bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis of Individual Ribosomal Proteins during a Nutritional Shift‐Up
Authors: Carpenter Graham , Sells Bruce H. 
Published: March 2005

Although individual ribosomal proteins are synthesized at the same rate during exponential growth, during a nutritional shift‐up, they are synthesized at different rates. Proteins S6, S10, S14, S20 and L2, L3, L4 and L13 are synthesized to the greatest extent. Proteins S2, S3, S4, S13, S17 and L7, L8, L9, L25, L29 and L30 are synthesized at the lowest rate. Although the average rate of synthesis of 50‐S protein is greater than that for 30‐S protein during a shift‐up, the data on rates of synthesis of individual proteins do not support such a distinction for each individual ribosomal protein.The extent of increased synthesis for the individual ribosomal proteins of the 30‐S subunits correlated well with the assembly order of these proteins. This correlation is not as striking for the 50‐S subunit.
Isopycnic Centrifugation of Free and Membrane‐Bound Polysomes from Rat Liver
Authors: Dissous Claude , Caner François , Krembel Jean 
Published: March 2005

Rat‐liver polysomes have been studied in CsCl buoyant density gradients.

Membrane‐bound polysomes band at a density of about 1.53 g/cm3, whereas free polysomes are recovered from the gradients at two higher densities (about 1.56 and 1.545 g/cm3, called, respectively, type I and II). Both types of free polysomes appear heterogeneous on sedimentation. They are found in all regions of the polysomal gradient.
Monoribosomes obtained after mild ribonuclease digestion of either free or membrane‐bound polysomes band at a unique density (1.55 g/cm3).
The two types of free polysomes have quite different sedimentation behaviors in the preparative discontinuous sucrose gradient. After 6 h of centrifugation at 113000 ×gav the polysomal pellet is mainly composed of the type I. Type II remains with rough membranes at the lower interphase and is recovered in the pellet only after a 20‐h centrifugation.
During starvation, it appears that the proportion of type I polysomes increases, while the proportion of type II decreases.

Possible interaction with membranes of the type II polysomes is discussed.
Immobilization of Urease by Thiol‐Disulphide Interchange with Concomitant Purification
Authors: Carlsson Jan , Axén Rolf , Brocklehurst Keith , Crook Eric M. 
Published: March 2005

Urease has been immobilized by covalent attachment to thiolated Sepharose via a mixed disulphide derivative of Sepharose, obtained by reacting thiolated Sepharose with 2,2′‐dipyridyl‐disulphide (2‐Py‐S‐S‐2‐Py). The immobilized urease was used for continuous hydrolysis of urea.
The immobilization procedure described accomplished a 6–10‐fold purification of the enzyme preparation used. Since the coupling is reversible, the technique can be used for purification of urease.
The chemical selectivity of the mixed disulphide derivative of Sepharose toward urease is discussed with the assumption of close analogy between the mixed disulphide of Sepharose and the reagent 2‐Py‐S‐S‐2‐Py. The reactivity of 2‐Py‐S‐S‐2‐Py, in contrast to other commonly used thiol reagents, appears to be directed preferentially toward thiol groups which are of importance for the urease activity.
Heterogeneity of Triglyceride Lipase of Rat Adipocytes
Authors: Chmelařaron;ová Marcela , Chmelař Miroslav 
Published: March 2005

