rats. The major sterols in this fraction were studied by gas‐liquid chromatography and gas chromatography‐mass spectrometry. Cholesterol, desmosterol, lathosterol, campesterol, methostenol, and β‐sitosterol were the predominant solvolyzable sterols in conventional animals, whereas cholesterol, desmosterol, and β‐sitosterol were the quantitatively most important sulphurylated sterols in germfree animals. The relative amounts of conjugated lathosterol was significantly higher in the conventional than in the germfree animals. Solvolyzable conjugates of sterols with a 3β, 5β‐configuration could not be found.
Glycerol Metabolism in Yeasts
Authors: Gancedo C. , Gancedo J. M. , Sols A.
Published: March 2005

The utilization of glycerol by Candida utilis has been studied. It has been found that this yeast has a permeability for glycerol and other three carbon compounds much greater than that of baker's yeast. This permeability allows the entrance of glycerol in Candida cells rapidly enough to permit its efficient utilization even at low concentrations. The inducibility of glycerol kinase has been established. An increase in the concentration of the mitochondrial L‐α‐glycerophosphate oxidase when the yeast is grown on glycerol has also been observed.

A model is presented for the substrate specificity pattern of glycerol kinase of C. mycoderma. It postulates the involvement of three hydroxyl groups in the spatial distribution corresponding to the formation of L‐α‐glycerophosphate from glycerol. This requirement can be met by aldo‐and ketotrioses in their respective hydrated forms.

The pathway of glycerol formation in S. cerevisiae has also been studied. Evidence is shown of the existence of a NADH dependent enzymatic activity reducing triose phosphate to α‐glycerophosphate which can roughly account for the glycerol production. A low ionic strength seems to be required for the activity of this enzyme. The α‐glycerophosphatase is specific for the L from, the efficiency of α‐glycerophosphatase on D‐α‐glycerophosphate being 1/30 of that on L‐α‐glycerophosphate. The concentration of the α‐glycerophosphatase in yeasts is higher when grown on hexoses than when grown on non‐sugar carbon sources.
Strukturveränderungen und Dissoziation von Leberribosomen in Abhängigkeit von der Mg++ ‐Konzentration
Authors: Bielka H. , Welfle H. , Böttger M. , Förster W.
Published: March 2005

The sedimentation properties of ribosomes and ribosomal subunits from rat liver have been examined in media containing Mg2+ ions at different concentrations.

Purified ribosomes contain 51.4% RNA and 48.6% protein; the phospholipid content is less than 0.1%.

At 10−3 M Mg2+ the S value of monomeric ribosomes is 80 S. At 10−4 M Mg2+ and in Mg2+‐free media there are present in addition many particles having a S value of 55 S or 49 S respectively. We have attempted to determine the nature of these particles. All the proteins which we regard as characteristic of 80 S ribosomes, can be extracted from the 55 S particles. The particles also contain two components of RNA sedimenting at a mass ratio of 2:1 at 33 and 18 S, respectively. By raising the concentration of Mg2+ to 10−3 M the 55 S particles revert to 80 S particles. In the presence of EDTA the 55 S particles are dissociated into particles with values of 48 and 28 S.

The separation of the ribosomes into their subunits can be achieved only when all the ribosomal Mg2+ is removed by equimolar amounts of EDTA. The results of short and long time sucrose gradient centrifugation of ribosomes, which were treated before with EDTA clearly show, that the 80 S ribosomes are in an equilibrium with their subunits which is largely on the side of the dissociation products. Complete separation of the subunits with gradient centrifugation is possible only by continuous displacement of the equilibrium during prolonged centrifugation. The subunits have S values of 49 and 29 S in the presence of EDTA, and contain 45.6% RNA.

Studies on the sedimentation characteristics of mixtures of 32P‐labelled small subunits with the unlabelled large subunits or with 80 S particles exclude the possibility that the 55 S particle fraction consists of mixture of the 49 S subunit with dimers of the 29 S subunit. Thus our evidence supports the idea that the 55 S and 80 S particles are interconvertible forms of an intact ribosomal particle.

