bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)42– and Au(CN)2–
Authors: Gunnarsson Per‐Olov , Pettersson Gösta , Zeppezauer Michael
Published: March 2005

Single and multiple‐inhibition experiments provide evidence that the complex anion Pt(CN)42‐ is bound at a single site per subunit of liver alcohol dehydrogenase in strict competition with coenzyme, ADP‐ribose, AMP and (less than 100 mM) chloride ion, but independently of 1,10‐phenanthroline. Binding of adenosine decreases the stability constant for the enzyme · Pt(CN)42‐ complex from 16 mM‐1 to 4 mM‐1.

Au(CN)2‐ is bound at two sites per subunit of the enzyme. Both interactions are competitive with coenzyme, ADP‐ribose and AMP, but independent of 1,10‐phenanthroline. Only one site is available for interaction with Au(CN)2‐ in the binary enzyme complexes formed with Pt(CN)42‐, chloride ion, or adenosine. Estimation of the corresponding binding constants for Au(CN)2‐ gave evidence for cooperativity between the two Au(CN)2‐ binding sites.

These results strongly indicate that Pt(CN)42‐ is bound at a site within the protein region occupied by the phosphate groups of the coenzyme, and it is suggested that arginine‐47 in the enzymatic amino‐acid sequence is part of a common binding site for negative charges on the coenzyme and coenzyme competitive anions such as Pt(CN)42‐, Au(CN)2‐, chloride and iodoacetate. The second binding site for Au(CN)2‐ appears to be situated within the protein region occupied by the adenosine part of the coenzyme.
Structural Investigations on the Salmonella T2 Lipopolysaccharide
Authors: Bruneteau Maud , Volk Wesley A. , Singh Prem P. , Lüderitz Otto
Published: March 2005

Comparative chemical analyses have been performed on the lipopolysaccharides of a Salmonella T2 form and of a T2− mutant derived from it and found to belong to the chemotype Ra. Methylation analysis showed that the T2 structure contains an N‐acylglucosamine residue linked to position 4 of the subterminal glucose II unit of the core polysaccharide. The N‐acyl group and a substituent which renders this glucosamine periodate resistant have not been identified. The structure of the T2‐specific region was further studied by the analysis of two fragments. One was obtained after Smith degradation and had the structure glucosaminyl‐1,4‐glucosyl‐glyceraldehyde, the amino sugar carrying the N‐acyl group and the unknown substituent. The other fragment obtained after hydrazinolysis and acid hydrolysis was the trisaccharide GlcN‐1,4‐(GlcN‐1,2)‐Glc. The results show that the T2‐specific structure is represented by a substituted N‐acylglucosamine unit which is attached to the core at the same position as the T1‐ and the O‐specific structures in the corresponding lipopolysaccharides. These results are in agreement with findings obtained with a T2, SR(4,12) form and the corresponding T2− mutant.
Thermosensitive Mutants of Escherichia coli Unable to Propagate RNA Phage at 42 °C and Altered in Protein Synthesis
Authors: Lupker Jan H. , Verschoor Gerrit J. , Glickman Barry W. , Rörsch Arthur , Bosch Leendert
Published: March 2005

A procedure is described which can be employed to isolate the thermosensitive mutants of Escherichia coli affected in protein synthesis. The technique involves the enrichment of cells unable to propagate MS2 phage at 42 °C and the subsequent screening for thermosensitive growth and protein synthesis. Two of the mutants isolated in this way, QL‐152 and QL‐199, were studied in some detail. The synthesis of glutamyl‐tRNA by QL‐152 supernatant enzyme appeared to be thermosensitive. In the case of QL‐199 a 65% reduction of the glutaminyl‐tRNA synthesis was measured. Moreover QL‐199 cells contain an elongation factor EF‐Tu which is more thermolabile than wild‐type EF‐Tu.
Characterization of the Single‐Strand‐Specific Nuclease S1 Activity on Double‐Stranded Supercoiled Polyoma DNA
Authors: Germond Jacques‐E. , Vogt Volker M. , Hirt Bernhard
Published: March 2005