Triglyceride and monoglyceride lipase activities were compared in different preparations from rat adipose tissue or isolated adipocytes: fat‐free homogenate, 105000 ×g fat‐free infranatant (soluble fraction) and 105000 ×g sediment (particulate fraction) were assayed. The ratio of monoolein to triolein cleavage was 6.8 in whole homogenate and 1.9 and 20 in particulate and soluble fractions, respectively. Lipoprotein lipase activity of these enzyme preparations was not measurable under the experimental conditions used.
Since specific activities especially of triglyceride lipase(s) were low, other substrates were tested to increase the sensitivity. Among them 2‐naphthol laurate was found to be hydrolyzed by the whole homogenate at a rate about 50 times higher than triolein.
2‐Naphthol laurate was readily hydrolyzed by lipases in all preparations studied. Dioxane and ethylene glycol, the typical inhibitors of monoglyceride lipase activity strongly inhibited its cleavage in the soluble fraction whereas the typical inhibitors of triglyceride lipase activity (Triton ×‐100, lanthanum nitrate, 2‐propanol) were effective in inhibiting 2‐naphthol laurate cleavage particularly in 105000 ×g sediment.
Heat inactivation, pH optima and starch‐block electrophoresis confirmed the difference between lipases in particulate and soluble fractions. Whereas in the soluble fraction these criteria did not reveal further heterogeneity, the particulate fraction was apparently heterogenous and exhibited two pH optima at 6.5 and 8, a non‐linear rate of heat inactivation with time and two electrophoretic fractions, thus suggesting the presence of two distinct triglyceride lipases.
The monoglyceride lipase character of the soluble lipase is supported by the kinetic evidence of competition between monoolein and 2‐naphthol laurate. The particulate lipases, on the other hand, exhibited competitive inhibition of 2‐naphthol laurate cleavage by triolein, which supports their triglyceride lipase character.
2‐Naphthol laurate might be thus a valuable tool in the study of both monoglyceride and triglyceride lipase activities in adipose tissue. The possibility of high dilution of enzyme preparation excludes the interference of endogenous substrate, the preparation of a stable substrate emulsion is easy and the estimation of the first product of the reaction is rapid and simple.
Sequence Analysis of Fluorescamine‐Stained Peptides and Proteins Purified on a Nanomole Scale
Authors: Vandekerckhove Joël , Van montagu Marc 
Published: March 2005

Some techniques, facilitating the determination of the amino‐acid sequence of proteins or protein fragments available in 10 nanomole amounts are presented.Fluorescamine was used to follow the purification of proteins and peptides without interfering in the determination of the amino‐acid composition and sequence.These techniques were applied on the coat protein of coliphage MS2 and on some peptides obtained from a chymotryptic digest of the assembly protein of MS2.
Quaternary‐Structure‐Dependent Proton Dissociation of Tyrosines, Lysines and Cysteines in Human Hemoglobin
Authors: Janssen Lambert H. M. , Willekens Frans L. A. , de Bruin Simon H. , Van Os Gerard A. J. 
Published: March 2005

The titration behaviour of tyrosines, lysines and the –SH groups of the β‐93 cysteines in human carboxy and deoxyhemoglobin was studied in the pH region 9 to 10.3 at an ionic strength of 0.1 and a temperature of 25°C. Normal acid‐base titrations of native hemoglobin as compared with those of hemoglobin modified by blocking the reactive –SH groups by iodoacetamide yielded information about the titration behaviour of the –SH groups. Combining these data with the results obtained from spectrophotometric tyrosine titration studies, the proton dissociation of the lysyl residues could be studied. In addition the titration behaviour of the tyrosines and lysines in the α and β‐chains was measured.The results of the spectrophotometric titrations showed that per heme in the free α‐chains all three tyrosines are titratable but only two in the β‐chains. In deoxyhemoglobin fewer tyrosines are titrated than in carboxyhemoglobin. The average number of titratable tyrosines in the isolated chains is larger than in carboxy or deoxyhemoglobin.The acid‐base titration curves of native carboxy‐ and deoxyhemoglobin are identical up to pH 10.3. Blocking the β‐93 –SH groups causes no changes in the pH region studied. This indicates that the pK of these groups is certainly higher than 11, both in carboxy and deoxyhemoglobin.The titration of the lysines appeared to be similar in the case of the isolated α and β‐chains. Also in carboxy and deoxyhemoglobin the lysines behaved nearly the same. However, the number of lysines titrated up to pH 10.3 is smaller than expected when compared to the behaviour of the isolated chains.
Evidence for a Proton‐Dependent Regulation of Mitochondrial Nicotinamide‐Nucleotide Transhydrogenase
Authors: Rydström Jan 
Published: March 2005

