bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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+.
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.
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.
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.