The DNA of polyoma virus consists of double‐stranded supercoiled circular molecules. The single‐strand‐specific nuclease S1 converts these molecules to unit length rods. The conversion occurs in a two‐step reaction, with nuclease S1 cutting first one of the two strands and then the intact strand opposite to this nick. Cleavage of intact polyoma DNA by nuclease S1 is a consequence of supercoiling. DNA molecules containing random single‐strand breaks introduced by physical or enzymatic means are also cut opposite to nicks.
Purification and Properties of NADP‐Dependent 17β‐Hydroxysteroid Dehydrogenase Solubilized from Porcine‐Testicular Microsomal Fraction
Authors: Inano Hiroshi , Tamaoki Bun‐ichi
Published: March 2005

17β‐Hydroxysteroid dehydrogenase, which catalyzes oxidoreduction between androstenedione and testosterone, has been solubilized by sonication from porcine testicular microsomal membrane for the first time. From the solubilized enzyme preparation, the dehydrogenase activity was precipitated by between 40 and 75% saturation of ammonium sulfate. After the precipitate was dissolved, the enzyme solution was applied to a Sephadex G‐100 column and the dehydrogenase activity emerged at 1.80 void volumes. For further purification, it was subjected to DEAE‐cellulose column chromatography; by eluting with a linear gradient of increasing phosphate concentration, the enzyme activity between 15 and 20 mM phosphate. Finally, when the partially purified enzyme preparation was applied to a Bio‐Gel P‐100 column, the dehydrogenase activity emerged at 1.46 void volumes. The final enzyme preparation was purified 1880‐fold in specific activity compared to the microsomal fraction.

The purified enzyme preparation approached apparent homogeneity, checked by discontinuous polyacrylamide gel electrophoresis at pH 8.3 and by thin‐layer gel chromatography. The final preparation was able to produce 67.7 nmol testosterone from androstenedione per min per mg protein at 37°C in the presence of NADPH, and could be stored at–20°C for 6 months with no significant loss of the enzyme activity. The molecular weight and molecular radius of the 17β‐hydroxysteroid dehydrogenase was estimated to be about 35500 and 2.37 nm, respectively, from the results of the gel filtration chromatography. In the phosphate buffer pH 7.4 maximal rate of reduction of androstenedione by the enzyme was observed at 50°C, and Q10 for the enzyme was 3.7, while the activation energy was 19500 cal. The rate of reduction of androstenedione at 37.5°C was about a half of the maximal one at 50°C in the phosphate buffer, whereas the reduction rate at 37°C was higher than the one at 50°C in the citric acid‐Na2HPO4 buffer pH 7.3. The enzyme for androstenedione showed a pH optimum between 6.5 and 7.5 in the presence of NADPH at 37°C but the optimal pH of the enzyme was shifted to 8.5 by raising the incubation temperature to 50°C. Estrone was the acceptor of the hydrogen released from NADPH by the 17β‐hydroxysteroid dehydrogenase and dehydroepiandrosterone served a similar purpose for androstenedione. The oxidation of testosterone by the enzyme in the presence of NADP+ proceeded at the same rate as the reduction of androstenedione. The purified enzyme preparation was devoid of any alcohol or 3‐hydroxyhexobarbital dehydrogenase activities or other enzymes related to testicular steroidogenesis.
The Self‐Association of Chymotrypsinogen A
Authors: Tung Ming Sung , Steiner Robert Frank
Published: March 2005

The self‐association of chymotrypsinogen A at pH 8.1 and at ionic strength of 0.021 has been examined by means of sedimentation equilibrium. The data have been analyzed by several methods and results compared. Application of the older methods available in the literature indicate that the concentration dependence of molecular weight may be well accounted for in terms of an indefinite association in which the consecutive stepwise association constants are equal and the second virial coefficient is small and positive.