The relationship between energy requirement, [NAD+] [NADPH]/[NADH] [NADP]+ ratio and velocity of the nicotinamide‐nucleotide transhydrogenase reaction, catalyzed by submitochondrial particles from beef heart, was investigated under conditions where the pH and the energy level were varied.
At low energy levels, obtained by decreasing the rate of oxidation of respiratory substrates, energy‐dependent anilinonaphthalene sulfanote fluorescence is a sensitive tool for estimating the relative energy requirement by the transhydrogenase reaction.
The velocity of the energy‐linked transhydrogenase reaction is directly proportional to the anilinonaphthalene sulfonate fluorescence.
The energy requirement is related to the total rate of the transhydrogenase reaction rather than to the degree of activation of the reaction by energy. Under conditions where the energy requirement and the total rate of the transhydrogenase reaction are constant, a decreased pH is accompanied by a decrease in the activation by energy.
It is concluded that the kinetic effect and the thermodynamic effect of energy on the transhydrogenase reaction may be separated by varying the pH of the reaction medium. Acidification in the absence of an energy source promotes the conversion of the transhydrogenase into an active form, presumably a protonated species of the enzyme, the kinetic properties of which are similar to those of the energy‐activated enzyme. In the presence of an energy source acidification may replace energization as the means for activation of the transhydrogenase.
The relationship between the proton‐activated and the energy‐activated forms of the transhydrogenase with regard to the coupling mechanism is discussed.
Histones from Baker's Yeast
Authors: Franco Luis , Johns Ernest W. , Navlet Jacinto M. 
Published: March 2005

Baker's yeast histones have been isolated either by acid extraction of purified chromatin or by salt‐dissociation of crude chromatin. These two methods gave similar results as judged by the amino‐acid composition and electrophoretic behaviour of histones.
Chemical fractionation methods have been used to isolate both, moderately lysine‐rich and arginine‐rich histones. Neither F1 nor F3 histones could be detected in yeast chromatin throughout these fractionation procedures. Results obtained in experiments of interactions between yeast whole histones ans calf thymus DNA also confirm the absence of a very lysine‐rich histone in yeast.
The fractionation of arginine‐rich histones was achieved by selective precipitation with acetone. F2A1 and F2A2 histones were obtained, purified and characterised by electrophoresis and amino acid analyses. The similarities between yeast and other organisms histone fractions are discussed.
Wheatgerm Hexokinase: Physical and Active‐Site Properties
Authors: Higgins Trevor J. C. , Easterby John S. 
Published: March 2005

The LII form of wheatgerm hexokinase has been purified and is homogeneous by criteria of electrophoresis and ultracentrifugation. The purified enzyme has a specific activity of 3.5 units/mg with respect to glucose and is a single polypeptide chain of 51000 molecular weight.On the basis of Km values, fructose appears to be the preferred hexose substrate in contrast to the hexokinases of mammals and yeast. Data from “dead‐end” inhibition studies suggest that a rapid‐equilibrium random mechanism obtains but end‐product inhibition indicates that the mechanism may show some additional complexities.The pH dependence of the alkylation and inactivation of the enzyme by iodoacetamide indicates the participation of a group of pKa 6.58 in these processes. This group is not itself alkylated, but its state of protonation determines the rate of the alkylation reaction. Substrates and products affect the rate of alkylation, and the dissociation constants of the enzyme · substrate and enzyme product · complexes are similar to the Km and Ki values for these ligands. These data also are consistent with a random mechanism. Studies on the pH dependence of photo‐oxidation of the enzyme show a bisphasic loss of enzyme activity with respect to time. Two ionizing groups with pKa values of 6.78 and 7.44 are involved in the first and second phases, respectively. Investigation of the effects of ligands on the rate of photooxidation shows that, for the first phase, the values for the dissociation constants are equal to the Km and Ki values for the substrates and products. However, for the second phase the values are greatly elevated. This may imply that a conformation change occurs in the enzyme following the initial phase of photo‐oxidation.A possible role for hexokinases in germinating cereal seeds is discussed.
Action of Thyrotropin on Phosphate Incorporation into Thyroid Proteins in vitro
Authors: Lamy Françoise , Dumont Jacques E. 
Published: March 2005