The results are interpreted as indicating that two processes result from a progressive reduction in the amount of Mg2+ bound to the 80 S ribosomes. Firstly, the structure of the subunits is changed and secondly, at the same time, the association of the subunits is loosened due to a stepwise cleavage of the Mg2+ dependent linkages between the subunits. Both processes change the structure of the ribosome such that its S value is reduced from 80 S to lower values. The 49 S and 29 S subunits become separated when the ribosomal Mg2+ is removed quantitatively.
Extraction, Purification and Preliminary Characterisation of the Epidermal Chalone:
Authors: Hondius Boldingh W. , Laurence E. B.
Published: March 2005

Pig skin powder, prepared in different ways, was extracted with water and fractionated for the purification of epidermal chalone.

Various fractionation methods were tried, e.g. ethanol fractionation, ion exchange chromatography, gel filtration and column electrophoresis. The most effective method proved to be ethanol fractionation followed by column electrophoresis at pH 3 and then dialysis.

Ethanol fractionation of a cod skin powder extract is also reported.

Although the biological tests involving depression of epidermal mitotic activity did not result in exact evaluation of the fractions, they did indicate that the active principle of crude pig skin extracts was purified about 2,000 times.

Characterisation studies show that the chalone obtained from pig skin is a protein or a glycoprotein which is stable at low pH, and has an isoelectric point between 5.2 and 6.0.

The most purified material shows in the ultracentrifuge a symmetrical peak corresponding to a molecular weight of 30,000 to 40,000. It may still be heterogeneous.
The Reaction of β‐Chloroglutamic Acid with Glutamate‐Aspartate Transaminase
Authors: Manning J. M. , Khomutov R. M. , Fasella P.
Published: March 2005

Porcine glutamate‐aspartate transaminase catalyzes a β‐elimination reaction with both the threo‐ and erythro‐isomers of β‐chloroglutamate; chloride, ammonia, and α‐ketoglutarate are formed in equimolar amounts. The latter product was characterized as the 2,4‐dinitrophenylhydrazone and by catalytic hydrogenation of this derivative to glutamic acid.

The β‐elimination reaction is catalyzed by highly purified glutamate‐aspartate transaminase; the reaction is not catalyzed by the phosphopyridoxamine form of the enzyme, the apoenzyme, or by free pyridoxal 5′‐phosphate. There is no detectable transamination between β‐chloroglutamate and either oxaloacetate or α‐ketoglutarate. Incubation of either the holoenzyme or the apoenzyme with β‐chloroglutamate does not result in any detectable loss of enzyme activity. In the presence of N‐ethylmaleimide much less α‐ketoglutarate than ammonia is formed; this is consistent with the idea that a reactive carbanion intermediate reacts with N‐ethylmaleimide.

The novel β‐elimination reaction with glutamate‐aspartate transaminase demonstrated here supports the idea that in some instances the specificity of an enzymatic reaction can be determined by the structure of the substrate.
General Properties of a Protease Inhibitor from the Seeds of Kidney Bean
Authors: Pusztai A.
Published: March 2005

A protein isolated from kidney bean was found to react and form a 1:1 enzyme‐inhibitor complex with trypsin, pancreatic elastase and, at low enzyme concentrations, with human plasmin. A similar reaction of the inhibitor with chymotrypsin occurred in two stages: at low enzyme concentrations a 1:1 complex formed while at high enzyme concentrations more chymotrypsin reacted in a non‐stoichiometric manner. The inhibitor had separate binding sites for trypsin and chymotrypsin. Plasmin in low concentrations was probably also bound at the trypsinreactive site by the inhibitor. The inhibitor had no effect on the activity of pepsin, papain, a protease from Bacillus subtilis and carboxypeptidase‐A but depressed the activity of a carboxypeptidase‐B and a thrombin preparation slightly. Complexes of the inhibitor with trypsin and chymotrypsin were prepared and isolated on a Sephadex G‐75 column and their properties were investigated. These complexes could be dissociated at or below pH 3. Free enzyme and inhibitor respectively were recovered by chromatography on a Sephadex G‐75 column from the dissociated chymotrypsin‐inhibitor complex. Under similar conditions, the extent of dissociation of a trypsininhibitor complex and the recovery of its components was much less. The inhibitor was found to be a small protein with a most probable molecular weight value of about 10,000. It was found to be unaffected by exposure to heating (up to 90°) or to pepsin and to low pH values (pH 2). Oxidation with performic acid or reduction with 2‐mercaptoethanol reduced the inhibitory activity to very low values. Re‐oxidation in air of the reduced and nearly inactive inhibitor failed to restore activity to any significant degree.
Kinetische Untersuchungen zum Mechanismus der Lactatdehydrogenase mit Tritium‐Isotopeneffekten
Authors: Palm D.
Published: March 2005