A new method has been developed, based on series expansion, which selects the values of the second virial coefficient and the set of statistically significant consecutive association constants which give the optimum least squares fit of the data. Application of this method indicates that the association extends at least to the tetramer.

The best model for this process is that of a stepwise association for which the stepwise constants are of similar magnitudes and for which the upper limit of association is undefined. This is essentially equivalent to the indefinite model.

Frontal analysis using gel filtration gives results compatible with the above model.
Primary Structure of Lobster‐Muscle Arginine Kinase
Authors: Regnouf Françoise , Kassab Ridha , Fattotum Abdellatif
Published: March 2005

Cleavage of the S‐carboxymethylated arginine kinase with cyanogen bromide gives rise to eight peptides of 11, 17, 23, 39, 60, 81, 87, 103 amino‐acid residues, one of which is an overlap. Ion‐exchange chromatography on sulfopropyl‐Sephadex and gel filtration on Sephadex G‐50 fine, in urea medium, as well as electrochromatography have been used with success for their separation. Their amino‐acid composition and end‐group structure have been determined and compared to those of the whole protein. All peptides have COOH‐terminal homoserine as expected from the presence of methionine at the carboxyl end of the native protein. One acetylated fragment was identified as the N‐terminal portion of the protein. The seven major fragments account for the amino‐acid composition of the entire protein; from the sum of their amino‐acid composition, a molecular weight of 37687 was calculated.
Methylglyoxal Formation during Glucose Catabolism by Pseudomonas saccharophila
Authors: Cooper Ronald A.
Published: March 2005

Methylglyoxal synthase, which forms methylglyoxal from dihydroxyacetone phosphate, has been detected in both glucose‐ and succinate‐grown Pseudomonas saccharophila but there was no evidence for methylglyoxal production from d‐glyceraldehyde. The enzyme was purified 100‐fold from glucose‐grown cells and found to be very similar to Escherichia coli methylglyoxal synthase. It has a pH optimum of 8.2, a molecular weight of approximately 67000 and a Km for dihydroxyacetone phosphate of 0.09 mM. Inorganic pyrophosphate, 3‐phosphoglycerate, phosphoenol‐pyruvate and inorganic orthophosphate were all potent inhibitors, the first three compounds being competitive with respect to dihydroxyacetone phosphate with K1 values of 0.048 mM, 0.029 mM and 0.096 mM, respectively. Inorganic orthophosphate inhibition appeared to be more complex and was overcome in a co‐operative manner by increasing dihydroxyacetone phosphate concentration. Hill plots suggested that in the presence of inorganic orthophosphate there were three dihydroxyacetone phosphate‐binding sites on the enzyme.
Purification and Properties of an Extracellular Proteinase of Psychrophilic Escherichia freundii
Authors: Nakajima Motoo , Mizusawa Kiyoshi , Yoshida Fumihiko
Published: March 2005

A proteinase was purified from the culture filtrate of psychrophilic Escherichia freundii, by a combination of procedures, such as precipitation with ammonium sulfate, batchwise treatment with CM‐cellulose, chromatography on DEAE‐Sephadex and gel filtration on Sephadex G‐100. The final enzyme preparation was homogeneous with respect to sedimentation in the ultracentrifuge, acrylamide‐gel electrophoresis and immunodiffusion. The enzyme was an alkaline proteinase with the optimum pH at 10.0. The optimum temperature was 25°C at pH 10.0. The proteinase of psychrophilic E. freundii was fairly thermolabile. The purified enzyme was inhibited by metal‐chelating agents but not by diisopropylphosphofluoridate and p‐chloromercuribenzoate.