The kinetics of [32P]phosphate incorporation into dog thyroid slices, in vitro, has been studied. Phosphate penetration was saturable which suggests a mediated transport. This transport appeared to be the limiting step in the slow incorporation of [32P]phosphate in the trichloroacetic‐acid‐soluble fraction of the slices. The incorporation of [32P]phosphate in thyroid proteins was a fast process (half equilibrium with the trichloroacetic‐acid‐soluble fraction radioactivity in 30 min). About 50%, of this incorporation took place in the nuclear proteins, 25%, of it in the histones (mostly in the arginine‐rich histones). The turnover of protein P was fast in all classes of proteins.Thyrotropin, in vitro, often enhanced the incorporation of [32P]phosphate in the trichloro‐acetic‐acid‐soluble fraction and into total proteins of the slices. However, this effect was delayed and rather small. It is ascribed mainly to an enhancement of phosphate penetration into the cells. This suggests, that, in dog thyroid cells, most of protein phosphorylation is independent of thyrotropin and cyclic AMP.Thyrotropin markedly enhanced the phosphorylation of F1 (lysine‐rich) histones. This effect was fast, specific and mimicked by dibutyryl cyclic AMP; it required higher concentrations of thyrotropin than the activation of iodide binding to proteins. The role of this action of thyrotropin is discussed; the hypothesis is proposed that it is related to thyrotropin‐induced growth of thyroid tissue.NaF (5 mM) enhanced total protein phosphorylation in the thyroid slices. This effect, which may be due to unspecific inhibition of protein phosphatases, may explain the fact that NaF mimics several effects of thyrotropin.
Cellular Macromolecule Synthesis in Escherichia coli Infected with Bacteriophage MS2
Authors: Berzin Valdis , Rosenthal Guntis , Gren Elmar J. 
Published: March 2005

In Escherichia coli cells infected with the bacteriophage MS2, pronounced inhibition of ribosomal RNA synthesis occurs amounting to 50%, of that of the uninfected control cells at 30 min post‐infection. Over the same period the synthesis of tRNA, mRNA and total protein remains close to the level of uninfected E. coli, yet the synthesis of host protein is gradually replaced by that of viral origin. After 30–35 min post‐infection the macromolecular synthesis of all the types under study significantly decreases.It is suggested that suppression of host protein synthesis is caused not by selective inhibition at transcription or translation level, but is rather a result of competition between E. coli mRNA and MS2 RNA. On the other hand, exclusive suppression of only ribosomal RNA synthesis may arise from changes in the regulation of transcription of ribosomal RNA locus.
Synthesis and Processing of Ribosomal Ribonucleic Acid in Salivary Gland Cells of Chironomus thummi
Authors: Serfling Edgar , Maximovsky Leonid F. , Wobus Ulrich 
Published: March 2005

Experimental data on the RNA content and RNA base composition in different microisolated cell compartments, and on the synthesis and processing of ribosomal RNA in cells of the Chironomus thummi salivary gland are presented.The nucleolus of a Chironomus salivary gland cell contains about 250 pg RNA representing nearly 50% of the RNA of chromosomal material. The RNA content of chromosomes I–III from 82, 79 and 73 pg corresponds well to their relative lengths. The base composition of nucleolar RNA (46.5% G + C) is similar to that of cytoplasmic RNA (47% G + C) but different from Balbiani ring RNA (37% G + C).The synthesis and processing of precursor and ribosomal RNA were studied in vitro by incubation of glands in Cannon's insect medium supplemented with [3H]uridine and [3H]cytidine, and in vivo by injection of the isotopes into animals followed by gel electrophoresis of RNA extracted from microisolated nuclear components (nucleolus, chromosomes, nuclear sap) and cytoplasm. During 1 h of incubation, mainly one RNA fraction with a molecular weight of about 3 × 106 (38‐S RNA) is synthesized within the nucleolus. This becomes converted into smaller RNA molecules of 1.66 ×106 (30‐S) and 0.96 × 106 (23‐S) during long‐term labelling (or under chase conditions). In some of the nucleolar RNA preparations 0.72 × 106 (18‐S) RNA was also detected. Low‐molecular‐weight RNA in the 4–5‐S range is present after all incubation times, but without a distinct 4‐S peak while 5‐S RNA increases in amount with time of incubation. An additional low‐molecular‐weight RNA possessing a molecular weight of about 0.77 × 105 was detected in preparations of nucleolar RNA labelled for at least 3 h.RNA from chromosomes I–III contains, besides HRNA and low‐molecular‐weight RNA, RNAs with molecular weights 1.55 × 106 (“30‐S”), 0.96 × 106 (23‐S) and/or 0.72 × 106 (18‐S) representing (pre)‐rRNA species. “30‐S” and 18‐S RNA are also present in the nuclear sap while a distinct 23‐S peak is absent. Although in whole larvae mature rRNAs can only be found after 1‐h labelling, clear rRNA peaks in cytoplasmic RNA from salivary glands are not found earlier than after a 6‐h incubation.An RNA of molecular weight 0.65 × 105 first observed in nucleolar RNA preparations and accumulated in cytoplasm is suggested to be (pre)‐rRNA formed during processing by an endonucleolytic activity.
Immunochemical Characterization of Naja naja siamensis Toxin and of a Chemically Modified Toxin
Authors: Aharonov Aharon , Fuchs Sara , Gurari Dalia 
Published: March 2005