The mechanism of action of rabbit‐muscle lactate dehydrogenase was studied earlier by Thomson with deuteriated NADH. Reexamination of the reverse reaction with tritium‐labelled [A‐4‐3H]NADH and study of the forward reaction with L‐[2‐3H] lactate require a new interpretation of the isotope effects.

L‐[2‐3H]lactate yields an isotope effect of kH/k3H= 2.5 (25°) which decreases slightly with decreasing temperature. This is in agreement with the rate limiting influence of two steps: isomerisation of enzyme‐NADH complexes and NADH dissociation from this complex; both steps may cause secondary isotope effects. Their size may be compared to those in the rate limiting NADH dissociation of the liver alcohol dehydrogenase catalyzed reactions (1.3–1.8).

The existence of an isotope effect in the reverse reaction (1.0–2.0 at 25°) excludes the ratelimiting enzyme‐coenzyme isomerisation or coenzyme dissociation found in the forward reaction. Despite the similarity in size between the deuterium and tritium NADH isotope effects, the latter effect is too small to support kinetically significant ternary complexes as proposed by Thomson. As in the forward reaction, the isotope effects are secondary effects, which can arise during NADH binding, enzyme‐NADH isomerisation, or eventually, lactate dissociation.

The results are in agreement with an iso‐Theorell‐Chance mechanism as proposed by Zewe and Fromm and which indeed requires an influence of enzyme‐NADH isomerisation in the forward and reverse reaction.

With increasing concentrations of pyruvate the isotope effect of the reverse reaction disappears, due to a now rate‐limiting dissociation of NAD from the inhibitory complex enzyme‐NAD‐pyruvate.
Physico‐Chemical Properties of Bovine Chymotrypsinogen B
Authors: Delaage M. , Abita J. P. , Lazdunski M.
Published: March 2005

A comparative structural study of chymotrypsinogens A and B at acidic pH has shown in both cases an isomerisation I′⇌II′ occurs due to the unmasking of an abnormal carboxylate ion.

Chymotrypsin Bπ does not bind the proflavine which is a competitive inhibitor for trypsin and chymotrypsin Aα. The acidic transition observed for the zymogens also exists for the corresponding enzymes (I⇌II). The unmasking of the abnormal carboxylate ions involves a dissociation of the chymotrypsin Aα or trypsin‐proflavine complexes.

The thermal transitions of chymotrypsinogen B have been studied and compared with those of chymotrypsinogen A, trypsinogen and chymotrypsin Bπ.

The kinetics of denaturation by urea of chymotrypsinogen B, neochymotrypsinogen B, chymotrypsins Bπ and Bα were studied at different pH. The transformations of chymotrypsinogen B into neochymotrypsinogen and of chymotrypsin Bπ into chymotrypsin Bα are accompanied by a decreased stability in urea. The activation of chymotrypsinogen B to chymotrypsin Bπ increases the stability of the molecule. However, the rates of denaturation of states II′ and II of chymotrypsinogen and chymotrypsin Bπ are much more similar than the rates of denaturation of states I′ and I. The implications of this result are discussed. Ca2+ binds specifically to chymotrypsinogens A and B and to trypsinogen. (pKCa2+= 3.0, 3.1, and 3.2 respectively), probably on identical sites. The protection afforded by this binding against urea denaturation is of the same magnitude for trypsinogen and chymotrypsinogen A; it is much lower for chymotrypsinogen B. This observation together with the experiments concerning proflavine binding shows that chymotrypsinogen A shares with trypsinogen certain functional similarities that it does not share with chymotrypsinogen B.