The molecular weight of the proteinase was estimated to be 45800 by sedimentation‐equilibrium studies, 45000 by gel‐filtration method and 51000 by polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulfate. The calculation of minimum molecular weight on the basis of zinc content yielded a value near 45400 which indicated the presence of 1 g‐atom zinc per mol enzyme. The proteinase had a sedimentation coefficient of 4.12 S, a diffusion coefficient of 77.4 μm2/s, a partial specific volume of 0.718 ml/g, an isoelectric point of pH 5.2, A1%1cm at 280 nm of 12.45, and an intrinsic viscosity of 0.048 dl/g. The amino‐acid composition indicated 429 residues per molecule and the absence of sulfhydryl groups and disulfide bridges. The NH2‐ terminal amino acid of the enzyme was shown to be glycine.

The extracellular Escherichia proteinase has been found to be a zinc‐containing alkaline metallo‐proteinase.
Isolation and Properties of a Staphylococcal Protease, Preferentially Cleaving Glutamoyl‐Peptide Bonds
Authors: Rydén Ann‐Christine , Phllipson Lennart , Rydén Lars
Published: March 2005

An extracellular protease of a mutant of strain 8325N of Staphylococcus aureus, was isolated by a three‐step procedure involving stepwise chromatography on DEAE‐cellulose, gradient chromatography on hydroxyapatite and gel filtration on Sephadex G‐150. The enzyme was purified 250 times to homogeneity with a recovery of 20–30%.

The protease has a molecular weight of about 29000 and consists of a single polypeptide chain as shown by gel filtration of native enzyme on calibrated columns of Sephadex G‐150 as well as gel filtration of denaturated enzyme on Sepharose 6‐B in 6 M guanidine‐HCl and dodecylsulfate‐polyacrylamide gel electrophoresis. Peptide maps also suggest a molecular weight around 30000. It is an acidic protein and is rich in aspartic acid and asparagine, but no cysteine or cystine can be detected. The N‐terminal sequence is Val‐Ile‐Leu‐Pro‐Asn‐Asn‐Asp. The enzyme contains no carbohydrate.

The protease preferentially hydrolyzes peptide bonds on the carboxyl‐terminal side of glutamic acid. In a few cases aspartoyl bonds were also cleaved. It has no activity towards dipeptides with N‐terminal glutamic acid. The activity was not affected by divalent cations or EDTA, but was completely abolished after incubation with diisopropylfluorophosphate suggesting that the enzyme is a serine protease.

The purified enzyme is stable between pH 3 and 10. It has maximum activity between pH 7.5 and 7.8 with casein as substrate and between pH 4.5 and 5.0 with hemoglobin as substrate.
Purification of DNA‐Dependent RNA Polymerases A and B from Ovaries of Xenopus laevis
Authors: Beebee Trevor J. C. , Butterworth Peter H. W.
Published: March 2005

A method was developed for preparing RNA polymerases A and B from ovaries of adult female Xenopus laevis, avoiding sonication procedures at high ionic strength. The technique involves homogenisation in low ionic strength glycerol buffers, precipitation of contaminants with streptomycin sulphate and concentration of enzymes by ammonium sulphate precipitation; this is followed by resuspension of the protein pellets, centrifugation through glycerol density gradients and chromatography on DEAE‐cellulose and phosphocellulose. A and B enzymes, separated at the DEAE‐cellulose step, were purified 90‐fold and 200‐fold respectively by these procedures and were free of ribonuclease and deoxyribonuclease contamination. The two enzymes exhibited properties known to be characteristic of the major eukaryotic RNA polymerases in their ionic strength optima, cation and template requirements and responses to α‐amanitin.
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.
Interaction of β‐Amylase with Substrates and Inhibitors with Comments on Koshland's Induced‐Fit Theory
Authors: Thoma John A.
Published: March 2005

Rebuttals are made to Marshall's criticism of our proposed binding mechanism for β‐amylase. Marshall's arguments are not soundly based in enzyme kinetics and are not convincingly supported by his own data.
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.
Delayed Expression of Neonatal Sexual Differentiation of Corticosteroid Patterns in Rat Bile
Authors: Gustafsson Jan‐Åke , Gustafsson Sven A.
Published: March 2005