High titers of antibodies against Naja naja siamensis toxin were produced in rabbits by repeating immunizations with the toxin in complete Freund's adjuvant. Antibody fractionation was achieved by using a toxin‐Sepharose immunoadsorbent. Naja naja siamensis toxin was radio‐actively labelled with 125I; the iodinated toxin had a specific activity of 7 Ci/mmol. Several sensitive immunological techniques were applied for the detection of the antibodies and determining their specificity, These included radioimmunoassay, microcomplement fixation and the modified phage technique. The toxin may be quantitatively determined in very minute amounts by utilizing these immunological assays. No immunological cross‐reactivity was detected between Naja naja siamensis toxin and α‐bungarotoxin.A chemically modified derivative of Naja naja siamensis toxin was prepared by polyalanylation. This modification resulted in partial decrease in the antigenic activity of the toxin.
Kinetics of the Inhibition of Citrate Synthase from Pig Heart by Substrate Analogues
Authors: JOHANSSON Carl‐Johan , PETTERSSON Gösta 
Published: March 2005

1. The kinetics of inhibition of pig‐heart citrate synthase by the substrate analogues ATP, propionyl‐CoA, and 2‐oxoglutarate have been investigated. Evidence is presented that the citrate‐synthase‐catalyzed condensation reaction between acetyl‐CoA and oxaloacetate proceeds by an effectively ordered ternary‐complex mechanism with oxaloacetate adding first to the enzyme.2. Inhibition of citrate synthase by 2‐oxoglutarate is competitive with oxaloacetate, and due to the formation of a binary complex with a stability constant of 0.19 mM−1. Inhibition by ATP and propionyl‐CoA is competitive with acetyl‐CoA. ATP binds equally strongly (a binding constant of 2.0 mM−1) to the free enzyme and the binary enzyme · oxaloacetate complex, whereas propionyl‐CoA inhibits mainly through the formation of a ternary enzyme · propionyl‐CoA · oxaloacetate complex with a stability constant of 150 mM−1.3. The affinity of citrate synthase for acetyl‐CoA and propionyl‐CoA increases about 20‐fold on the binding of oxaloacetate to the enzyme. This enhancement of the binding‐strength is suggested to be due to an oxaloacetate‐induced interaction between enzyme or enzyme‐bound oxaloacetate and the acyl moiety of acetyl‐CoA and propionyl‐CoA, which has the function of accommodating the acyl group properly in the active site.
Investigation of the α‐Galactosidase Deficiency in Fabry's Disease Using Antibodies against the Purified Enzyme
Authors: RIETRA Peter J. G. M. , TAGER Joseph M. , BORST Piet , MOLENAAR Jacques L. , HAMERS Michiel N. 
Published: March 2005

1. α‐Galactosidase A, the enzyme deficient in Fabry's disease, was purified from normal human urine. The final preparation hydrolysed about 50 μmol p‐nitrophenyl‐α‐galactoside per h per mg protein at 37 °C. An antiserum against this enzyme was raised in rabbits. Preincubation of preparations of normal kidney and urine with the antiserum, followed by centrifugation, led to a marked reduction of the α‐galactosidase activity of the preparations.2. There was no influence of the antiserum on the residual α‐galactosidase activity in Fabry kidney or urine, or on an α‐galactosidase B preparation from normal urine.3. Pretreatment of the antiserum with urine or kidney preparations from a Fabry patient did not result in detectable loss of antibodies reacting with α‐galactosidase A, as shown by the unimpaired ability of the antiserum to diminish α‐galactosidase activity in normal kidney and urine. Furthermore, double immunodiffusion of the pretreated antiserum with a partially purified α‐galactosidase preparation, showed that antibodies not absorbable by the Fabry material were still present in the antiserum.4. Incubation of normal urinary α‐galactosidase with antiserum, followed by centrifugation and enzyme assay in the supernatant, led to the same reduction of α‐galactosidase activity, regardless of whether a Fabry urine or kidney preparation was simultaneously present or not.5. It is concluded that no detectable cross‐reacting material was present in this case of Fabry's disease.