The reactivity of phenol side‐chains has been studied by titration and specific modification with acetylimidazole. The localisation of the normal and abnormal tyrosines in the sequences of chymotrypsinogens A and B is discussed.

The reactivity of indole side‐chains was examined with 2‐hydroxy‐5‐nitrobenzyl bromide. The four indoles of trypsinogen and the eight indoles of chymotrypsinogens A and B are masked in the native forms (I′).
Kompartimentierte Verteilung von Enzymen in Rattenlebermitochondrien
Authors: Brdiczka D. , Pette D. , Brunner G. , Miller F.
Published: March 2005

Mitochondria with broken outer membrane were prepared from isolated rat liver mitochondria by incubation with digitonin according to the method of Schnaitman et al. [2]. Electron microscopic controls of these preparations revealed results similar to those obtained by a second method which is based on rapid freezing of mitochondria in a slightly hypertonic medium: the external mitochondrial membrane is ruptured or is partially stripped off, whereas the inner membrane remains intact and the mitochondrial matrix shows normal electronmicroscopic density.

The activities of the following enzymes were determined in mitochondria which were prepared either by the digitonin or by the freezing method: adenylate‐kinase, monoamine oxidase, malate dehydrogenase, TPN‐specific isocitrate dehydrogenase, condensing enzyme, succinate dehydrogenase, glutamate dehydrogenase, β‐hydroxyacyl‐CoA dehydrogenase.

The enzymological data prove that only adenylate‐kinase is eluted from the mitochondria when their outer membrane is opened by one or the other method. The extracted activity of adenylate‐kinase is quantitatively recovered in the supernatant.

The soluble activities of citric acid cycle enzymes as well as the activities of β‐hydroxyacyl‐CoA dehydrogenase and glutamate dehydrogenase can only be extracted after mechanical disruption of both mitochondrial membranes.

Small amounts of soluble citric acid cycle enzymes, of glutamate dehydrogenase and of β‐hydroxyacyl‐CoA dehydrogenase which are found in the supernatant after digitonin treatment of intact mitochondria result from a small portion of completely destroyed mitochondria as proved electromicroscopically and enzymologically by the detection of the structure bound activity of succinate dehydrogenase in the supernatant of digitonin treated mitochondria. Moreover, the activities of succinate dehydrogenase and soluble citric acid cycle enzymes which are present in the supernatant of digitonin treated mitochondria are found in the same proportions as in homogenates of sedimented intact mitochondria.

From the data obtained, it is concluded that citric acid cycle enzymes, glutamate dehydrogenase and β‐hydroxyacyl‐CoA dehydrogenase are located within the mitochondrial matrix and are not present within the outer mitochondrial membrane, that is the intracristal space. On the contrary, the total mitochondrial activity of adenylate kinase is present within the intracristal space.

Incubations of mitochondria with phospholipase‐A according to the method of Bachmann et al. [8] reveal no difference between the extractability of adenylate‐kinase and citric acid cycle enzymes. It is therefore suggested that Phospholipase‐A causes an opening of the intracristal space as well as of the mitochondrial matrix.
The Inhibition of Unsaturated Fatty Acid Biosynthesis in Plants by Sterculic Acid
Authors: James A. T. , Harris P. , Bezard J.
Published: March 2005

Sterculic acid, 8‐(2‐octyl‐1‐cyclopropenyl)octanoic acid, is a potent inhibitor of the enzyme system converting stearic acid to oleic acid in Chlorella vulgaris. No inhibition of oleic acid for mation from acetate, is, however, observed. With the C10, C12 or C14 acids as precursor, inhibition increases with chain length. The enzyme system converting oleic acid to linoleic acid is also less sensitive than the stearate‐oleate system. Stearic acid generated internally in leaf preparations anaerobically from acetate is still converted to oleic acid on transference to air in the presence of sterculic acid. Desaturation of added palmitic acid is readily inhibited by sterculic acid, so far as formation of the Δ7 and Δ9 acids are concerned. Desaturation of palmitic acid to the 3‐trans‐hexadecenoic acid is unaffected by sterculic acid.