The excretion of corticosterone metabolites in bile from male and female rats 21 to 56 days of age was studied. No sexual differences were observed in rats 21 and 28 days of age; in these animals 3β, 11β,21‐trihydroxy‐5α‐pregnan‐20‐one disulfate was the major corticosteroid but significant amounts of monosulfurylated 3α,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 3α,11β,15α,21‐tetra‐hydroxy‐5α‐pregnan‐20‐one were also excreted. From 35 days of age, sexual differences became successively more apparent in the biliary steroid excretion patterns and at 56 days of age female rats had developed a characteristic pattern of mono‐ and disulfurylated 3α,11β,15α,21‐tetra‐hydroxy‐5α‐pregnan‐20‐one whereas the male biliary steroid pattern was characterized by the complete absence of 15‐hydroxylated corticosteroids, by relatively large amounts of 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one disulfate and by the sex‐specific metabolite 5α‐pregnane‐3β,11β, 20β,21‐tetrol disulfate. It is concluded that the “programming” or “imprinting” of hepatic corticosteroid metabolism that occurs in neonatal male rats and results in the absence of 15‐hydroxy corticosterone metabolites and the presence of 5α‐pregnane‐3β, 11β,20β,21‐tetrol in bile of adult male rats is not expressed until after puberty.
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.
The Amino‐Acid Sequence of Plastocyanin from Sambucus nigra L. (Elder)
Authors: Scawen Michael D. , Boulter Donald , Brown Richard H. , Ramshaw John A. M.
Published: March 2005

The complete amino acid sequence of Sambucus nigra L. plastocyanin has been determined. The protein consists of a single polypeptide chain of 99 amino acids and one atom of copper, giving a molecular weight of 10517. The sequence shows considerable similarity to that of Chlorella fusca plastocyanin.
Antamanide. Relaxation Study of Conformational Equilibria
Authors: Burgermeister Wolfgang , Wieland Theodor , Winkler Ruthild
Published: March 2005

Ultrasonic absorption of antamanide in different solvents was measured in the frequency range of 0.5–100 MHz. Three relaxation effects could be resolved, each occurring at a given frequency which remains unchanged for the different solvents. The slowest effect disappears upon Na+‐complex formation. It is ascribed to a conformational transition of the peptide backbone. Rate constants of this effect are in the range of 106 s−1. The two other relaxation effects may be caused by rotational isomerism of amino‐acid side chains. The solvent dependence of the structural conformations is discussed.
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.
Tryptophan Synthase from Yeast
Authors: Wolf Dieter H. , Hoffmann Marion
Published: March 2005

Tryptophan synthase was purified from Saccharomyces cerevisiae with the aid of affinity chromatography up to a specific activity of 525 units/g protein. Electrophoresis on polyacrylamide gel showed one main protein component. Gel filtration on Sephadex G‐150 indicates an average molecluar weight of 143000. Dissociation with dodecylsulfate followed by dodecyl‐sulfate‐acrylamide gel electrophoresis leads to the conclusion that the enzyme is probably composed of four subunits of equal size. The Km‐values with respect to its substrates indoleglycerol phosphate, indole and l‐serine were estimated. l‐Serine was shown to increase the Km‐value for indoleglycerol phosphate about 7‐fold. Several indole analogues inhibit the tryptophan synthase reaction (B) (i.e. indole + serine → tryptophan); indoleacrylic acid is the most effective. The pH‐optimum of tryptophan synthase is around 6.9 to 7.0. Sulfhydryl modification by 5,5′‐dithiobis(2‐nitrobenzoic acid) and p‐chloromercuriphenylsulfonic acid results in a large loss of tryptophan synthase activity, which cannot be prevented by the substrates l‐serine and indole. The tryptophan synthase inactivating system from yeast affects the catalysis of both reactions (A) (i.e. indoleglycerol phosphate → indole + glyceraldehyde 3‐phosphate) and (B).
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.
The Kinetics of Substrate‐Competitive Inhibition in Ternary‐Complex Mechanisms for Enzyme Reactions Involving Two Substrates
Authors: PETTERSSON Gösta
Published: March 2005