Two possible schemes to account for the effects of sterculic acid on stearate desaturation are suggested.
Glucose Induced Inactivation of Malate Dehydrogenase in Intact Yeast Cells
Authors: Duntze W. , Neumann D. , Holzer H.
Published: March 2005

The glucose‐dependent inactivation of malate dehydrogenase (l‐malate: NAD oxidoreductase, EC 1.1.1.37) in acetate‐grown yeast cells was studied in vivo. Among different yeast species inactivation was observed only in strains of the genus Saccharomyces. The phenomenon is initiated by the addition of glucose or related hexoses to the medium and is reversibly interrupted at 0°. By use of a yeast mutant requiring tryptophan, it was shown that malate dehydrogenase inactivation is not influenced by inhibition of protein synthesis, whereas recovery of enzyme activity in inactivated cells requires the presence of an energy source and tryptophan and is presumably due to de novo protein synthesis. The reported results provide evidence that a metabolite formed in the first steps of glycolysis might be responsible for initiation of malate dehydrogenase inactivation.
A Model for Sulfhydryl Groups in Proteins. Hydrophobic Interactions of the Cysteine Side Chain in Micelles
Authors: Heitmann P.
Published: March 2005

A number of long chain N‐acyl‐glycines, N‐acyl‐dl‐serines, and N‐acyl‐dl‐cysteines have been synthesized by means of the mixed anhydride method. The critical micelle concentrations of the sodium salts of these compounds have been determined with the dye method. These values in the cysteine series are only half as high as in the glycine and serine series, i.e. the micelles of the cysteine derivatives are more stable. From these observations one may conclude that the cysteine side chain participates in the hydrophobic bonding system of the micelle. Moreover, the quantitative comparison of the values of the critical micelle concentrations leads to the conclusion that the CH2SH group has the same effect on the micelle stability as a methylene group of the hydrocarbon tail of the compounds investigated. Therefore, there is justification for the recently voiced assumptions of the hydrophobic nature of the cysteine side chain in proteins.
A Kinetic Investigation on the Allosteric Effects in Intestinal Sucrase
Authors: Semenza G.
Published: March 2005

This paper is a part of a project on the mechanism of activation of intestinal sucrase and of intestinal sugar transport by Na+. It reports a formal kinetic analysis of the following aspects:

Investigation on the apparent Km for sucrose hydrolysis in intestinal rings and homogenates does not indicate the existence of any gross diffusion barrier for sucrose between lumen and brush‐border bound sucrase. Therefore, it is possible to compare kinetic parameters of solu‐bilized sucrase with kinetics parameters of the intestinal sugar uptake system.

Intestinal sucrases from hamster, rat, Greek turtle, man and rabbit (the latter two reported in previous papers) are all activated by Na+. The modes of activation and the apparent Michaelis constants for Na+ are different for each species considered.

K+ and Li+ have effects similar to those of Na+.

The Kapps= f ([Na+]) functions indicate a non‐compulsory reaction sequence in Na+‐activation of sucrase.

Hamster and rat sucrases show a cooperative interaction between homologous sites. Papain solubilization does not make it disappear.

The pH‐activity curves are different in each species. In some species the percent activation by Na+ depends on the pH.

The Kochsaft of small intestine contains low molecular weight substance(s) which decrease the color yield in the glucose‐oxidase/peroxidase assay for glucose.
Change of Conformation of DNA by Association with Proteins
Authors: Cattan D. , Bourgoin D. , Joly M.
Published: March 2005

A comparison of the conformation in solution of DNA‐protein associations with that of free DNA has been carried out for lysozyme, ribonuclease and bovine serum albumin. The solutions have been studied simultaneously by ultracentrifugation, light‐scattering, spectropolarimetry and viscometry.

From the data it can be concluded:

For DNA‐ribonuclease and DNA‐bovine serum albumin complexes, the interaction forces between complex particles themselves or between particles and solvent, decrease with increasing amounts of bound protein since the viscosity decreases markedly while there is only a small variation in the radius of gyration. For DNA‐lysozyme complexes, the slight increase in viscosity for low amounts of bound protein results from a simultaneous decrease of the interaction forces and a thickening of the filament.

The molecular weight of DNA‐ribonuclease complexes increases as expected for a normal association. Those of DNA‐lysozyme complexes reveal at first a disaggregation of DNA under the influence of a low amount of protein, then with higher amounts of protein a normal increase in the molecular weight occurs up to the lysozyme concentration where aggregation sets in.

In the case of DNA‐bovine serum albumin complexes, the apparent molecular weight of DNA decreases continuously with increasing amount of bound bovine serum albumin.

The variation of the radius of gyration is dependent on the protein associated to DNA.

In no case has a variation of secondary structure of the components been induced by their association.
The Binding of Peptidyl‐tRNA and Acylaminoacyl‐tRNA to E. coli Ribosomes
Authors: de Groot N. , Fry‐Shafrir I. , Lapidot Y.
Published: March 2005

The non‐enzymatic binding of aminoacyl‐tRNA, N‐blocked aminoacyl‐tRNA, peptidyl‐tRNA and N‐blocked peptidyl‐tRNA to E. coli ribosomes was measured at 0.01 M magnesium ion concentration in the presence of poly U. It was found that the maximal amounts of phenylalanyl‐tRNA and peptidyl‐tRNA, that can be bound to the ribosomes are the same, but the amounts of N‐blocked phenylalanyl‐tRNA and N‐blocked peptidyl‐tRNA, that can maximally be bound to the ribosomes are much less.

Phenylalanyl‐tRNA and peptidyl‐tRNA can be bound to ribosomes which are saturated in respect to N‐blocked phenylalanyl‐tRNA or N‐blocked peptidyl‐tRNA; however, the presence of the N‐blocked substrates on the ribosomes partially inhibits the additional binding of phenylalanyl‐tRNA or peptidyl‐tRNA. Aminoacyl‐tRNA added to ribosomes saturated with respect to peptidyl‐tRNA, partially displaces the peptidyl‐tRNA from its ribosomal binding site. Under the same conditions, peptidyl‐tRNA exchanges with prebound aminoacyl‐tRNA considerably less.
Structure et activité de la polynucléotide phosphorylase d'Escherichia coli: une espèce à faible poids moléculaire
Authors: Thang M. N. , Thang D. C. , Grunberg‐Manago M.
Published: March 2005

A polynucleotide phosphorylase with a molecular weight of 100,000 has been isolated from Escherichia coli Q13 mutant and partially purified. This mutant is particularly rich in this low molecular species which can, however, also be found in small proportion in wild strains of E. coli B and K12.

Low molecular weight polynucleotide phosphorylase catalyzes only one of the reactions catalyzed by native polynucleotide phosphorylase (molecular weight 200,000), i. e. the phosphoro‐lysis of polyribonucleotides, and is unable to polymerize nucleoside diphosphates. However, in so far as the phosphorolysis reaction is concerned, there are some marked differences between the 100,000 polynucleotide phosphorylase and the 200,000 enzyme isolated from wild strains: reaction only with short chains polymers, higher requirement for Mg++ ions, non‐lineartity of the reaction as a function of protein concentration.

The Q13 mutant also contains a polynucleotide phosphorylase with a molecular weight of 200,000 but this enzyme is markedly different from the enzyme isolated from wild strains: in particular, it requires Mn++ for the polymerization reaction and has a higher Km for poly A in the phosphorolysis; for this latter reaction Mg++ and not Mn++ is required.

The oligomeric structure of polynucleotide phosphorylase and the relation between structure and activities is discussed.