Generalized relationships for interpretation of the kinetics of competitive inhibition in ternary‐complex mechanisms for enzyme reactions involving two substrates have been derived. These relationships can be applied to any special case of the random mechanism indicated by Scheme I, including rapid‐equilibrium and compulsory‐order cases, and are also valid for more extended ternary‐complex mechanisms (Scheme II) in which abortive enzyme‐product‐substrate complexes and ternary enzyme‐product‐inhibitor complexes are formed.
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.
Properties of Basic Amino‐Acid Residues
Authors: WAGNER Karl G. , ARFMANN Hans‐Adolf
Published: March 2005

The interaction of four basic polyamino acids, poly(l‐lysine), poly(l‐ornithine), poly(l‐arginine) and poly(l‐homoarginine) with nucleotides was investigated using equilibrium dialysis and circular dichroism. Polyhomoarginine was prepared by guanidization of polylysine. The interaction of 5′‐GMP exhibited equal values for the binding cooperativity in all cases, indicating that nucleotide‐nucleotide interaction, i.e. the stacking tendency of the guanine base, was the origin of this affinity. The affinity parameters of 5′‐GMP and other nucleotides directed towards the basic polypeptides, however, decrease in the series: polyarginine > polyhomoarginine > polyornithine > polylysine. This indicates that the ion bond between guanidinium and phosphate is stronger than that between ammonium and phosphate, and that decreasing the length of the poly(amino acid) side chains by one CH2 group results in an increase of affinity. The binding of 3′‐GMP and 5′‐GMP by polylysine and polyornithine induces a strong negative circular dichroism band in the absorption region of the base and in the case of polylysine also changes at shorter wavelength. Temperature‐dependent measurements of these circular dichroic spectra revealed that polylysine fits best to and forms the most stable complexes with helical arrangements of bound GMP molecules.
Influence of Ligands on the Coenzyme Dissociation Constants in Octopine Dehydrogenase
Authors: BAICI Antonio , LUISI Pier Luigi , OLOMUCKI Anna , DOUBLET Marie‐Odile , KLINCAK Jozef
Published: March 2005

Various ligands influence the affinity of coenzymes to octopine dehydrogenase. When the enzyme is saturated by the alternative substrate l‐arginine plus pyruvate or d‐octopine, the dissociation constants of NAD and NADH decrease more than one order of magnitude. Studies with stereoisomeric substrate analogues show that the configuration of the asymmetric carbon atom of the arginine moiety has a profound influence on the binding, but changing the configuration of the alanine moiety does not prevent good binding. Changes in pH and ionic strength do not appreciably alter the dissociation constants of NAD and NADH, which is taken as an indication that electrostatic forces are not very important for the binding process and that the driving forces for the coenzyme binding are essentially hydrophobic in nature. The values of the coenzyme dissociation constants in the presence of various ligands are consistent with a mechanism of compulsory order for coenzyme binding and release. The binding of nicotinamide 1:N6‐ethanoadenine dinucleotide (ɛ‐NAD, an analog of NAD with a fluorescent tag on the adenine ring) is accompanied by a remarkable enhancement of the coenzyme fluorescence and by a blue shift of the fluorescent maximum, showing that the adenosine moiety must be directly involved in the coenzyme binding, also that the coenzyme probably binds in an open conformation to the enzyme. The binding data obtained by following the enhancement of the ɛ‐NAD fluorescence are in agreement with those obtained by monitoring the quenching of the protein fluorescence.
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.